US20030131376A1 - Nucleic acid sequences encoding cell wall-degrading enzymes and use to engineer resistance to fusarium and other pathogens - Google Patents
Nucleic acid sequences encoding cell wall-degrading enzymes and use to engineer resistance to fusarium and other pathogens Download PDFInfo
- Publication number
- US20030131376A1 US20030131376A1 US10/316,754 US31675402A US2003131376A1 US 20030131376 A1 US20030131376 A1 US 20030131376A1 US 31675402 A US31675402 A US 31675402A US 2003131376 A1 US2003131376 A1 US 2003131376A1
- Authority
- US
- United States
- Prior art keywords
- nucleic acid
- nucleotide
- polypeptide
- seq
- plant
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- 150000007523 nucleic acids Chemical group 0.000 title claims abstract description 248
- 241000223218 Fusarium Species 0.000 title abstract description 53
- 102000004190 Enzymes Human genes 0.000 title abstract description 40
- 108090000790 Enzymes Proteins 0.000 title abstract description 40
- UHPMCKVQTMMPCG-UHFFFAOYSA-N 5,8-dihydroxy-2-methoxy-6-methyl-7-(2-oxopropyl)naphthalene-1,4-dione Chemical compound CC1=C(CC(C)=O)C(O)=C2C(=O)C(OC)=CC(=O)C2=C1O UHPMCKVQTMMPCG-UHFFFAOYSA-N 0.000 title abstract description 36
- 244000052769 pathogen Species 0.000 title abstract description 16
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 256
- 229920001184 polypeptide Polymers 0.000 claims abstract description 255
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 255
- 108010022172 Chitinases Proteins 0.000 claims abstract description 218
- 102000012286 Chitinases Human genes 0.000 claims abstract description 199
- 101000895977 Amycolatopsis orientalis Exo-beta-D-glucosaminidase Proteins 0.000 claims abstract description 153
- 230000000694 effects Effects 0.000 claims abstract description 134
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 103
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 103
- 238000000034 method Methods 0.000 claims abstract description 96
- 230000014509 gene expression Effects 0.000 claims abstract description 54
- 241000196324 Embryophyta Species 0.000 claims description 198
- 239000002773 nucleotide Substances 0.000 claims description 176
- 125000003729 nucleotide group Chemical group 0.000 claims description 176
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 139
- 239000012634 fragment Substances 0.000 claims description 90
- 239000013612 plasmid Substances 0.000 claims description 67
- 235000021307 Triticum Nutrition 0.000 claims description 66
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 51
- 150000001413 amino acids Chemical class 0.000 claims description 39
- 241000209510 Liliopsida Species 0.000 claims description 33
- 230000000295 complement effect Effects 0.000 claims description 22
- 238000004519 manufacturing process Methods 0.000 claims description 21
- 244000053095 fungal pathogen Species 0.000 claims description 8
- 241000209140 Triticum Species 0.000 claims description 4
- 230000001131 transforming effect Effects 0.000 claims description 4
- 230000002708 enhancing effect Effects 0.000 claims 1
- 239000013598 vector Substances 0.000 abstract description 63
- 108700005088 Fungal Genes Proteins 0.000 abstract description 6
- 239000002299 complementary DNA Substances 0.000 description 172
- 210000004027 cell Anatomy 0.000 description 162
- 108090000623 proteins and genes Proteins 0.000 description 125
- 108020004414 DNA Proteins 0.000 description 112
- 108091026890 Coding region Proteins 0.000 description 73
- 244000098338 Triticum aestivum Species 0.000 description 71
- 238000003752 polymerase chain reaction Methods 0.000 description 71
- 241000567178 Fusarium venenatum Species 0.000 description 66
- 239000000047 product Substances 0.000 description 66
- 210000001519 tissue Anatomy 0.000 description 47
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 44
- 102000004169 proteins and genes Human genes 0.000 description 44
- 235000018102 proteins Nutrition 0.000 description 43
- 238000012163 sequencing technique Methods 0.000 description 41
- 108700019146 Transgenes Proteins 0.000 description 40
- 108020004999 messenger RNA Proteins 0.000 description 39
- 235000001014 amino acid Nutrition 0.000 description 37
- 230000009261 transgenic effect Effects 0.000 description 36
- 229940024606 amino acid Drugs 0.000 description 34
- 229920002101 Chitin Polymers 0.000 description 32
- 239000002609 medium Substances 0.000 description 31
- 239000013604 expression vector Substances 0.000 description 30
- 239000000523 sample Substances 0.000 description 29
- 238000003757 reverse transcription PCR Methods 0.000 description 27
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 26
- 241000233866 Fungi Species 0.000 description 26
- 240000005979 Hordeum vulgare Species 0.000 description 26
- 108020004635 Complementary DNA Proteins 0.000 description 25
- 230000002538 fungal effect Effects 0.000 description 25
- 238000009396 hybridization Methods 0.000 description 25
- 230000009466 transformation Effects 0.000 description 25
- 241000223192 Fusarium sporotrichioides Species 0.000 description 24
- 235000007340 Hordeum vulgare Nutrition 0.000 description 24
- 235000013339 cereals Nutrition 0.000 description 23
- 230000036961 partial effect Effects 0.000 description 23
- 240000008042 Zea mays Species 0.000 description 22
- 210000002421 cell wall Anatomy 0.000 description 22
- 239000000203 mixture Substances 0.000 description 22
- 210000000056 organ Anatomy 0.000 description 22
- 108091008146 restriction endonucleases Proteins 0.000 description 22
- 239000011780 sodium chloride Substances 0.000 description 22
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 21
- 108091081024 Start codon Proteins 0.000 description 21
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 21
- 230000000692 anti-sense effect Effects 0.000 description 21
- 240000007594 Oryza sativa Species 0.000 description 20
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 20
- 235000007164 Oryza sativa Nutrition 0.000 description 19
- 230000004048 modification Effects 0.000 description 19
- 238000012986 modification Methods 0.000 description 19
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 18
- 235000016383 Zea mays subsp huehuetenangensis Nutrition 0.000 description 18
- 230000001580 bacterial effect Effects 0.000 description 18
- 238000010367 cloning Methods 0.000 description 18
- 235000009973 maize Nutrition 0.000 description 18
- 235000009566 rice Nutrition 0.000 description 18
- 239000000243 solution Substances 0.000 description 18
- 241000588724 Escherichia coli Species 0.000 description 17
- 108010076504 Protein Sorting Signals Proteins 0.000 description 17
- 229920001503 Glucan Polymers 0.000 description 16
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 16
- 238000012408 PCR amplification Methods 0.000 description 16
- 239000011543 agarose gel Substances 0.000 description 16
- 238000006243 chemical reaction Methods 0.000 description 16
- 238000006467 substitution reaction Methods 0.000 description 16
- 241000223195 Fusarium graminearum Species 0.000 description 15
- 241000208125 Nicotiana Species 0.000 description 15
- 230000004927 fusion Effects 0.000 description 15
- 238000013518 transcription Methods 0.000 description 15
- 230000035897 transcription Effects 0.000 description 15
- 235000002637 Nicotiana tabacum Nutrition 0.000 description 14
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 14
- 238000004458 analytical method Methods 0.000 description 14
- 210000002257 embryonic structure Anatomy 0.000 description 14
- 241000894007 species Species 0.000 description 14
- 241000282326 Felis catus Species 0.000 description 13
- 230000012010 growth Effects 0.000 description 13
- 229960000318 kanamycin Drugs 0.000 description 13
- 244000075850 Avena orientalis Species 0.000 description 12
- 125000000539 amino acid group Chemical group 0.000 description 12
- 230000003321 amplification Effects 0.000 description 12
- 239000003550 marker Substances 0.000 description 12
- 244000005700 microbiome Species 0.000 description 12
- 238000003199 nucleic acid amplification method Methods 0.000 description 12
- 229920000642 polymer Polymers 0.000 description 12
- 235000007319 Avena orientalis Nutrition 0.000 description 11
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 11
- 206010020649 Hyperkeratosis Diseases 0.000 description 11
- SITLTJHOQZFJGG-UHFFFAOYSA-N N-L-alpha-glutamyl-L-valine Natural products CC(C)C(C(O)=O)NC(=O)C(N)CCC(O)=O SITLTJHOQZFJGG-UHFFFAOYSA-N 0.000 description 11
- NJGMALCNYAMYCB-JRQIVUDYSA-N Thr-Tyr-Asn Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(N)=O)C(O)=O NJGMALCNYAMYCB-JRQIVUDYSA-N 0.000 description 11
- GINJFDRNADDBIN-FXQIFTODSA-N bilanafos Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCP(C)(O)=O GINJFDRNADDBIN-FXQIFTODSA-N 0.000 description 11
- 238000003776 cleavage reaction Methods 0.000 description 11
- 230000000875 corresponding effect Effects 0.000 description 11
- 238000005516 engineering process Methods 0.000 description 11
- 108010034529 leucyl-lysine Proteins 0.000 description 11
- 238000011160 research Methods 0.000 description 11
- 230000007017 scission Effects 0.000 description 11
- 238000013519 translation Methods 0.000 description 11
- 230000014616 translation Effects 0.000 description 11
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 10
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 10
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 10
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 10
- 108010092854 aspartyllysine Proteins 0.000 description 10
- 230000002068 genetic effect Effects 0.000 description 10
- 238000002955 isolation Methods 0.000 description 10
- 239000007788 liquid Substances 0.000 description 10
- KWGKDLIKAYFUFQ-UHFFFAOYSA-M lithium chloride Chemical compound [Li+].[Cl-] KWGKDLIKAYFUFQ-UHFFFAOYSA-M 0.000 description 10
- 108010064235 lysylglycine Proteins 0.000 description 10
- 238000000636 Northern blotting Methods 0.000 description 9
- 241000209056 Secale Species 0.000 description 9
- 230000000977 initiatory effect Effects 0.000 description 9
- 229930027917 kanamycin Natural products 0.000 description 9
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 9
- 229930182823 kanamycin A Natural products 0.000 description 9
- 239000000463 material Substances 0.000 description 9
- 239000011159 matrix material Substances 0.000 description 9
- 229910052757 nitrogen Inorganic materials 0.000 description 9
- 230000002441 reversible effect Effects 0.000 description 9
- 230000002103 transcriptional effect Effects 0.000 description 9
- 238000011144 upstream manufacturing Methods 0.000 description 9
- 229920000936 Agarose Polymers 0.000 description 8
- 241000223221 Fusarium oxysporum Species 0.000 description 8
- FZIJIFCXUCZHOL-CIUDSAMLSA-N Lys-Ala-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCCCN FZIJIFCXUCZHOL-CIUDSAMLSA-N 0.000 description 8
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 8
- 235000007238 Secale cereale Nutrition 0.000 description 8
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 8
- 108010024078 alanyl-glycyl-serine Proteins 0.000 description 8
- 108010040443 aspartyl-aspartic acid Proteins 0.000 description 8
- 238000002474 experimental method Methods 0.000 description 8
- 229910001629 magnesium chloride Inorganic materials 0.000 description 8
- 108010061238 threonyl-glycine Proteins 0.000 description 8
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 7
- 241000894006 Bacteria Species 0.000 description 7
- 241000287937 Colinus Species 0.000 description 7
- 241000223194 Fusarium culmorum Species 0.000 description 7
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 7
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 7
- KCXUCYYZNZFGLL-SRVKXCTJSA-N Lys-Ala-Leu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(O)=O KCXUCYYZNZFGLL-SRVKXCTJSA-N 0.000 description 7
- 101000763602 Manilkara zapota Thaumatin-like protein 1 Proteins 0.000 description 7
- 101000763586 Manilkara zapota Thaumatin-like protein 1a Proteins 0.000 description 7
- 101000966653 Musa acuminata Glucan endo-1,3-beta-glucosidase Proteins 0.000 description 7
- AJHCSUXXECOXOY-UHFFFAOYSA-N N-glycyl-L-tryptophan Natural products C1=CC=C2C(CC(NC(=O)CN)C(O)=O)=CNC2=C1 AJHCSUXXECOXOY-UHFFFAOYSA-N 0.000 description 7
- 108700026244 Open Reading Frames Proteins 0.000 description 7
- 244000062793 Sorghum vulgare Species 0.000 description 7
- 238000002105 Southern blotting Methods 0.000 description 7
- 108010086434 alanyl-seryl-glycine Proteins 0.000 description 7
- 230000000843 anti-fungal effect Effects 0.000 description 7
- 108010038633 aspartylglutamate Proteins 0.000 description 7
- 239000000872 buffer Substances 0.000 description 7
- 239000013599 cloning vector Substances 0.000 description 7
- LINOMUASTDIRTM-QGRHZQQGSA-N deoxynivalenol Chemical compound C([C@@]12[C@@]3(C[C@@H](O)[C@H]1O[C@@H]1C=C(C([C@@H](O)[C@@]13CO)=O)C)C)O2 LINOMUASTDIRTM-QGRHZQQGSA-N 0.000 description 7
- 201000010099 disease Diseases 0.000 description 7
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 7
- XKUKSGPZAADMRA-UHFFFAOYSA-N glycyl-glycyl-glycine Natural products NCC(=O)NCC(=O)NCC(O)=O XKUKSGPZAADMRA-UHFFFAOYSA-N 0.000 description 7
- 230000010354 integration Effects 0.000 description 7
- 230000001717 pathogenic effect Effects 0.000 description 7
- 108010083476 phenylalanyltryptophan Proteins 0.000 description 7
- 238000002360 preparation method Methods 0.000 description 7
- 238000003259 recombinant expression Methods 0.000 description 7
- 230000010076 replication Effects 0.000 description 7
- LINOMUASTDIRTM-UHFFFAOYSA-N vomitoxin hydrate Natural products OCC12C(O)C(=O)C(C)=CC1OC1C(O)CC2(C)C11CO1 LINOMUASTDIRTM-UHFFFAOYSA-N 0.000 description 7
- AMBKWKJGMIHTJR-UHFFFAOYSA-N 2-[2-[2-[(2-azaniumyl-3-methylbutanoyl)amino]propanoylamino]propanoylamino]-3-phenylpropanoate Chemical compound CC(C)C(N)C(=O)NC(C)C(=O)NC(C)C(=O)NC(C(O)=O)CC1=CC=CC=C1 AMBKWKJGMIHTJR-UHFFFAOYSA-N 0.000 description 6
- 241000589158 Agrobacterium Species 0.000 description 6
- RZZMZYZXNJRPOJ-BJDJZHNGSA-N Ala-Ile-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](C)N RZZMZYZXNJRPOJ-BJDJZHNGSA-N 0.000 description 6
- 108010011667 Ala-Phe-Ala Proteins 0.000 description 6
- CYBJZLQSUJEMAS-LFSVMHDDSA-N Ala-Phe-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)NC(=O)[C@H](C)N)O CYBJZLQSUJEMAS-LFSVMHDDSA-N 0.000 description 6
- HPNDBHLITCHRSO-WHFBIAKZSA-N Asp-Ala-Gly Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(=O)NCC(O)=O HPNDBHLITCHRSO-WHFBIAKZSA-N 0.000 description 6
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 6
- 241000427940 Fusarium solani Species 0.000 description 6
- XOZOSAUOGRPCES-STECZYCISA-N Ile-Pro-Tyr Chemical compound CC[C@H](C)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 XOZOSAUOGRPCES-STECZYCISA-N 0.000 description 6
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 6
- 125000003412 L-alanyl group Chemical group [H]N([H])[C@@](C([H])([H])[H])(C(=O)[*])[H] 0.000 description 6
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 6
- WNGVUZWBXZKQES-YUMQZZPRSA-N Leu-Ala-Gly Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C)C(=O)NCC(O)=O WNGVUZWBXZKQES-YUMQZZPRSA-N 0.000 description 6
- HKCCVDWHHTVVPN-CIUDSAMLSA-N Lys-Asp-Ala Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(O)=O HKCCVDWHHTVVPN-CIUDSAMLSA-N 0.000 description 6
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 6
- SQPZCTBSLIIMBL-BPUTZDHNSA-N Met-Trp-Ser Chemical compound CSCC[C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)N[C@@H](CO)C(=O)O)N SQPZCTBSLIIMBL-BPUTZDHNSA-N 0.000 description 6
- BQVUABVGYYSDCJ-UHFFFAOYSA-N Nalpha-L-Leucyl-L-tryptophan Natural products C1=CC=C2C(CC(NC(=O)C(N)CC(C)C)C(O)=O)=CNC2=C1 BQVUABVGYYSDCJ-UHFFFAOYSA-N 0.000 description 6
- 108020004711 Nucleic Acid Probes Proteins 0.000 description 6
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 6
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 6
- 206010039509 Scab Diseases 0.000 description 6
- MIJWOJAXARLEHA-WDSKDSINSA-N Ser-Gly-Glu Chemical compound OC[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCC(O)=O MIJWOJAXARLEHA-WDSKDSINSA-N 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 235000011684 Sorghum saccharatum Nutrition 0.000 description 6
- OHAJHDJOCKKJLV-LKXGYXEUSA-N Thr-Asp-Ser Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(O)=O OHAJHDJOCKKJLV-LKXGYXEUSA-N 0.000 description 6
- 241000223260 Trichoderma harzianum Species 0.000 description 6
- CKHQKYHIZCRTAP-SOUVJXGZSA-N Tyr-Gln-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CC2=CC=C(C=C2)O)N)C(=O)O CKHQKYHIZCRTAP-SOUVJXGZSA-N 0.000 description 6
- 108010001271 arginyl-glutamyl-arginine Proteins 0.000 description 6
- 230000015572 biosynthetic process Effects 0.000 description 6
- 230000029087 digestion Effects 0.000 description 6
- 238000004520 electroporation Methods 0.000 description 6
- 239000000284 extract Substances 0.000 description 6
- VPZXBVLAVMBEQI-UHFFFAOYSA-N glycyl-DL-alpha-alanine Natural products OC(=O)C(C)NC(=O)CN VPZXBVLAVMBEQI-UHFFFAOYSA-N 0.000 description 6
- 108010037850 glycylvaline Proteins 0.000 description 6
- 230000003301 hydrolyzing effect Effects 0.000 description 6
- 208000015181 infectious disease Diseases 0.000 description 6
- 108010044311 leucyl-glycyl-glycine Proteins 0.000 description 6
- 108010054155 lysyllysine Proteins 0.000 description 6
- 239000012528 membrane Substances 0.000 description 6
- 239000002853 nucleic acid probe Substances 0.000 description 6
- 239000002245 particle Substances 0.000 description 6
- 239000008188 pellet Substances 0.000 description 6
- 108010051242 phenylalanylserine Proteins 0.000 description 6
- 102000040430 polynucleotide Human genes 0.000 description 6
- 108091033319 polynucleotide Proteins 0.000 description 6
- 239000002157 polynucleotide Substances 0.000 description 6
- 230000008929 regeneration Effects 0.000 description 6
- 238000011069 regeneration method Methods 0.000 description 6
- 238000002741 site-directed mutagenesis Methods 0.000 description 6
- 238000000638 solvent extraction Methods 0.000 description 6
- 108010073969 valyllysine Proteins 0.000 description 6
- SMCGQGDVTPFXKB-XPUUQOCRSA-N Ala-Gly-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@H](C)N SMCGQGDVTPFXKB-XPUUQOCRSA-N 0.000 description 5
- VNYMOTCMNHJGTG-JBDRJPRFSA-N Ala-Ile-Ser Chemical compound [H]N[C@@H](C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(O)=O VNYMOTCMNHJGTG-JBDRJPRFSA-N 0.000 description 5
- OPZJWMJPCNNZNT-DCAQKATOSA-N Ala-Leu-Met Chemical compound C[C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(=O)O)N OPZJWMJPCNNZNT-DCAQKATOSA-N 0.000 description 5
- NINQYGGNRIBFSC-CIUDSAMLSA-N Ala-Lys-Ser Chemical compound NCCCC[C@H](NC(=O)[C@@H](N)C)C(=O)N[C@@H](CO)C(O)=O NINQYGGNRIBFSC-CIUDSAMLSA-N 0.000 description 5
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 5
- 241000206602 Eukaryota Species 0.000 description 5
- 241000233732 Fusarium verticillioides Species 0.000 description 5
- 229920002148 Gellan gum Polymers 0.000 description 5
- 102000053187 Glucuronidase Human genes 0.000 description 5
- 108010060309 Glucuronidase Proteins 0.000 description 5
- RPLLQZBOVIVGMX-QWRGUYRKSA-N Gly-Asp-Phe Chemical compound [H]NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O RPLLQZBOVIVGMX-QWRGUYRKSA-N 0.000 description 5
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 5
- 241000238631 Hexapoda Species 0.000 description 5
- UDLAWRKOVFDKFL-PEFMBERDSA-N Ile-Asp-Gln Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N UDLAWRKOVFDKFL-PEFMBERDSA-N 0.000 description 5
- RIVKTKFVWXRNSJ-GRLWGSQLSA-N Ile-Ile-Gln Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N RIVKTKFVWXRNSJ-GRLWGSQLSA-N 0.000 description 5
- BQIIHAGJIYOQBP-YFYLHZKVSA-N Ile-Trp-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)N3CCC[C@@H]3C(=O)O)N BQIIHAGJIYOQBP-YFYLHZKVSA-N 0.000 description 5
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 5
- 241000880493 Leptailurus serval Species 0.000 description 5
- QNTJIDXQHWUBKC-BZSNNMDCSA-N Leu-Lys-Phe Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O QNTJIDXQHWUBKC-BZSNNMDCSA-N 0.000 description 5
- PPGBXYKMUMHFBF-KATARQTJSA-N Leu-Ser-Thr Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(O)=O PPGBXYKMUMHFBF-KATARQTJSA-N 0.000 description 5
- PRSBSVAVOQOAMI-BJDJZHNGSA-N Lys-Ile-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@@H](N)CCCCN PRSBSVAVOQOAMI-BJDJZHNGSA-N 0.000 description 5
- ZRACLHJYVRBJFC-ULQDDVLXSA-N Met-Lys-Phe Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 ZRACLHJYVRBJFC-ULQDDVLXSA-N 0.000 description 5
- 208000031888 Mycoses Diseases 0.000 description 5
- 108010002311 N-glycylglutamic acid Proteins 0.000 description 5
- IAOZOFPONWDXNT-IXOXFDKPSA-N Phe-Ser-Thr Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(O)=O IAOZOFPONWDXNT-IXOXFDKPSA-N 0.000 description 5
- HFNPOYOKIPGAEI-SRVKXCTJSA-N Pro-Leu-Glu Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H]1CCCN1 HFNPOYOKIPGAEI-SRVKXCTJSA-N 0.000 description 5
- FIODMZKLZFLYQP-GUBZILKMSA-N Pro-Val-Ser Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(O)=O FIODMZKLZFLYQP-GUBZILKMSA-N 0.000 description 5
- 108700008625 Reporter Genes Proteins 0.000 description 5
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 5
- 238000012300 Sequence Analysis Methods 0.000 description 5
- JFWDJFULOLKQFY-QWRGUYRKSA-N Ser-Gly-Phe Chemical compound [H]N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O JFWDJFULOLKQFY-QWRGUYRKSA-N 0.000 description 5
- WGDYNRCOQRERLZ-KKUMJFAQSA-N Ser-Lys-Phe Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CO)N WGDYNRCOQRERLZ-KKUMJFAQSA-N 0.000 description 5
- KKKVOZNCLALMPV-XKBZYTNZSA-N Ser-Thr-Glu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(O)=O)C(O)=O KKKVOZNCLALMPV-XKBZYTNZSA-N 0.000 description 5
- PURRNJBBXDDWLX-ZDLURKLDSA-N Ser-Thr-Gly Chemical compound C[C@H]([C@@H](C(=O)NCC(=O)O)NC(=O)[C@H](CO)N)O PURRNJBBXDDWLX-ZDLURKLDSA-N 0.000 description 5
- NIEWSKWFURSECR-FOHZUACHSA-N Thr-Gly-Asp Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(O)=O NIEWSKWFURSECR-FOHZUACHSA-N 0.000 description 5
- CRZNCABIJLRFKZ-IUKAMOBKSA-N Thr-Ile-Asp Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)O)NC(=O)[C@H]([C@@H](C)O)N CRZNCABIJLRFKZ-IUKAMOBKSA-N 0.000 description 5
- OGOYMQWIWHGTGH-KZVJFYERSA-N Thr-Val-Ala Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C)C(O)=O OGOYMQWIWHGTGH-KZVJFYERSA-N 0.000 description 5
- UEFHVUQBYNRNQC-SFJXLCSZSA-N Trp-Phe-Thr Chemical compound C([C@@H](C(=O)N[C@@H]([C@H](O)C)C(O)=O)NC(=O)[C@@H](N)CC=1C2=CC=CC=C2NC=1)C1=CC=CC=C1 UEFHVUQBYNRNQC-SFJXLCSZSA-N 0.000 description 5
- OKDNSNWJEXAMSU-IRXDYDNUSA-N Tyr-Phe-Gly Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)NCC(O)=O)C1=CC=C(O)C=C1 OKDNSNWJEXAMSU-IRXDYDNUSA-N 0.000 description 5
- 108090000848 Ubiquitin Proteins 0.000 description 5
- 102000044159 Ubiquitin Human genes 0.000 description 5
- COYSIHFOCOMGCF-UHFFFAOYSA-N Val-Arg-Gly Natural products CC(C)C(N)C(=O)NC(C(=O)NCC(O)=O)CCCN=C(N)N COYSIHFOCOMGCF-UHFFFAOYSA-N 0.000 description 5
- ZXAGTABZUOMUDO-GVXVVHGQSA-N Val-Glu-Lys Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CCCCN)C(=O)O)N ZXAGTABZUOMUDO-GVXVVHGQSA-N 0.000 description 5
- RWOGENDAOGMHLX-DCAQKATOSA-N Val-Lys-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C(C)C)N RWOGENDAOGMHLX-DCAQKATOSA-N 0.000 description 5
- BGXVHVMJZCSOCA-AVGNSLFASA-N Val-Pro-Lys Chemical compound CC(C)[C@@H](C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCCN)C(=O)O)N BGXVHVMJZCSOCA-AVGNSLFASA-N 0.000 description 5
- 238000009825 accumulation Methods 0.000 description 5
- 230000002378 acidificating effect Effects 0.000 description 5
- 238000007792 addition Methods 0.000 description 5
- 238000013019 agitation Methods 0.000 description 5
- 238000003556 assay Methods 0.000 description 5
- 101150103518 bar gene Proteins 0.000 description 5
- 230000008901 benefit Effects 0.000 description 5
- 229940098773 bovine serum albumin Drugs 0.000 description 5
- 238000004422 calculation algorithm Methods 0.000 description 5
- 239000012677 causal agent Substances 0.000 description 5
- 238000005119 centrifugation Methods 0.000 description 5
- 230000000593 degrading effect Effects 0.000 description 5
- 229930002954 deoxynivalenol Natural products 0.000 description 5
- 108010057083 glutamyl-aspartyl-leucine Proteins 0.000 description 5
- 108010049041 glutamylalanine Proteins 0.000 description 5
- 230000002363 herbicidal effect Effects 0.000 description 5
- 239000004009 herbicide Substances 0.000 description 5
- 108010036413 histidylglycine Proteins 0.000 description 5
- 238000000338 in vitro Methods 0.000 description 5
- 230000001965 increasing effect Effects 0.000 description 5
- 238000011534 incubation Methods 0.000 description 5
- 238000003780 insertion Methods 0.000 description 5
- 230000037431 insertion Effects 0.000 description 5
- 108010025153 lysyl-alanyl-alanine Proteins 0.000 description 5
- 210000001161 mammalian embryo Anatomy 0.000 description 5
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 5
- 108010079317 prolyl-tyrosine Proteins 0.000 description 5
- 210000001938 protoplast Anatomy 0.000 description 5
- 230000001105 regulatory effect Effects 0.000 description 5
- 230000001568 sexual effect Effects 0.000 description 5
- 230000030118 somatic embryogenesis Effects 0.000 description 5
- 239000000758 substrate Substances 0.000 description 5
- 238000012546 transfer Methods 0.000 description 5
- 238000011282 treatment Methods 0.000 description 5
- 108010084932 tryptophyl-proline Proteins 0.000 description 5
- 241000228158 x Triticosecale Species 0.000 description 5
- KDZIGQIDPXKMBA-UHFFFAOYSA-N 2-[[2-[[2-[(2-amino-3-methylbutanoyl)amino]acetyl]amino]-3-hydroxypropanoyl]amino]pentanedioic acid Chemical compound CC(C)C(N)C(=O)NCC(=O)NC(CO)C(=O)NC(C(O)=O)CCC(O)=O KDZIGQIDPXKMBA-UHFFFAOYSA-N 0.000 description 4
- 229920001817 Agar Polymers 0.000 description 4
- JBVSSSZFNTXJDX-YTLHQDLWSA-N Ala-Ala-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@H](C)N JBVSSSZFNTXJDX-YTLHQDLWSA-N 0.000 description 4
- TTXMOJWKNRJWQJ-FXQIFTODSA-N Ala-Arg-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)C)CCCN=C(N)N TTXMOJWKNRJWQJ-FXQIFTODSA-N 0.000 description 4
- YAXNATKKPOWVCP-ZLUOBGJFSA-N Ala-Asn-Ala Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(O)=O YAXNATKKPOWVCP-ZLUOBGJFSA-N 0.000 description 4
- ZEXDYVGDZJBRMO-ACZMJKKPSA-N Ala-Asn-Gln Chemical compound C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N ZEXDYVGDZJBRMO-ACZMJKKPSA-N 0.000 description 4
- HMRWQTHUDVXMGH-GUBZILKMSA-N Ala-Glu-Lys Chemical compound C[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@H](C(O)=O)CCCCN HMRWQTHUDVXMGH-GUBZILKMSA-N 0.000 description 4
- FAJIYNONGXEXAI-CQDKDKBSSA-N Ala-His-Phe Chemical compound C([C@H](NC(=O)[C@@H](N)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CNC=N1 FAJIYNONGXEXAI-CQDKDKBSSA-N 0.000 description 4
- IYKVSFNGSWTTNZ-GUBZILKMSA-N Ala-Val-Arg Chemical compound C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N IYKVSFNGSWTTNZ-GUBZILKMSA-N 0.000 description 4
- WESHVRNMNFMVBE-FXQIFTODSA-N Arg-Asn-Asp Chemical compound C(C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CC(=O)O)C(=O)O)N)CN=C(N)N WESHVRNMNFMVBE-FXQIFTODSA-N 0.000 description 4
- HKRXJBBCQBAGIM-FXQIFTODSA-N Arg-Asp-Ser Chemical compound C(C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CO)C(=O)O)N)CN=C(N)N HKRXJBBCQBAGIM-FXQIFTODSA-N 0.000 description 4
- VDCIPFYVCICPEC-FXQIFTODSA-N Asn-Arg-Ala Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(O)=O VDCIPFYVCICPEC-FXQIFTODSA-N 0.000 description 4
- MFFOYNGMOYFPBD-DCAQKATOSA-N Asn-Arg-Leu Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(O)=O MFFOYNGMOYFPBD-DCAQKATOSA-N 0.000 description 4
- UYCPJVYQYARFGB-YDHLFZDLSA-N Asn-Phe-Val Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](C(C)C)C(O)=O UYCPJVYQYARFGB-YDHLFZDLSA-N 0.000 description 4
- KSZHWTRZPOTIGY-AVGNSLFASA-N Asn-Tyr-Gln Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)O)NC(=O)[C@H](CC(=O)N)N)O KSZHWTRZPOTIGY-AVGNSLFASA-N 0.000 description 4
- ZAESWDKAMDVHLL-RCOVLWMOSA-N Asn-Val-Gly Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)NCC(O)=O ZAESWDKAMDVHLL-RCOVLWMOSA-N 0.000 description 4
- SDHFVYLZFBDSQT-DCAQKATOSA-N Asp-Arg-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(=O)O)N SDHFVYLZFBDSQT-DCAQKATOSA-N 0.000 description 4
- DGKCOYGQLNWNCJ-ACZMJKKPSA-N Asp-Glu-Ser Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(O)=O DGKCOYGQLNWNCJ-ACZMJKKPSA-N 0.000 description 4
- DTNUIAJCPRMNBT-WHFBIAKZSA-N Asp-Gly-Ala Chemical compound [H]N[C@@H](CC(O)=O)C(=O)NCC(=O)N[C@@H](C)C(O)=O DTNUIAJCPRMNBT-WHFBIAKZSA-N 0.000 description 4
- KTTCQQNRRLCIBC-GHCJXIJMSA-N Asp-Ile-Ala Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(O)=O KTTCQQNRRLCIBC-GHCJXIJMSA-N 0.000 description 4
- KLYPOCBLKMPBIQ-GHCJXIJMSA-N Asp-Ile-Ser Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)O)NC(=O)[C@H](CC(=O)O)N KLYPOCBLKMPBIQ-GHCJXIJMSA-N 0.000 description 4
- XLILXFRAKOYEJX-GUBZILKMSA-N Asp-Leu-Gln Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O XLILXFRAKOYEJX-GUBZILKMSA-N 0.000 description 4
- OZBXOELNJBSJOA-UBHSHLNASA-N Asp-Ser-Trp Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(=O)O)N OZBXOELNJBSJOA-UBHSHLNASA-N 0.000 description 4
- 235000007558 Avena sp Nutrition 0.000 description 4
- 241000223205 Coccidioides immitis Species 0.000 description 4
- CWHKESLHINPNBX-XIRDDKMYSA-N Cys-Lys-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@@H](NC(=O)[C@@H](N)CS)CCCCN)C(O)=O)=CNC2=C1 CWHKESLHINPNBX-XIRDDKMYSA-N 0.000 description 4
- MSWBLPLBSLQVME-XIRDDKMYSA-N Cys-Trp-Leu Chemical compound C1=CC=C2C(C[C@@H](C(=O)N[C@@H](CC(C)C)C(O)=O)NC(=O)[C@@H](N)CS)=CNC2=C1 MSWBLPLBSLQVME-XIRDDKMYSA-N 0.000 description 4
- 238000007399 DNA isolation Methods 0.000 description 4
- 108091060211 Expressed sequence tag Proteins 0.000 description 4
- 241000221779 Fusarium sambucinum Species 0.000 description 4
- 108700007698 Genetic Terminator Regions Proteins 0.000 description 4
- RMOCFPBLHAOTDU-ACZMJKKPSA-N Gln-Asn-Ser Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(O)=O RMOCFPBLHAOTDU-ACZMJKKPSA-N 0.000 description 4
- JKGHMESJHRTHIC-SIUGBPQLSA-N Gln-Ile-Tyr Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)O)NC(=O)[C@H](CCC(=O)N)N JKGHMESJHRTHIC-SIUGBPQLSA-N 0.000 description 4
- QENSHQJGWGRPQS-QEJZJMRPSA-N Gln-Ser-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)N)C(O)=O)=CNC2=C1 QENSHQJGWGRPQS-QEJZJMRPSA-N 0.000 description 4
- WPJDPEOQUIXXOY-AVGNSLFASA-N Gln-Tyr-Asn Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](CCC(=O)N)N)O WPJDPEOQUIXXOY-AVGNSLFASA-N 0.000 description 4
- WZZSKAJIHTUUSG-ACZMJKKPSA-N Glu-Ala-Asp Chemical compound OC(=O)C[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCC(O)=O WZZSKAJIHTUUSG-ACZMJKKPSA-N 0.000 description 4
- NKLRYVLERDYDBI-FXQIFTODSA-N Glu-Glu-Asp Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O NKLRYVLERDYDBI-FXQIFTODSA-N 0.000 description 4
- DXVOKNVIKORTHQ-GUBZILKMSA-N Glu-Pro-Glu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(O)=O DXVOKNVIKORTHQ-GUBZILKMSA-N 0.000 description 4
- WXONSNSSBYQGNN-AVGNSLFASA-N Glu-Ser-Tyr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O WXONSNSSBYQGNN-AVGNSLFASA-N 0.000 description 4
- GPSHCSTUYOQPAI-JHEQGTHGSA-N Glu-Thr-Gly Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O GPSHCSTUYOQPAI-JHEQGTHGSA-N 0.000 description 4
- QIZJOTQTCAGKPU-KWQFWETISA-N Gly-Ala-Tyr Chemical compound [NH3+]CC(=O)N[C@@H](C)C(=O)N[C@H](C([O-])=O)CC1=CC=C(O)C=C1 QIZJOTQTCAGKPU-KWQFWETISA-N 0.000 description 4
- CLODWIOAKCSBAN-BQBZGAKWSA-N Gly-Arg-Asp Chemical compound NC(N)=NCCC[C@H](NC(=O)CN)C(=O)N[C@@H](CC(O)=O)C(O)=O CLODWIOAKCSBAN-BQBZGAKWSA-N 0.000 description 4
- ULZCYBYDTUMHNF-IUCAKERBSA-N Gly-Leu-Glu Chemical compound NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O ULZCYBYDTUMHNF-IUCAKERBSA-N 0.000 description 4
- FXLVSYVJDPCIHH-STQMWFEESA-N Gly-Phe-Arg Chemical compound [H]NCC(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O FXLVSYVJDPCIHH-STQMWFEESA-N 0.000 description 4
- MTBIKIMYHUWBRX-QWRGUYRKSA-N Gly-Phe-Asn Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)CN MTBIKIMYHUWBRX-QWRGUYRKSA-N 0.000 description 4
- IEGFSKKANYKBDU-QWHCGFSZSA-N Gly-Phe-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC2=CC=CC=C2)NC(=O)CN)C(=O)O IEGFSKKANYKBDU-QWHCGFSZSA-N 0.000 description 4
- GGAPHLIUUTVYMX-QWRGUYRKSA-N Gly-Phe-Ser Chemical compound OC[C@@H](C([O-])=O)NC(=O)[C@@H](NC(=O)C[NH3+])CC1=CC=CC=C1 GGAPHLIUUTVYMX-QWRGUYRKSA-N 0.000 description 4
- JJGBXTYGTKWGAT-YUMQZZPRSA-N Gly-Pro-Glu Chemical compound NCC(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(O)=O JJGBXTYGTKWGAT-YUMQZZPRSA-N 0.000 description 4
- WTUSRDZLLWGYAT-KCTSRDHCSA-N Gly-Trp-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)NC(=O)CN WTUSRDZLLWGYAT-KCTSRDHCSA-N 0.000 description 4
- BDFCIKANUNMFGB-PMVVWTBXSA-N His-Gly-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CC1=CN=CN1 BDFCIKANUNMFGB-PMVVWTBXSA-N 0.000 description 4
- RPZFUIQVAPZLRH-GHCJXIJMSA-N Ile-Asp-Ala Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](C)C(=O)O)N RPZFUIQVAPZLRH-GHCJXIJMSA-N 0.000 description 4
- GECLQMBTZCPAFY-PEFMBERDSA-N Ile-Gln-Asp Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CC(=O)O)C(=O)O)N GECLQMBTZCPAFY-PEFMBERDSA-N 0.000 description 4
- NZOCIWKZUVUNDW-ZKWXMUAHSA-N Ile-Gly-Ala Chemical compound CC[C@H](C)[C@H](N)C(=O)NCC(=O)N[C@@H](C)C(O)=O NZOCIWKZUVUNDW-ZKWXMUAHSA-N 0.000 description 4
- AKOYRLRUFBZOSP-BJDJZHNGSA-N Ile-Lys-Ser Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(=O)O)N AKOYRLRUFBZOSP-BJDJZHNGSA-N 0.000 description 4
- IITVUURPOYGCTD-NAKRPEOUSA-N Ile-Pro-Ala Chemical compound CC[C@H](C)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C)C(O)=O IITVUURPOYGCTD-NAKRPEOUSA-N 0.000 description 4
- SAEWJTCJQVZQNZ-IUKAMOBKSA-N Ile-Thr-Asn Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(=O)N)C(=O)O)N SAEWJTCJQVZQNZ-IUKAMOBKSA-N 0.000 description 4
- 108010065920 Insulin Lispro Proteins 0.000 description 4
- FADYJNXDPBKVCA-UHFFFAOYSA-N L-Phenylalanyl-L-lysin Natural products NCCCCC(C(O)=O)NC(=O)C(N)CC1=CC=CC=C1 FADYJNXDPBKVCA-UHFFFAOYSA-N 0.000 description 4
- RCFDOSNHHZGBOY-UHFFFAOYSA-N L-isoleucyl-L-alanine Natural products CCC(C)C(N)C(=O)NC(C)C(O)=O RCFDOSNHHZGBOY-UHFFFAOYSA-N 0.000 description 4
- MJOZZTKJZQFKDK-GUBZILKMSA-N Leu-Ala-Gln Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCC(N)=O MJOZZTKJZQFKDK-GUBZILKMSA-N 0.000 description 4
- HASRFYOMVPJRPU-SRVKXCTJSA-N Leu-Arg-Glu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCC(O)=O)C(O)=O HASRFYOMVPJRPU-SRVKXCTJSA-N 0.000 description 4
- QUAAUWNLWMLERT-IHRRRGAJSA-N Leu-Arg-Leu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CC(C)C)C(O)=O QUAAUWNLWMLERT-IHRRRGAJSA-N 0.000 description 4
- QCSFMCFHVGTLFF-NHCYSSNCSA-N Leu-Asp-Val Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O QCSFMCFHVGTLFF-NHCYSSNCSA-N 0.000 description 4
- KAFOIVJDVSZUMD-DCAQKATOSA-N Leu-Gln-Gln Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O KAFOIVJDVSZUMD-DCAQKATOSA-N 0.000 description 4
- KAFOIVJDVSZUMD-UHFFFAOYSA-N Leu-Gln-Gln Natural products CC(C)CC(N)C(=O)NC(CCC(N)=O)C(=O)NC(CCC(N)=O)C(O)=O KAFOIVJDVSZUMD-UHFFFAOYSA-N 0.000 description 4
- DPWGZWUMUUJQDT-IUCAKERBSA-N Leu-Gln-Gly Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)NCC(O)=O DPWGZWUMUUJQDT-IUCAKERBSA-N 0.000 description 4
- LXKNSJLSGPNHSK-KKUMJFAQSA-N Leu-Leu-Lys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)O)N LXKNSJLSGPNHSK-KKUMJFAQSA-N 0.000 description 4
- MJWVXZABPOKJJF-ACRUOGEOSA-N Leu-Phe-Phe Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O MJWVXZABPOKJJF-ACRUOGEOSA-N 0.000 description 4
- JIHDFWWRYHSAQB-GUBZILKMSA-N Leu-Ser-Glu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCC(O)=O JIHDFWWRYHSAQB-GUBZILKMSA-N 0.000 description 4
- VHFFQUSNFFIZBT-CIUDSAMLSA-N Lys-Ala-Cys Chemical compound C[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CCCCN)N VHFFQUSNFFIZBT-CIUDSAMLSA-N 0.000 description 4
- KNKHAVVBVXKOGX-JXUBOQSCSA-N Lys-Ala-Thr Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O KNKHAVVBVXKOGX-JXUBOQSCSA-N 0.000 description 4
- FACUGMGEFUEBTI-SRVKXCTJSA-N Lys-Asn-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](N)CCCCN FACUGMGEFUEBTI-SRVKXCTJSA-N 0.000 description 4
- LCMWVZLBCUVDAZ-IUCAKERBSA-N Lys-Gly-Glu Chemical compound [NH3+]CCCC[C@H]([NH3+])C(=O)NCC(=O)N[C@H](C([O-])=O)CCC([O-])=O LCMWVZLBCUVDAZ-IUCAKERBSA-N 0.000 description 4
- RFQATBGBLDAKGI-VHSXEESVSA-N Lys-Gly-Pro Chemical compound C1C[C@@H](N(C1)C(=O)CNC(=O)[C@H](CCCCN)N)C(=O)O RFQATBGBLDAKGI-VHSXEESVSA-N 0.000 description 4
- PGLGNCVOWIORQE-SRVKXCTJSA-N Lys-His-Ser Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CO)C(O)=O PGLGNCVOWIORQE-SRVKXCTJSA-N 0.000 description 4
- PYFNONMJYNJENN-AVGNSLFASA-N Lys-Lys-Gln Chemical compound C(CCN)C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N PYFNONMJYNJENN-AVGNSLFASA-N 0.000 description 4
- YCJCEMKOZOYBEF-OEAJRASXSA-N Lys-Thr-Phe Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O YCJCEMKOZOYBEF-OEAJRASXSA-N 0.000 description 4
- VKCPHIOZDWUFSW-ONGXEEELSA-N Lys-Val-Gly Chemical compound OC(=O)CNC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CCCCN VKCPHIOZDWUFSW-ONGXEEELSA-N 0.000 description 4
- UZWMJZSOXGOVIN-LURJTMIESA-N Met-Gly-Gly Chemical compound CSCC[C@H](N)C(=O)NCC(=O)NCC(O)=O UZWMJZSOXGOVIN-LURJTMIESA-N 0.000 description 4
- MNGBICITWAPGAS-BPUTZDHNSA-N Met-Ser-Trp Chemical compound [H]N[C@@H](CCSC)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O MNGBICITWAPGAS-BPUTZDHNSA-N 0.000 description 4
- VEKRTVRZDMUOQN-AVGNSLFASA-N Met-Val-His Chemical compound CSCC[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CC1=CNC=N1 VEKRTVRZDMUOQN-AVGNSLFASA-N 0.000 description 4
- WUGMRIBZSVSJNP-UHFFFAOYSA-N N-L-alanyl-L-tryptophan Natural products C1=CC=C2C(CC(NC(=O)C(N)C)C(O)=O)=CNC2=C1 WUGMRIBZSVSJNP-UHFFFAOYSA-N 0.000 description 4
- 239000004677 Nylon Substances 0.000 description 4
- 241000286209 Phasianidae Species 0.000 description 4
- SWZKMTDPQXLQRD-XVSYOHENSA-N Phe-Asp-Thr Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O SWZKMTDPQXLQRD-XVSYOHENSA-N 0.000 description 4
- VLZGUAUYZGQKPM-DRZSPHRISA-N Phe-Gln-Ala Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C)C(O)=O VLZGUAUYZGQKPM-DRZSPHRISA-N 0.000 description 4
- RYQWALWYQWBUKN-FHWLQOOXSA-N Phe-Phe-Glu Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCC(O)=O)C(O)=O RYQWALWYQWBUKN-FHWLQOOXSA-N 0.000 description 4
- GTMSCDVFQLNEOY-BZSNNMDCSA-N Phe-Tyr-Asn Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CC2=CC=C(C=C2)O)C(=O)N[C@@H](CC(=O)N)C(=O)O)N GTMSCDVFQLNEOY-BZSNNMDCSA-N 0.000 description 4
- FCCBQBZXIAZNIG-LSJOCFKGSA-N Pro-Ala-His Chemical compound C[C@H](NC(=O)[C@@H]1CCCN1)C(=O)N[C@@H](Cc1cnc[nH]1)C(O)=O FCCBQBZXIAZNIG-LSJOCFKGSA-N 0.000 description 4
- LANQLYHLMYDWJP-SRVKXCTJSA-N Pro-Gln-Lys Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CCCCN)C(=O)O LANQLYHLMYDWJP-SRVKXCTJSA-N 0.000 description 4
- RUDOLGWDSKQQFF-DCAQKATOSA-N Pro-Leu-Asn Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O RUDOLGWDSKQQFF-DCAQKATOSA-N 0.000 description 4
- OFSZYRZOUMNCCU-BZSNNMDCSA-N Pro-Trp-Met Chemical compound N([C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(O)=O)C(=O)[C@@H]1CCCN1 OFSZYRZOUMNCCU-BZSNNMDCSA-N 0.000 description 4
- OFOBLEOULBTSOW-UHFFFAOYSA-N Propanedioic acid Natural products OC(=O)CC(O)=O OFOBLEOULBTSOW-UHFFFAOYSA-N 0.000 description 4
- 108020004511 Recombinant DNA Proteins 0.000 description 4
- GXXTUIUYTWGPMV-FXQIFTODSA-N Ser-Arg-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(O)=O GXXTUIUYTWGPMV-FXQIFTODSA-N 0.000 description 4
- CTRHXXXHUJTTRZ-ZLUOBGJFSA-N Ser-Asp-Cys Chemical compound C([C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CO)N)C(=O)O CTRHXXXHUJTTRZ-ZLUOBGJFSA-N 0.000 description 4
- SFTZWNJFZYOLBD-ZDLURKLDSA-N Ser-Gly-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CO SFTZWNJFZYOLBD-ZDLURKLDSA-N 0.000 description 4
- UBRMZSHOOIVJPW-SRVKXCTJSA-N Ser-Leu-Lys Chemical compound OC[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(O)=O UBRMZSHOOIVJPW-SRVKXCTJSA-N 0.000 description 4
- YUJLIIRMIAGMCQ-CIUDSAMLSA-N Ser-Leu-Ser Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O YUJLIIRMIAGMCQ-CIUDSAMLSA-N 0.000 description 4
- BYCVMHKULKRVPV-GUBZILKMSA-N Ser-Lys-Gln Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(N)=O)C(O)=O BYCVMHKULKRVPV-GUBZILKMSA-N 0.000 description 4
- XUDRHBPSPAPDJP-SRVKXCTJSA-N Ser-Lys-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CO XUDRHBPSPAPDJP-SRVKXCTJSA-N 0.000 description 4
- RTXKJFWHEBTABY-IHPCNDPISA-N Ser-Trp-Tyr Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CC3=CC=C(C=C3)O)C(=O)O)NC(=O)[C@H](CO)N RTXKJFWHEBTABY-IHPCNDPISA-N 0.000 description 4
- 235000002595 Solanum tuberosum Nutrition 0.000 description 4
- 244000061456 Solanum tuberosum Species 0.000 description 4
- DWYAUVCQDTZIJI-VZFHVOOUSA-N Thr-Ala-Ser Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O DWYAUVCQDTZIJI-VZFHVOOUSA-N 0.000 description 4
- XDARBNMYXKUFOJ-GSSVUCPTSA-N Thr-Asp-Thr Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O XDARBNMYXKUFOJ-GSSVUCPTSA-N 0.000 description 4
- VGYVVSQFSSKZRJ-OEAJRASXSA-N Thr-Phe-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)[C@H](O)C)CC1=CC=CC=C1 VGYVVSQFSSKZRJ-OEAJRASXSA-N 0.000 description 4
- CJEHCEOXPLASCK-MEYUZBJRSA-N Thr-Tyr-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)[C@H](O)C)CC1=CC=C(O)C=C1 CJEHCEOXPLASCK-MEYUZBJRSA-N 0.000 description 4
- 235000019714 Triticale Nutrition 0.000 description 4
- 239000013504 Triton X-100 Substances 0.000 description 4
- 229920004890 Triton X-100 Polymers 0.000 description 4
- IUFQHOCOKQIOMC-XIRDDKMYSA-N Trp-Asn-Lys Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CCCCN)C(=O)O)N IUFQHOCOKQIOMC-XIRDDKMYSA-N 0.000 description 4
- XZSJDSBPEJBEFZ-QRTARXTBSA-N Trp-Asn-Val Chemical compound [H]N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C(C)C)C(O)=O XZSJDSBPEJBEFZ-QRTARXTBSA-N 0.000 description 4
- DQDXHYIEITXNJY-BPUTZDHNSA-N Trp-Gln-Gln Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N DQDXHYIEITXNJY-BPUTZDHNSA-N 0.000 description 4
- UUIYFDAWNBSWPG-IHPCNDPISA-N Trp-Lys-Lys Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)O)N UUIYFDAWNBSWPG-IHPCNDPISA-N 0.000 description 4
- IKUMWSDCGQVGHC-UMPQAUOISA-N Trp-Pro-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@@H]1CCCN1C(=O)[C@H](CC2=CNC3=CC=CC=C32)N)O IKUMWSDCGQVGHC-UMPQAUOISA-N 0.000 description 4
- YCQXZDHDSUHUSG-FJHTZYQYSA-N Trp-Thr-Ala Chemical compound C1=CC=C2C(C[C@H](N)C(=O)N[C@@H]([C@H](O)C)C(=O)N[C@@H](C)C(O)=O)=CNC2=C1 YCQXZDHDSUHUSG-FJHTZYQYSA-N 0.000 description 4
- RKISDJMICOREEL-QRTARXTBSA-N Trp-Val-Asp Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)O)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)N RKISDJMICOREEL-QRTARXTBSA-N 0.000 description 4
- XQMGDVVKFRLQKH-BBRMVZONSA-N Trp-Val-Gly Chemical compound C1=CC=C2C(C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)NCC(O)=O)=CNC2=C1 XQMGDVVKFRLQKH-BBRMVZONSA-N 0.000 description 4
- UOXPLPBMEPLZBW-WDSOQIARSA-N Trp-Val-Lys Chemical compound C1=CC=C2C(C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(O)=O)=CNC2=C1 UOXPLPBMEPLZBW-WDSOQIARSA-N 0.000 description 4
- HTHCZRWCFXMENJ-KKUMJFAQSA-N Tyr-Arg-Glu Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(O)=O HTHCZRWCFXMENJ-KKUMJFAQSA-N 0.000 description 4
- FGVFBDZSGQTYQX-UFYCRDLUSA-N Tyr-Phe-Val Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](C(C)C)C(O)=O FGVFBDZSGQTYQX-UFYCRDLUSA-N 0.000 description 4
- QHONGSVIVOFKAC-ULQDDVLXSA-N Tyr-Pro-His Chemical compound N[C@@H](Cc1ccc(O)cc1)C(=O)N1CCC[C@H]1C(=O)N[C@@H](Cc1cnc[nH]1)C(O)=O QHONGSVIVOFKAC-ULQDDVLXSA-N 0.000 description 4
- VYQQQIRHIFALGE-UWJYBYFXSA-N Tyr-Ser-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 VYQQQIRHIFALGE-UWJYBYFXSA-N 0.000 description 4
- LABUITCFCAABSV-UHFFFAOYSA-N Val-Ala-Tyr Natural products CC(C)C(N)C(=O)NC(C)C(=O)NC(C(O)=O)CC1=CC=C(O)C=C1 LABUITCFCAABSV-UHFFFAOYSA-N 0.000 description 4
- COYSIHFOCOMGCF-WPRPVWTQSA-N Val-Arg-Gly Chemical compound CC(C)[C@H](N)C(=O)N[C@H](C(=O)NCC(O)=O)CCCN=C(N)N COYSIHFOCOMGCF-WPRPVWTQSA-N 0.000 description 4
- JLFKWDAZBRYCGX-ZKWXMUAHSA-N Val-Asn-Ser Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CO)C(=O)O)N JLFKWDAZBRYCGX-ZKWXMUAHSA-N 0.000 description 4
- XTDDIVQWDXMRJL-IHRRRGAJSA-N Val-Leu-His Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@H](C(C)C)N XTDDIVQWDXMRJL-IHRRRGAJSA-N 0.000 description 4
- MLADEWAIYAPAAU-IHRRRGAJSA-N Val-Lys-His Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N MLADEWAIYAPAAU-IHRRRGAJSA-N 0.000 description 4
- USLVEJAHTBLSIL-CYDGBPFRSA-N Val-Pro-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)C(C)C USLVEJAHTBLSIL-CYDGBPFRSA-N 0.000 description 4
- JVGDAEKKZKKZFO-RCWTZXSCSA-N Val-Val-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](C(C)C)N)O JVGDAEKKZKKZFO-RCWTZXSCSA-N 0.000 description 4
- 239000008272 agar Substances 0.000 description 4
- 108010047495 alanylglycine Proteins 0.000 description 4
- -1 aromatic amino acids Chemical class 0.000 description 4
- 108010047857 aspartylglycine Proteins 0.000 description 4
- 230000027455 binding Effects 0.000 description 4
- 230000003115 biocidal effect Effects 0.000 description 4
- 238000010804 cDNA synthesis Methods 0.000 description 4
- 229940041514 candida albicans extract Drugs 0.000 description 4
- 229960003669 carbenicillin Drugs 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 4
- 235000013399 edible fruits Nutrition 0.000 description 4
- 230000007613 environmental effect Effects 0.000 description 4
- 241001233957 eudicotyledons Species 0.000 description 4
- 238000000605 extraction Methods 0.000 description 4
- 239000000499 gel Substances 0.000 description 4
- 230000035784 germination Effects 0.000 description 4
- 108010051307 glycyl-glycyl-proline Proteins 0.000 description 4
- 108010020688 glycylhistidine Proteins 0.000 description 4
- 238000006460 hydrolysis reaction Methods 0.000 description 4
- OOYGSFOGFJDDHP-KMCOLRRFSA-N kanamycin A sulfate Chemical compound OS(O)(=O)=O.O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N OOYGSFOGFJDDHP-KMCOLRRFSA-N 0.000 description 4
- 229960002064 kanamycin sulfate Drugs 0.000 description 4
- 108010083708 leucyl-aspartyl-valine Proteins 0.000 description 4
- VZCYOOQTPOCHFL-UPHRSURJSA-N maleic acid Chemical compound OC(=O)\C=C/C(O)=O VZCYOOQTPOCHFL-UPHRSURJSA-N 0.000 description 4
- 239000011976 maleic acid Substances 0.000 description 4
- 229920001778 nylon Polymers 0.000 description 4
- 244000000003 plant pathogen Species 0.000 description 4
- 230000008488 polyadenylation Effects 0.000 description 4
- 239000001103 potassium chloride Substances 0.000 description 4
- 235000011164 potassium chloride Nutrition 0.000 description 4
- 230000000306 recurrent effect Effects 0.000 description 4
- 108010048397 seryl-lysyl-leucine Proteins 0.000 description 4
- 108010026333 seryl-proline Proteins 0.000 description 4
- 239000000377 silicon dioxide Substances 0.000 description 4
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 4
- 239000012064 sodium phosphate buffer Substances 0.000 description 4
- 239000002689 soil Substances 0.000 description 4
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 description 4
- 239000012138 yeast extract Substances 0.000 description 4
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 3
- 241000589155 Agrobacterium tumefaciens Species 0.000 description 3
- VHVVPYOJIIQCKS-QEJZJMRPSA-N Ala-Leu-Phe Chemical compound C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 VHVVPYOJIIQCKS-QEJZJMRPSA-N 0.000 description 3
- DIIGDGJKTMLQQW-IHRRRGAJSA-N Arg-Lys-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCCN=C(N)N)N DIIGDGJKTMLQQW-IHRRRGAJSA-N 0.000 description 3
- DNBMCNQKNOKOSD-DCAQKATOSA-N Arg-Pro-Gln Chemical compound NC(N)=NCCC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(O)=O DNBMCNQKNOKOSD-DCAQKATOSA-N 0.000 description 3
- IYVSIZAXNLOKFQ-BYULHYEWSA-N Asn-Asp-Val Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O IYVSIZAXNLOKFQ-BYULHYEWSA-N 0.000 description 3
- JBDLMLZNDRLDIX-HJGDQZAQSA-N Asn-Thr-Leu Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(O)=O JBDLMLZNDRLDIX-HJGDQZAQSA-N 0.000 description 3
- IVPNEDNYYYFAGI-GARJFASQSA-N Asp-Leu-Pro Chemical compound CC(C)C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CC(=O)O)N IVPNEDNYYYFAGI-GARJFASQSA-N 0.000 description 3
- LIVXPXUVXFRWNY-CIUDSAMLSA-N Asp-Lys-Ala Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(O)=O LIVXPXUVXFRWNY-CIUDSAMLSA-N 0.000 description 3
- 241000123650 Botrytis cinerea Species 0.000 description 3
- 240000002791 Brassica napus Species 0.000 description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 3
- 241000701489 Cauliflower mosaic virus Species 0.000 description 3
- 108020004705 Codon Proteins 0.000 description 3
- 102100027819 Cytosolic beta-glucosidase Human genes 0.000 description 3
- 101100437498 Escherichia coli (strain K12) uidA gene Proteins 0.000 description 3
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 3
- YPHPEHMXOYTEQG-LAEOZQHASA-N Glu-Val-Asp Chemical compound OC(=O)C[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CCC(O)=O YPHPEHMXOYTEQG-LAEOZQHASA-N 0.000 description 3
- VIPDPMHGICREIS-GVXVVHGQSA-N Glu-Val-Leu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O VIPDPMHGICREIS-GVXVVHGQSA-N 0.000 description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 3
- JSNNHGHYGYMVCK-XVKPBYJWSA-N Gly-Glu-Val Chemical compound [H]NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O JSNNHGHYGYMVCK-XVKPBYJWSA-N 0.000 description 3
- UESJMAMHDLEHGM-NHCYSSNCSA-N Gly-Ile-Leu Chemical compound NCC(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(C)C)C(O)=O UESJMAMHDLEHGM-NHCYSSNCSA-N 0.000 description 3
- ZWRDOVYMQAAISL-UWVGGRQHSA-N Gly-Met-Lys Chemical compound CSCC[C@H](NC(=O)CN)C(=O)N[C@H](C(O)=O)CCCCN ZWRDOVYMQAAISL-UWVGGRQHSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- JBCLFWXMTIKCCB-UHFFFAOYSA-N H-Gly-Phe-OH Natural products NCC(=O)NC(C(O)=O)CC1=CC=CC=C1 JBCLFWXMTIKCCB-UHFFFAOYSA-N 0.000 description 3
- PSFDQSOCUJVVGF-UHFFFAOYSA-N Harman Natural products C12=CC=CC=C2NC2=C1C=CN=C2C PSFDQSOCUJVVGF-UHFFFAOYSA-N 0.000 description 3
- 101000859692 Homo sapiens Cytosolic beta-glucosidase Proteins 0.000 description 3
- 101000997662 Homo sapiens Lysosomal acid glucosylceramidase Proteins 0.000 description 3
- DSDPLOODKXISDT-XUXIUFHCSA-N Ile-Leu-Val Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(O)=O DSDPLOODKXISDT-XUXIUFHCSA-N 0.000 description 3
- DTPGSUQHUMELQB-GVARAGBVSA-N Ile-Tyr-Ala Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](C)C(O)=O)CC1=CC=C(O)C=C1 DTPGSUQHUMELQB-GVARAGBVSA-N 0.000 description 3
- 125000000570 L-alpha-aspartyl group Chemical group [H]OC(=O)C([H])([H])[C@]([H])(N([H])[H])C(*)=O 0.000 description 3
- 108091026898 Leader sequence (mRNA) Proteins 0.000 description 3
- VWHGTYCRDRBSFI-ZETCQYMHSA-N Leu-Gly-Gly Chemical compound CC(C)C[C@H](N)C(=O)NCC(=O)NCC(O)=O VWHGTYCRDRBSFI-ZETCQYMHSA-N 0.000 description 3
- XQXGNBFMAXWIGI-MXAVVETBSA-N Leu-His-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)CC(C)C)CC1=CN=CN1 XQXGNBFMAXWIGI-MXAVVETBSA-N 0.000 description 3
- WFCKERTZVCQXKH-KBPBESRZSA-N Leu-Tyr-Gly Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)NCC(O)=O WFCKERTZVCQXKH-KBPBESRZSA-N 0.000 description 3
- 239000006142 Luria-Bertani Agar Substances 0.000 description 3
- 235000007688 Lycopersicon esculentum Nutrition 0.000 description 3
- SWWCDAGDQHTKIE-RHYQMDGZSA-N Lys-Arg-Thr Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(O)=O SWWCDAGDQHTKIE-RHYQMDGZSA-N 0.000 description 3
- GJJQCBVRWDGLMQ-GUBZILKMSA-N Lys-Glu-Ala Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(O)=O GJJQCBVRWDGLMQ-GUBZILKMSA-N 0.000 description 3
- UIJVKVHLCQSPOJ-XIRDDKMYSA-N Lys-Ser-Trp Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](Cc1c[nH]c2ccccc12)C(O)=O UIJVKVHLCQSPOJ-XIRDDKMYSA-N 0.000 description 3
- 231100000678 Mycotoxin Toxicity 0.000 description 3
- SXJGROGVINAYSH-AVGNSLFASA-N Phe-Gln-Asp Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CC(=O)O)C(=O)O)N SXJGROGVINAYSH-AVGNSLFASA-N 0.000 description 3
- CSDMCMITJLKBAH-SOUVJXGZSA-N Phe-Glu-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CC2=CC=CC=C2)N)C(=O)O CSDMCMITJLKBAH-SOUVJXGZSA-N 0.000 description 3
- KUSYCSMTTHSZOA-DZKIICNBSA-N Phe-Val-Gln Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)N KUSYCSMTTHSZOA-DZKIICNBSA-N 0.000 description 3
- 240000004713 Pisum sativum Species 0.000 description 3
- 235000010582 Pisum sativum Nutrition 0.000 description 3
- NHDVNAKDACFHPX-GUBZILKMSA-N Pro-Arg-Ala Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(O)=O NHDVNAKDACFHPX-GUBZILKMSA-N 0.000 description 3
- PEYNRYREGPAOAK-LSJOCFKGSA-N Pro-His-Ala Chemical compound C([C@@H](C(=O)N[C@@H](C)C([O-])=O)NC(=O)[C@H]1[NH2+]CCC1)C1=CN=CN1 PEYNRYREGPAOAK-LSJOCFKGSA-N 0.000 description 3
- RMODQFBNDDENCP-IHRRRGAJSA-N Pro-Lys-Leu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(O)=O RMODQFBNDDENCP-IHRRRGAJSA-N 0.000 description 3
- 238000002123 RNA extraction Methods 0.000 description 3
- 241000813090 Rhizoctonia solani Species 0.000 description 3
- JIPVNVNKXJLFJF-BJDJZHNGSA-N Ser-Ile-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CO)N JIPVNVNKXJLFJF-BJDJZHNGSA-N 0.000 description 3
- MUJQWSAWLLRJCE-KATARQTJSA-N Ser-Leu-Thr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O MUJQWSAWLLRJCE-KATARQTJSA-N 0.000 description 3
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 3
- 240000003768 Solanum lycopersicum Species 0.000 description 3
- OLFOOYQTTQSSRK-UNQGMJICSA-N Thr-Pro-Phe Chemical compound C[C@@H](O)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 OLFOOYQTTQSSRK-UNQGMJICSA-N 0.000 description 3
- 239000007983 Tris buffer Substances 0.000 description 3
- PEYSVKMXSLPQRU-FJHTZYQYSA-N Trp-Ala-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](C)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)N)O PEYSVKMXSLPQRU-FJHTZYQYSA-N 0.000 description 3
- HMPMGPISLMLHSI-JBACZVJFSA-N Tyr-Trp-Gln Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)O)NC(=O)[C@H](CC3=CC=C(C=C3)O)N HMPMGPISLMLHSI-JBACZVJFSA-N 0.000 description 3
- VMRFIKXKOFNMHW-GUBZILKMSA-N Val-Arg-Ser Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CO)C(=O)O)N VMRFIKXKOFNMHW-GUBZILKMSA-N 0.000 description 3
- XBJKAZATRJBDCU-GUBZILKMSA-N Val-Pro-Ala Chemical compound CC(C)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C)C(O)=O XBJKAZATRJBDCU-GUBZILKMSA-N 0.000 description 3
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 3
- 230000009471 action Effects 0.000 description 3
- 238000000246 agarose gel electrophoresis Methods 0.000 description 3
- 108010005233 alanylglutamic acid Proteins 0.000 description 3
- 229960000723 ampicillin Drugs 0.000 description 3
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 3
- 229960001230 asparagine Drugs 0.000 description 3
- 108010077245 asparaginyl-proline Proteins 0.000 description 3
- 239000011324 bead Substances 0.000 description 3
- 239000012472 biological sample Substances 0.000 description 3
- 230000000903 blocking effect Effects 0.000 description 3
- 238000009395 breeding Methods 0.000 description 3
- 230000001488 breeding effect Effects 0.000 description 3
- FPPNZSSZRUTDAP-UWFZAAFLSA-N carbenicillin Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)C(C(O)=O)C1=CC=CC=C1 FPPNZSSZRUTDAP-UWFZAAFLSA-N 0.000 description 3
- 229910052799 carbon Inorganic materials 0.000 description 3
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 108010050949 chitinase C Proteins 0.000 description 3
- 230000001794 chitinolytic effect Effects 0.000 description 3
- 235000005822 corn Nutrition 0.000 description 3
- 238000012217 deletion Methods 0.000 description 3
- 230000037430 deletion Effects 0.000 description 3
- 230000001419 dependent effect Effects 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 230000000408 embryogenic effect Effects 0.000 description 3
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 description 3
- 229960005542 ethidium bromide Drugs 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 108010078144 glutaminyl-glycine Proteins 0.000 description 3
- 108010050848 glycylleucine Proteins 0.000 description 3
- 230000007062 hydrolysis Effects 0.000 description 3
- 238000010348 incorporation Methods 0.000 description 3
- 230000001939 inductive effect Effects 0.000 description 3
- 230000002401 inhibitory effect Effects 0.000 description 3
- 108010053037 kyotorphin Proteins 0.000 description 3
- 238000002372 labelling Methods 0.000 description 3
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 3
- 235000019341 magnesium sulphate Nutrition 0.000 description 3
- 230000000877 morphologic effect Effects 0.000 description 3
- 239000002636 mycotoxin Substances 0.000 description 3
- 229950006780 n-acetylglucosamine Drugs 0.000 description 3
- 230000037361 pathway Effects 0.000 description 3
- 230000008121 plant development Effects 0.000 description 3
- 238000001556 precipitation Methods 0.000 description 3
- 239000002243 precursor Substances 0.000 description 3
- 108010029020 prolylglycine Proteins 0.000 description 3
- 229920006395 saturated elastomer Polymers 0.000 description 3
- 230000003248 secreting effect Effects 0.000 description 3
- 230000028327 secretion Effects 0.000 description 3
- 239000001632 sodium acetate Substances 0.000 description 3
- 235000017281 sodium acetate Nutrition 0.000 description 3
- 239000001509 sodium citrate Substances 0.000 description 3
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 230000001052 transient effect Effects 0.000 description 3
- 230000014621 translational initiation Effects 0.000 description 3
- 210000003934 vacuole Anatomy 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 2
- 239000005631 2,4-Dichlorophenoxyacetic acid Substances 0.000 description 2
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 2
- STACJSVFHSEZJV-GHCJXIJMSA-N Ala-Asn-Ile Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O STACJSVFHSEZJV-GHCJXIJMSA-N 0.000 description 2
- BTYTYHBSJKQBQA-GCJQMDKQSA-N Ala-Asp-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](C)N)O BTYTYHBSJKQBQA-GCJQMDKQSA-N 0.000 description 2
- CWEAKSWWKHGTRJ-BQBZGAKWSA-N Ala-Gly-Met Chemical compound [H]N[C@@H](C)C(=O)NCC(=O)N[C@@H](CCSC)C(O)=O CWEAKSWWKHGTRJ-BQBZGAKWSA-N 0.000 description 2
- LDLSENBXQNDTPB-DCAQKATOSA-N Ala-Lys-Arg Chemical compound NCCCC[C@H](NC(=O)[C@@H](N)C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N LDLSENBXQNDTPB-DCAQKATOSA-N 0.000 description 2
- PEIBBAXIKUAYGN-UBHSHLNASA-N Ala-Phe-Arg Chemical compound NC(N)=NCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)C)CC1=CC=CC=C1 PEIBBAXIKUAYGN-UBHSHLNASA-N 0.000 description 2
- RTZCUEHYUQZIDE-WHFBIAKZSA-N Ala-Ser-Gly Chemical compound C[C@H](N)C(=O)N[C@@H](CO)C(=O)NCC(O)=O RTZCUEHYUQZIDE-WHFBIAKZSA-N 0.000 description 2
- YNOCMHZSWJMGBB-GCJQMDKQSA-N Ala-Thr-Asp Chemical compound [H]N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(O)=O)C(O)=O YNOCMHZSWJMGBB-GCJQMDKQSA-N 0.000 description 2
- LSMDIAAALJJLRO-XQXXSGGOSA-N Ala-Thr-Glu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(O)=O)C(O)=O LSMDIAAALJJLRO-XQXXSGGOSA-N 0.000 description 2
- 101710083587 Antifungal protein Proteins 0.000 description 2
- 241000726091 Aphanocladium album Species 0.000 description 2
- 241000219195 Arabidopsis thaliana Species 0.000 description 2
- DPXDVGDLWJYZBH-GUBZILKMSA-N Arg-Asn-Arg Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O DPXDVGDLWJYZBH-GUBZILKMSA-N 0.000 description 2
- SYAUZLVLXCDRSH-IUCAKERBSA-N Arg-Gly-Met Chemical compound CSCC[C@@H](C(=O)O)NC(=O)CNC(=O)[C@H](CCCN=C(N)N)N SYAUZLVLXCDRSH-IUCAKERBSA-N 0.000 description 2
- DNUKXVMPARLPFN-XUXIUFHCSA-N Arg-Leu-Ile Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O DNUKXVMPARLPFN-XUXIUFHCSA-N 0.000 description 2
- NGTYEHIRESTSRX-UWVGGRQHSA-N Arg-Lys-Gly Chemical compound NCCCC[C@@H](C(=O)NCC(O)=O)NC(=O)[C@@H](N)CCCN=C(N)N NGTYEHIRESTSRX-UWVGGRQHSA-N 0.000 description 2
- ADPACBMPYWJJCE-FXQIFTODSA-N Arg-Ser-Asp Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(O)=O ADPACBMPYWJJCE-FXQIFTODSA-N 0.000 description 2
- WCZXPVPHUMYLMS-VEVYYDQMSA-N Arg-Thr-Asp Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(O)=O)C(O)=O WCZXPVPHUMYLMS-VEVYYDQMSA-N 0.000 description 2
- XHFXZQHTLJVZBN-FXQIFTODSA-N Asn-Arg-Asn Chemical compound C(C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](CC(=O)N)N)CN=C(N)N XHFXZQHTLJVZBN-FXQIFTODSA-N 0.000 description 2
- GFFRWIJAFFMQGM-NUMRIWBASA-N Asn-Glu-Thr Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O GFFRWIJAFFMQGM-NUMRIWBASA-N 0.000 description 2
- BXUHCIXDSWRSBS-CIUDSAMLSA-N Asn-Leu-Asp Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O BXUHCIXDSWRSBS-CIUDSAMLSA-N 0.000 description 2
- GLWFAWNYGWBMOC-SRVKXCTJSA-N Asn-Leu-Leu Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O GLWFAWNYGWBMOC-SRVKXCTJSA-N 0.000 description 2
- YVXRYLVELQYAEQ-SRVKXCTJSA-N Asn-Leu-Lys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC(=O)N)N YVXRYLVELQYAEQ-SRVKXCTJSA-N 0.000 description 2
- FHETWELNCBMRMG-HJGDQZAQSA-N Asn-Leu-Thr Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O FHETWELNCBMRMG-HJGDQZAQSA-N 0.000 description 2
- IPPFAOCLQSGHJV-WFBYXXMGSA-N Asn-Trp-Ala Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](C)C(O)=O IPPFAOCLQSGHJV-WFBYXXMGSA-N 0.000 description 2
- KRXIWXCXOARFNT-ZLUOBGJFSA-N Asp-Ala-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC(O)=O KRXIWXCXOARFNT-ZLUOBGJFSA-N 0.000 description 2
- NECWUSYTYSIFNC-DLOVCJGASA-N Asp-Ala-Phe Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 NECWUSYTYSIFNC-DLOVCJGASA-N 0.000 description 2
- GWTLRDMPMJCNMH-WHFBIAKZSA-N Asp-Asn-Gly Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(O)=O GWTLRDMPMJCNMH-WHFBIAKZSA-N 0.000 description 2
- PDECQIHABNQRHN-GUBZILKMSA-N Asp-Glu-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)CC(O)=O PDECQIHABNQRHN-GUBZILKMSA-N 0.000 description 2
- WBDWQKRLTVCDSY-WHFBIAKZSA-N Asp-Gly-Asp Chemical compound OC(=O)C[C@H](N)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(O)=O WBDWQKRLTVCDSY-WHFBIAKZSA-N 0.000 description 2
- MFTVXYMXSAQZNL-DJFWLOJKSA-N Asp-Ile-His Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@H](CC(=O)O)N MFTVXYMXSAQZNL-DJFWLOJKSA-N 0.000 description 2
- RRUWMFBLFLUZSI-LPEHRKFASA-N Asp-Met-Pro Chemical compound CSCC[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CC(=O)O)N RRUWMFBLFLUZSI-LPEHRKFASA-N 0.000 description 2
- LTCKTLYKRMCFOC-KKUMJFAQSA-N Asp-Phe-Leu Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(C)C)C(O)=O LTCKTLYKRMCFOC-KKUMJFAQSA-N 0.000 description 2
- KESWRFKUZRUTAH-FXQIFTODSA-N Asp-Pro-Asp Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(O)=O)C(O)=O KESWRFKUZRUTAH-FXQIFTODSA-N 0.000 description 2
- JSHWXQIZOCVWIA-ZKWXMUAHSA-N Asp-Ser-Val Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(O)=O JSHWXQIZOCVWIA-ZKWXMUAHSA-N 0.000 description 2
- JSNWZMFSLIWAHS-HJGDQZAQSA-N Asp-Thr-Leu Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)O)NC(=O)[C@H](CC(=O)O)N)O JSNWZMFSLIWAHS-HJGDQZAQSA-N 0.000 description 2
- XWKPSMRPIKKDDU-RCOVLWMOSA-N Asp-Val-Gly Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)NCC(O)=O XWKPSMRPIKKDDU-RCOVLWMOSA-N 0.000 description 2
- 241000351920 Aspergillus nidulans Species 0.000 description 2
- 101100004029 Avena sativa P60B gene Proteins 0.000 description 2
- 101800003223 Cecropin-A Proteins 0.000 description 2
- 108010059892 Cellulase Proteins 0.000 description 2
- 235000009854 Cucurbita moschata Nutrition 0.000 description 2
- 240000001980 Cucurbita pepo Species 0.000 description 2
- OTXLNICGSXPGQF-KBIXCLLPSA-N Cys-Ile-Glu Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(O)=O)C(O)=O OTXLNICGSXPGQF-KBIXCLLPSA-N 0.000 description 2
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 2
- 238000001712 DNA sequencing Methods 0.000 description 2
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 2
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 2
- 108010067770 Endopeptidase K Proteins 0.000 description 2
- 241000620209 Escherichia coli DH5[alpha] Species 0.000 description 2
- 241000701959 Escherichia virus Lambda Species 0.000 description 2
- 101710112457 Exoglucanase Proteins 0.000 description 2
- 108700024394 Exon Proteins 0.000 description 2
- 241000567163 Fusarium cerealis Species 0.000 description 2
- 241001149959 Fusarium sp. Species 0.000 description 2
- 101150086513 GLU2 gene Proteins 0.000 description 2
- 108700028146 Genetic Enhancer Elements Proteins 0.000 description 2
- OETQLUYCMBARHJ-CIUDSAMLSA-N Gln-Asn-Arg Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O OETQLUYCMBARHJ-CIUDSAMLSA-N 0.000 description 2
- QYTKAVBFRUGYAU-ACZMJKKPSA-N Gln-Asp-Asn Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O QYTKAVBFRUGYAU-ACZMJKKPSA-N 0.000 description 2
- XKBASPWPBXNVLQ-WDSKDSINSA-N Gln-Gly-Asn Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)NCC(=O)N[C@@H](CC(N)=O)C(O)=O XKBASPWPBXNVLQ-WDSKDSINSA-N 0.000 description 2
- ORYMMTRPKVTGSJ-XVKPBYJWSA-N Gln-Gly-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CCC(N)=O ORYMMTRPKVTGSJ-XVKPBYJWSA-N 0.000 description 2
- KSKFIECUYMYWNS-AVGNSLFASA-N Gln-Lys-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCC(=O)N)N KSKFIECUYMYWNS-AVGNSLFASA-N 0.000 description 2
- LVRKAFPPFJRIOF-GARJFASQSA-N Gln-Met-Pro Chemical compound CSCC[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CCC(=O)N)N LVRKAFPPFJRIOF-GARJFASQSA-N 0.000 description 2
- XKPACHRGOWQHFH-IRIUXVKKSA-N Gln-Thr-Tyr Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O XKPACHRGOWQHFH-IRIUXVKKSA-N 0.000 description 2
- UTKUTMJSWKKHEM-WDSKDSINSA-N Glu-Ala-Gly Chemical compound OC(=O)CNC(=O)[C@H](C)NC(=O)[C@@H](N)CCC(O)=O UTKUTMJSWKKHEM-WDSKDSINSA-N 0.000 description 2
- ZOXBSICWUDAOHX-GUBZILKMSA-N Glu-Asn-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](N)CCC(O)=O ZOXBSICWUDAOHX-GUBZILKMSA-N 0.000 description 2
- CGOHAEBMDSEKFB-FXQIFTODSA-N Glu-Glu-Ala Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(O)=O CGOHAEBMDSEKFB-FXQIFTODSA-N 0.000 description 2
- LKOAAMXDJGEYMS-ZPFDUUQYSA-N Glu-Met-Ile Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O LKOAAMXDJGEYMS-ZPFDUUQYSA-N 0.000 description 2
- RGJKYNUINKGPJN-RWRJDSDZSA-N Glu-Thr-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(=O)O)N RGJKYNUINKGPJN-RWRJDSDZSA-N 0.000 description 2
- 108010022769 Glucan 1,3-beta-Glucosidase Proteins 0.000 description 2
- KKBWDNZXYLGJEY-UHFFFAOYSA-N Gly-Arg-Pro Natural products NCC(=O)NC(CCNC(=N)N)C(=O)N1CCCC1C(=O)O KKBWDNZXYLGJEY-UHFFFAOYSA-N 0.000 description 2
- BYYNJRSNDARRBX-YFKPBYRVSA-N Gly-Gln-Gly Chemical compound NCC(=O)N[C@@H](CCC(N)=O)C(=O)NCC(O)=O BYYNJRSNDARRBX-YFKPBYRVSA-N 0.000 description 2
- HHSOPSCKAZKQHQ-PEXQALLHSA-N Gly-His-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC1=CN=CN1)NC(=O)CN HHSOPSCKAZKQHQ-PEXQALLHSA-N 0.000 description 2
- NNCSJUBVFBDDLC-YUMQZZPRSA-N Gly-Leu-Ser Chemical compound NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O NNCSJUBVFBDDLC-YUMQZZPRSA-N 0.000 description 2
- LHYJCVCQPWRMKZ-WEDXCCLWSA-N Gly-Leu-Thr Chemical compound [H]NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O LHYJCVCQPWRMKZ-WEDXCCLWSA-N 0.000 description 2
- LOEANKRDMMVOGZ-YUMQZZPRSA-N Gly-Lys-Asp Chemical compound NCCCC[C@H](NC(=O)CN)C(=O)N[C@@H](CC(O)=O)C(O)=O LOEANKRDMMVOGZ-YUMQZZPRSA-N 0.000 description 2
- GLACUWHUYFBSPJ-FJXKBIBVSA-N Gly-Pro-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)CN GLACUWHUYFBSPJ-FJXKBIBVSA-N 0.000 description 2
- IMRNSEPSPFQNHF-STQMWFEESA-N Gly-Ser-Trp Chemical compound NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CNC2=CC=CC=C12)C(=O)O IMRNSEPSPFQNHF-STQMWFEESA-N 0.000 description 2
- FKESCSGWBPUTPN-FOHZUACHSA-N Gly-Thr-Asn Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(O)=O FKESCSGWBPUTPN-FOHZUACHSA-N 0.000 description 2
- SBVMXEZQJVUARN-XPUUQOCRSA-N Gly-Val-Ser Chemical compound NCC(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(O)=O SBVMXEZQJVUARN-XPUUQOCRSA-N 0.000 description 2
- COZMNNJEGNPDED-HOCLYGCPSA-N Gly-Val-Trp Chemical compound [H]NCC(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O COZMNNJEGNPDED-HOCLYGCPSA-N 0.000 description 2
- KSOBNUBCYHGUKH-UWVGGRQHSA-N Gly-Val-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)CN KSOBNUBCYHGUKH-UWVGGRQHSA-N 0.000 description 2
- ZIMTWPHIKZEHSE-UWVGGRQHSA-N His-Arg-Gly Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(O)=O ZIMTWPHIKZEHSE-UWVGGRQHSA-N 0.000 description 2
- MPXGJGBXCRQQJE-MXAVVETBSA-N His-Ile-Leu Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(C)C)C(O)=O MPXGJGBXCRQQJE-MXAVVETBSA-N 0.000 description 2
- LVWIJITYHRZHBO-IXOXFDKPSA-N His-Leu-Thr Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O LVWIJITYHRZHBO-IXOXFDKPSA-N 0.000 description 2
- TVMNTHXFRSXZGR-IHRRRGAJSA-N His-Lys-Val Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(O)=O TVMNTHXFRSXZGR-IHRRRGAJSA-N 0.000 description 2
- 101000940063 Homo sapiens Ubiquitin-conjugating enzyme E2 variant 2 Proteins 0.000 description 2
- NULSANWBUWLTKN-NAKRPEOUSA-N Ile-Arg-Ser Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CO)C(=O)O)N NULSANWBUWLTKN-NAKRPEOUSA-N 0.000 description 2
- JRYQSFOFUFXPTB-RWRJDSDZSA-N Ile-Gln-Thr Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H]([C@@H](C)O)C(=O)O)N JRYQSFOFUFXPTB-RWRJDSDZSA-N 0.000 description 2
- CDGLBYSAZFIIJO-RCOVLWMOSA-N Ile-Gly-Gly Chemical compound CC[C@H](C)[C@H]([NH3+])C(=O)NCC(=O)NCC([O-])=O CDGLBYSAZFIIJO-RCOVLWMOSA-N 0.000 description 2
- RMJWFINHACYKJI-SIUGBPQLSA-N Ile-Tyr-Glu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N RMJWFINHACYKJI-SIUGBPQLSA-N 0.000 description 2
- PMGDADKJMCOXHX-UHFFFAOYSA-N L-Arginyl-L-glutamin-acetat Natural products NC(=N)NCCCC(N)C(=O)NC(CCC(N)=O)C(O)=O PMGDADKJMCOXHX-UHFFFAOYSA-N 0.000 description 2
- 125000003440 L-leucyl group Chemical group O=C([*])[C@](N([H])[H])([H])C([H])([H])C(C([H])([H])[H])([H])C([H])([H])[H] 0.000 description 2
- KFKWRHQBZQICHA-STQMWFEESA-N L-leucyl-L-phenylalanine Natural products CC(C)C[C@H](N)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 KFKWRHQBZQICHA-STQMWFEESA-N 0.000 description 2
- 125000002842 L-seryl group Chemical group O=C([*])[C@](N([H])[H])([H])C([H])([H])O[H] 0.000 description 2
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 2
- LZDNBBYBDGBADK-UHFFFAOYSA-N L-valyl-L-tryptophan Natural products C1=CC=C2C(CC(NC(=O)C(N)C(C)C)C(O)=O)=CNC2=C1 LZDNBBYBDGBADK-UHFFFAOYSA-N 0.000 description 2
- JKGHDYGZRDWHGA-SRVKXCTJSA-N Leu-Asn-Leu Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(O)=O JKGHDYGZRDWHGA-SRVKXCTJSA-N 0.000 description 2
- JLWZLIQRYCTYBD-IHRRRGAJSA-N Leu-Lys-Arg Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O JLWZLIQRYCTYBD-IHRRRGAJSA-N 0.000 description 2
- LZHJZLHSRGWBBE-IHRRRGAJSA-N Leu-Lys-Val Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(O)=O LZHJZLHSRGWBBE-IHRRRGAJSA-N 0.000 description 2
- PKKMDPNFGULLNQ-AVGNSLFASA-N Leu-Met-Arg Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O PKKMDPNFGULLNQ-AVGNSLFASA-N 0.000 description 2
- UHNQRAFSEBGZFZ-YESZJQIVSA-N Leu-Phe-Pro Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N2CCC[C@@H]2C(=O)O)N UHNQRAFSEBGZFZ-YESZJQIVSA-N 0.000 description 2
- HGLKOTPFWOMPOB-MEYUZBJRSA-N Leu-Thr-Tyr Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 HGLKOTPFWOMPOB-MEYUZBJRSA-N 0.000 description 2
- VUBIPAHVHMZHCM-KKUMJFAQSA-N Leu-Tyr-Ser Chemical compound CC(C)C[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](CO)C(O)=O)CC1=CC=C(O)C=C1 VUBIPAHVHMZHCM-KKUMJFAQSA-N 0.000 description 2
- 239000006137 Luria-Bertani broth Substances 0.000 description 2
- IRNSXVOWSXSULE-DCAQKATOSA-N Lys-Ala-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCCCN IRNSXVOWSXSULE-DCAQKATOSA-N 0.000 description 2
- CLBGMWIYPYAZPR-AVGNSLFASA-N Lys-Arg-Arg Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O CLBGMWIYPYAZPR-AVGNSLFASA-N 0.000 description 2
- RZHLIPMZXOEJTL-AVGNSLFASA-N Lys-Gln-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CCCCN)N RZHLIPMZXOEJTL-AVGNSLFASA-N 0.000 description 2
- PAMDBWYMLWOELY-SDDRHHMPSA-N Lys-Glu-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CCCCN)N)C(=O)O PAMDBWYMLWOELY-SDDRHHMPSA-N 0.000 description 2
- JQSIGLHQNSZZRL-KKUMJFAQSA-N Lys-Lys-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCCCN)N JQSIGLHQNSZZRL-KKUMJFAQSA-N 0.000 description 2
- 239000007993 MOPS buffer Substances 0.000 description 2
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 2
- 229930195725 Mannitol Natural products 0.000 description 2
- CAODKDAPYGUMLK-FXQIFTODSA-N Met-Asn-Ser Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(O)=O CAODKDAPYGUMLK-FXQIFTODSA-N 0.000 description 2
- CHQWUYSNAOABIP-ZPFDUUQYSA-N Met-Glu-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CCSC)N CHQWUYSNAOABIP-ZPFDUUQYSA-N 0.000 description 2
- MSSJHBAKDDIRMJ-SRVKXCTJSA-N Met-Lys-Gln Chemical compound [H]N[C@@H](CCSC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(N)=O)C(O)=O MSSJHBAKDDIRMJ-SRVKXCTJSA-N 0.000 description 2
- QQPMHUCGDRJFQK-RHYQMDGZSA-N Met-Thr-Leu Chemical compound CSCC[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@H](C(O)=O)CC(C)C QQPMHUCGDRJFQK-RHYQMDGZSA-N 0.000 description 2
- YBAFDPFAUTYYRW-UHFFFAOYSA-N N-L-alpha-glutamyl-L-leucine Natural products CC(C)CC(C(O)=O)NC(=O)C(N)CCC(O)=O YBAFDPFAUTYYRW-UHFFFAOYSA-N 0.000 description 2
- OVRNDRQMDRJTHS-UHFFFAOYSA-N N-acelyl-D-glucosamine Natural products CC(=O)NC1C(O)OC(CO)C(O)C1O OVRNDRQMDRJTHS-UHFFFAOYSA-N 0.000 description 2
- OVRNDRQMDRJTHS-FMDGEEDCSA-N N-acetyl-beta-D-glucosamine Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-FMDGEEDCSA-N 0.000 description 2
- MBLBDJOUHNCFQT-LXGUWJNJSA-N N-acetylglucosamine Natural products CC(=O)N[C@@H](C=O)[C@@H](O)[C@H](O)[C@H](O)CO MBLBDJOUHNCFQT-LXGUWJNJSA-N 0.000 description 2
- KZNQNBZMBZJQJO-UHFFFAOYSA-N N-glycyl-L-proline Natural products NCC(=O)N1CCCC1C(O)=O KZNQNBZMBZJQJO-UHFFFAOYSA-N 0.000 description 2
- 101710096342 Pathogenesis-related protein Proteins 0.000 description 2
- 108010002747 Pfu DNA polymerase Proteins 0.000 description 2
- 235000010627 Phaseolus vulgaris Nutrition 0.000 description 2
- 244000046052 Phaseolus vulgaris Species 0.000 description 2
- WSXKXSBOJXEZDV-DLOVCJGASA-N Phe-Ala-Asn Chemical compound NC(=O)C[C@@H](C([O-])=O)NC(=O)[C@H](C)NC(=O)[C@@H]([NH3+])CC1=CC=CC=C1 WSXKXSBOJXEZDV-DLOVCJGASA-N 0.000 description 2
- ZENDEDYRYVHBEG-SRVKXCTJSA-N Phe-Asp-Asp Chemical compound OC(=O)C[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](N)CC1=CC=CC=C1 ZENDEDYRYVHBEG-SRVKXCTJSA-N 0.000 description 2
- PSBJZLMFFTULDX-IXOXFDKPSA-N Phe-Cys-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H](CC1=CC=CC=C1)N)O PSBJZLMFFTULDX-IXOXFDKPSA-N 0.000 description 2
- AUJWXNGCAQWLEI-KBPBESRZSA-N Phe-Lys-Gly Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCCCN)C(=O)NCC(O)=O AUJWXNGCAQWLEI-KBPBESRZSA-N 0.000 description 2
- 241000233614 Phytophthora Species 0.000 description 2
- 108700001094 Plant Genes Proteins 0.000 description 2
- 239000004698 Polyethylene Substances 0.000 description 2
- 229920001213 Polysorbate 20 Polymers 0.000 description 2
- DBALDZKOTNSBFM-FXQIFTODSA-N Pro-Ala-Asn Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](C)C(=O)N[C@@H](CC(N)=O)C(O)=O DBALDZKOTNSBFM-FXQIFTODSA-N 0.000 description 2
- SZZBUDVXWZZPDH-BQBZGAKWSA-N Pro-Cys-Gly Chemical compound OC(=O)CNC(=O)[C@H](CS)NC(=O)[C@@H]1CCCN1 SZZBUDVXWZZPDH-BQBZGAKWSA-N 0.000 description 2
- 239000013614 RNA sample Substances 0.000 description 2
- 101500018493 Rhizopus oryzae Glucoamylase 3 Proteins 0.000 description 2
- 241000235347 Schizosaccharomyces pombe Species 0.000 description 2
- 101100179093 Schizosaccharomyces pombe (strain 972 / ATCC 24843) idh2 gene Proteins 0.000 description 2
- WDXYVIIVDIDOSX-DCAQKATOSA-N Ser-Arg-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)CO)CCCN=C(N)N WDXYVIIVDIDOSX-DCAQKATOSA-N 0.000 description 2
- VDVYTKZBMFADQH-AVGNSLFASA-N Ser-Gln-Tyr Chemical compound OC[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 VDVYTKZBMFADQH-AVGNSLFASA-N 0.000 description 2
- WLJPJRGQRNCIQS-ZLUOBGJFSA-N Ser-Ser-Asn Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(O)=O WLJPJRGQRNCIQS-ZLUOBGJFSA-N 0.000 description 2
- AXKJPUBALUNJEO-UBHSHLNASA-N Ser-Trp-Asn Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CC(N)=O)C(O)=O AXKJPUBALUNJEO-UBHSHLNASA-N 0.000 description 2
- FVFUOQIYDPAIJR-XIRDDKMYSA-N Ser-Trp-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)NC(=O)[C@H](CO)N FVFUOQIYDPAIJR-XIRDDKMYSA-N 0.000 description 2
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 2
- 101000866347 Solanum lycopersicum Glucan endo-1,3-beta-glucosidase A Proteins 0.000 description 2
- 208000037065 Subacute sclerosing leukoencephalitis Diseases 0.000 description 2
- 206010042297 Subacute sclerosing panencephalitis Diseases 0.000 description 2
- RZCIEJXAILMSQK-JXOAFFINSA-N TTP Chemical compound O=C1NC(=O)C(C)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 RZCIEJXAILMSQK-JXOAFFINSA-N 0.000 description 2
- SKHPKKYKDYULDH-HJGDQZAQSA-N Thr-Asn-Leu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(O)=O SKHPKKYKDYULDH-HJGDQZAQSA-N 0.000 description 2
- GKMYGVQDGVYCPC-IUKAMOBKSA-N Thr-Asp-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H]([C@@H](C)O)N GKMYGVQDGVYCPC-IUKAMOBKSA-N 0.000 description 2
- WBCCCPZIJIJTSD-TUBUOCAGSA-N Thr-His-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC1=CN=CN1)NC(=O)[C@H]([C@@H](C)O)N WBCCCPZIJIJTSD-TUBUOCAGSA-N 0.000 description 2
- BVOVIGCHYNFJBZ-JXUBOQSCSA-N Thr-Leu-Ala Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(O)=O BVOVIGCHYNFJBZ-JXUBOQSCSA-N 0.000 description 2
- KZSYAEWQMJEGRZ-RHYQMDGZSA-N Thr-Leu-Val Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(O)=O KZSYAEWQMJEGRZ-RHYQMDGZSA-N 0.000 description 2
- OETOOJXFNSEYHQ-WFBYXXMGSA-N Trp-Ala-Asp Chemical compound C1=CC=C2C(C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(O)=O)C(O)=O)=CNC2=C1 OETOOJXFNSEYHQ-WFBYXXMGSA-N 0.000 description 2
- LHHDBONOFZDWMW-AAEUAGOBSA-N Trp-Asp-Gly Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)NCC(=O)O)N LHHDBONOFZDWMW-AAEUAGOBSA-N 0.000 description 2
- UJGDFQRPYGJBEH-AAEUAGOBSA-N Trp-Ser-Gly Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CO)C(=O)NCC(=O)O)N UJGDFQRPYGJBEH-AAEUAGOBSA-N 0.000 description 2
- YXSSXUIBUJGHJY-SFJXLCSZSA-N Trp-Thr-Phe Chemical compound C([C@H](NC(=O)[C@@H](NC(=O)[C@@H](N)CC=1C2=CC=CC=C2NC=1)[C@H](O)C)C(O)=O)C1=CC=CC=C1 YXSSXUIBUJGHJY-SFJXLCSZSA-N 0.000 description 2
- DVLHKUWLNKDINO-PMVMPFDFSA-N Trp-Tyr-Leu Chemical compound [H]N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(C)C)C(O)=O DVLHKUWLNKDINO-PMVMPFDFSA-N 0.000 description 2
- XMNDQSYABVWZRK-BZSNNMDCSA-N Tyr-Asn-Phe Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O XMNDQSYABVWZRK-BZSNNMDCSA-N 0.000 description 2
- LTSIAOZUVISRAQ-QWRGUYRKSA-N Tyr-Gly-Cys Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)NCC(=O)N[C@@H](CS)C(=O)O)N)O LTSIAOZUVISRAQ-QWRGUYRKSA-N 0.000 description 2
- FNWGDMZVYBVAGJ-XEGUGMAKSA-N Tyr-Gly-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)CNC(=O)[C@H](CC1=CC=C(C=C1)O)N FNWGDMZVYBVAGJ-XEGUGMAKSA-N 0.000 description 2
- ZMKDQRJLMRZHRI-ACRUOGEOSA-N Tyr-Phe-His Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)NC(=O)[C@H](CC3=CC=C(C=C3)O)N ZMKDQRJLMRZHRI-ACRUOGEOSA-N 0.000 description 2
- RCMWNNJFKNDKQR-UFYCRDLUSA-N Tyr-Pro-Phe Chemical compound C([C@H](N)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=C(O)C=C1 RCMWNNJFKNDKQR-UFYCRDLUSA-N 0.000 description 2
- BCOBSVIZMQXKFY-KKUMJFAQSA-N Tyr-Ser-His Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)N)O BCOBSVIZMQXKFY-KKUMJFAQSA-N 0.000 description 2
- HRHYJNLMIJWGLF-BZSNNMDCSA-N Tyr-Ser-Phe Chemical compound C([C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=C(O)C=C1 HRHYJNLMIJWGLF-BZSNNMDCSA-N 0.000 description 2
- SYFHQHYTNCQCCN-MELADBBJSA-N Tyr-Ser-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CO)NC(=O)[C@H](CC2=CC=C(C=C2)O)N)C(=O)O SYFHQHYTNCQCCN-MELADBBJSA-N 0.000 description 2
- 102100031122 Ubiquitin-conjugating enzyme E2 variant 2 Human genes 0.000 description 2
- VJOWWOGRNXRQMF-UVBJJODRSA-N Val-Ala-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@H](C)NC(=O)[C@@H](N)C(C)C)C(O)=O)=CNC2=C1 VJOWWOGRNXRQMF-UVBJJODRSA-N 0.000 description 2
- QPZMOUMNTGTEFR-ZKWXMUAHSA-N Val-Asn-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](C(C)C)N QPZMOUMNTGTEFR-ZKWXMUAHSA-N 0.000 description 2
- KVRLNEILGGVBJX-IHRRRGAJSA-N Val-His-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)C(C)C)CC1=CN=CN1 KVRLNEILGGVBJX-IHRRRGAJSA-N 0.000 description 2
- WNZSAUMKZQXHNC-UKJIMTQDSA-N Val-Ile-Gln Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)O)NC(=O)[C@H](C(C)C)N WNZSAUMKZQXHNC-UKJIMTQDSA-N 0.000 description 2
- UMPVMAYCLYMYGA-ONGXEEELSA-N Val-Leu-Gly Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)NCC(O)=O UMPVMAYCLYMYGA-ONGXEEELSA-N 0.000 description 2
- JVGHIFMSFBZDHH-WPRPVWTQSA-N Val-Met-Gly Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)O)N JVGHIFMSFBZDHH-WPRPVWTQSA-N 0.000 description 2
- UFCHCOKFAGOQSF-BQFCYCMXSA-N Val-Trp-Glu Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N UFCHCOKFAGOQSF-BQFCYCMXSA-N 0.000 description 2
- OEVFFOBAXHBXKM-HSHDSVGOSA-N Val-Trp-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)NC(=O)[C@H](C(C)C)N)O OEVFFOBAXHBXKM-HSHDSVGOSA-N 0.000 description 2
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 2
- 241000607479 Yersinia pestis Species 0.000 description 2
- 108700041896 Zea mays Ubi-1 Proteins 0.000 description 2
- 108010081404 acein-2 Proteins 0.000 description 2
- 108010045350 alanyl-tyrosyl-alanine Proteins 0.000 description 2
- 108010070944 alanylhistidine Proteins 0.000 description 2
- 108010087924 alanylproline Proteins 0.000 description 2
- KOSRFJWDECSPRO-UHFFFAOYSA-N alpha-L-glutamyl-L-glutamic acid Natural products OC(=O)CCC(N)C(=O)NC(CCC(O)=O)C(O)=O KOSRFJWDECSPRO-UHFFFAOYSA-N 0.000 description 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 2
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 2
- 235000011130 ammonium sulphate Nutrition 0.000 description 2
- 229940121375 antifungal agent Drugs 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 239000006286 aqueous extract Substances 0.000 description 2
- 108010008355 arginyl-glutamine Proteins 0.000 description 2
- 108010068380 arginylarginine Proteins 0.000 description 2
- 108010069205 aspartyl-phenylalanine Proteins 0.000 description 2
- 108010068265 aspartyltyrosine Proteins 0.000 description 2
- 229960003237 betaine Drugs 0.000 description 2
- 239000003139 biocide Substances 0.000 description 2
- HCQPHKMLKXOJSR-IRCPFGJUSA-N cecropin-a Chemical compound C([C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@H](C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@H](C(=O)NCC(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)NCC(=O)N1[C@@H](CCC1)C(=O)N[C@@H](C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C(C)C)C(=O)NCC(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(N)=O)[C@@H](C)CC)C(C)C)[C@@H](C)CC)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@@H](N)CCCCN)C1=CC=CC=C1 HCQPHKMLKXOJSR-IRCPFGJUSA-N 0.000 description 2
- 230000032823 cell division Effects 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 229930002875 chlorophyll Natural products 0.000 description 2
- 235000019804 chlorophyll Nutrition 0.000 description 2
- ATNHDLDRLWWWCB-AENOIHSZSA-M chlorophyll a Chemical compound C1([C@@H](C(=O)OC)C(=O)C2=C3C)=C2N2C3=CC(C(CC)=C3C)=[N+]4C3=CC3=C(C=C)C(C)=C5N3[Mg-2]42[N+]2=C1[C@@H](CCC(=O)OC\C=C(/C)CCC[C@H](C)CCC[C@H](C)CCCC(C)C)[C@H](C)C2=C5 ATNHDLDRLWWWCB-AENOIHSZSA-M 0.000 description 2
- 229960001231 choline Drugs 0.000 description 2
- OEYIOHPDSNJKLS-UHFFFAOYSA-N choline Chemical compound C[N+](C)(C)CCO OEYIOHPDSNJKLS-UHFFFAOYSA-N 0.000 description 2
- 210000000349 chromosome Anatomy 0.000 description 2
- 239000002131 composite material Substances 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000004590 computer program Methods 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 230000001276 controlling effect Effects 0.000 description 2
- 244000038559 crop plants Species 0.000 description 2
- 210000000805 cytoplasm Anatomy 0.000 description 2
- SUYVUBYJARFZHO-RRKCRQDMSA-N dATP Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-RRKCRQDMSA-N 0.000 description 2
- SUYVUBYJARFZHO-UHFFFAOYSA-N dATP Natural products C1=NC=2C(N)=NC=NC=2N1C1CC(O)C(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-UHFFFAOYSA-N 0.000 description 2
- RGWHQCVHVJXOKC-SHYZEUOFSA-J dCTP(4-) Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)C1 RGWHQCVHVJXOKC-SHYZEUOFSA-J 0.000 description 2
- HAAZLUGHYHWQIW-KVQBGUIXSA-N dGTP Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 HAAZLUGHYHWQIW-KVQBGUIXSA-N 0.000 description 2
- 230000004665 defense response Effects 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 238000001952 enzyme assay Methods 0.000 description 2
- 239000011536 extraction buffer Substances 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 238000010353 genetic engineering Methods 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 108010055341 glutamyl-glutamic acid Proteins 0.000 description 2
- KWIUHFFTVRNATP-UHFFFAOYSA-N glycine betaine Chemical compound C[N+](C)(C)CC([O-])=O KWIUHFFTVRNATP-UHFFFAOYSA-N 0.000 description 2
- 108010067216 glycyl-glycyl-glycine Proteins 0.000 description 2
- 108010089804 glycyl-threonine Proteins 0.000 description 2
- 108010081551 glycylphenylalanine Proteins 0.000 description 2
- 108010084389 glycyltryptophan Proteins 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 239000011539 homogenization buffer Substances 0.000 description 2
- 230000002209 hydrophobic effect Effects 0.000 description 2
- 230000028644 hyphal growth Effects 0.000 description 2
- WQYVRQLZKVEZGA-UHFFFAOYSA-N hypochlorite Chemical compound Cl[O-] WQYVRQLZKVEZGA-UHFFFAOYSA-N 0.000 description 2
- 238000007654 immersion Methods 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 108010044374 isoleucyl-tyrosine Proteins 0.000 description 2
- 108010051673 leucyl-glycyl-phenylalanine Proteins 0.000 description 2
- 108010044056 leucyl-phenylalanine Proteins 0.000 description 2
- 108010057821 leucylproline Proteins 0.000 description 2
- 238000007834 ligase chain reaction Methods 0.000 description 2
- 230000000670 limiting effect Effects 0.000 description 2
- 244000144972 livestock Species 0.000 description 2
- 239000000594 mannitol Substances 0.000 description 2
- 235000010355 mannitol Nutrition 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 238000000520 microinjection Methods 0.000 description 2
- 238000013508 migration Methods 0.000 description 2
- 230000005012 migration Effects 0.000 description 2
- 239000004570 mortar (masonry) Substances 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 238000007899 nucleic acid hybridization Methods 0.000 description 2
- 210000004940 nucleus Anatomy 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- 108010074082 phenylalanyl-alanyl-lysine Proteins 0.000 description 2
- 108010089198 phenylalanyl-prolyl-arginine Proteins 0.000 description 2
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 2
- 230000037039 plant physiology Effects 0.000 description 2
- 239000013600 plasmid vector Substances 0.000 description 2
- 229920001223 polyethylene glycol Polymers 0.000 description 2
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 2
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 2
- SCVFZCLFOSHCOH-UHFFFAOYSA-M potassium acetate Chemical compound [K+].CC([O-])=O SCVFZCLFOSHCOH-UHFFFAOYSA-M 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 108010077112 prolyl-proline Proteins 0.000 description 2
- 230000000644 propagated effect Effects 0.000 description 2
- 239000011541 reaction mixture Substances 0.000 description 2
- 239000012492 regenerant Substances 0.000 description 2
- 230000003938 response to stress Effects 0.000 description 2
- 239000011833 salt mixture Substances 0.000 description 2
- 229920006298 saran Polymers 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- ATHGHQPFGPMSJY-UHFFFAOYSA-N spermidine Chemical compound NCCCCNCCCN ATHGHQPFGPMSJY-UHFFFAOYSA-N 0.000 description 2
- PFNFFQXMRSDOHW-UHFFFAOYSA-N spermine Chemical compound NCCCNCCCCNCCCN PFNFFQXMRSDOHW-UHFFFAOYSA-N 0.000 description 2
- 230000002195 synergetic effect Effects 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 description 2
- 229960000344 thiamine hydrochloride Drugs 0.000 description 2
- 235000019190 thiamine hydrochloride Nutrition 0.000 description 2
- 239000011747 thiamine hydrochloride Substances 0.000 description 2
- 230000005026 transcription initiation Effects 0.000 description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 2
- 239000012137 tryptone Substances 0.000 description 2
- 108010020532 tyrosyl-proline Proteins 0.000 description 2
- 108010003137 tyrosyltyrosine Proteins 0.000 description 2
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 2
- DBTMGCOVALSLOR-DEVYUCJPSA-N (2s,3r,4s,5r,6r)-4-[(2s,3r,4s,5r,6r)-3,5-dihydroxy-6-(hydroxymethyl)-4-[(2s,3r,4s,5s,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyoxan-2-yl]oxy-6-(hydroxymethyl)oxane-2,3,5-triol Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](CO)O[C@H](O)[C@@H]2O)O)O[C@H](CO)[C@H]1O DBTMGCOVALSLOR-DEVYUCJPSA-N 0.000 description 1
- FQVLRGLGWNWPSS-BXBUPLCLSA-N (4r,7s,10s,13s,16r)-16-acetamido-13-(1h-imidazol-5-ylmethyl)-10-methyl-6,9,12,15-tetraoxo-7-propan-2-yl-1,2-dithia-5,8,11,14-tetrazacycloheptadecane-4-carboxamide Chemical compound N1C(=O)[C@@H](NC(C)=O)CSSC[C@@H](C(N)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](C)NC(=O)[C@@H]1CC1=CN=CN1 FQVLRGLGWNWPSS-BXBUPLCLSA-N 0.000 description 1
- ZBMRKNMTMPPMMK-UHFFFAOYSA-N 2-amino-4-[hydroxy(methyl)phosphoryl]butanoic acid;azane Chemical compound [NH4+].CP(O)(=O)CCC(N)C([O-])=O ZBMRKNMTMPPMMK-UHFFFAOYSA-N 0.000 description 1
- NKDFYOWSKOHCCO-YPVLXUMRSA-N 20-hydroxyecdysone Chemical compound C1[C@@H](O)[C@@H](O)C[C@]2(C)[C@@H](CC[C@@]3([C@@H]([C@@](C)(O)[C@H](O)CCC(C)(O)C)CC[C@]33O)C)C3=CC(=O)[C@@H]21 NKDFYOWSKOHCCO-YPVLXUMRSA-N 0.000 description 1
- 101710168820 2S seed storage albumin protein Proteins 0.000 description 1
- 101150103244 ACT1 gene Proteins 0.000 description 1
- RZVAJINKPMORJF-UHFFFAOYSA-N Acetaminophen Chemical compound CC(=O)NC1=CC=C(O)C=C1 RZVAJINKPMORJF-UHFFFAOYSA-N 0.000 description 1
- 108010055851 Acetylglucosaminidase Proteins 0.000 description 1
- 108010085238 Actins Proteins 0.000 description 1
- 101150021974 Adh1 gene Proteins 0.000 description 1
- 241000589156 Agrobacterium rhizogenes Species 0.000 description 1
- DVWVZSJAYIJZFI-FXQIFTODSA-N Ala-Arg-Asn Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(N)=O)C(O)=O DVWVZSJAYIJZFI-FXQIFTODSA-N 0.000 description 1
- JBGSZRYCXBPWGX-BQBZGAKWSA-N Ala-Arg-Gly Chemical compound OC(=O)CNC(=O)[C@@H](NC(=O)[C@@H](N)C)CCCN=C(N)N JBGSZRYCXBPWGX-BQBZGAKWSA-N 0.000 description 1
- LWUWMHIOBPTZBA-DCAQKATOSA-N Ala-Arg-Lys Chemical compound NC(=N)NCCC[C@H](NC(=O)[C@@H](N)C)C(=O)N[C@@H](CCCCN)C(O)=O LWUWMHIOBPTZBA-DCAQKATOSA-N 0.000 description 1
- PXKLCFFSVLKOJM-ACZMJKKPSA-N Ala-Asn-Glu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O PXKLCFFSVLKOJM-ACZMJKKPSA-N 0.000 description 1
- NXSFUECZFORGOG-CIUDSAMLSA-N Ala-Asn-Leu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(O)=O NXSFUECZFORGOG-CIUDSAMLSA-N 0.000 description 1
- KIUYPHAMDKDICO-WHFBIAKZSA-N Ala-Asp-Gly Chemical compound C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(O)=O KIUYPHAMDKDICO-WHFBIAKZSA-N 0.000 description 1
- LSLIRHLIUDVNBN-CIUDSAMLSA-N Ala-Asp-Lys Chemical compound C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CCCCN LSLIRHLIUDVNBN-CIUDSAMLSA-N 0.000 description 1
- DAEFQZCYZKRTLR-ZLUOBGJFSA-N Ala-Cys-Asp Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(O)=O)C(O)=O DAEFQZCYZKRTLR-ZLUOBGJFSA-N 0.000 description 1
- XAGIMRPOEJSYER-CIUDSAMLSA-N Ala-Cys-Lys Chemical compound C[C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CCCCN)C(=O)O)N XAGIMRPOEJSYER-CIUDSAMLSA-N 0.000 description 1
- VIGKUFXFTPWYER-BIIVOSGPSA-N Ala-Cys-Pro Chemical compound C[C@@H](C(=O)N[C@@H](CS)C(=O)N1CCC[C@@H]1C(=O)O)N VIGKUFXFTPWYER-BIIVOSGPSA-N 0.000 description 1
- ZODMADSIQZZBSQ-FXQIFTODSA-N Ala-Gln-Glu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O ZODMADSIQZZBSQ-FXQIFTODSA-N 0.000 description 1
- YIGLXQRFQVWFEY-NRPADANISA-N Ala-Gln-Val Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C(C)C)C(O)=O YIGLXQRFQVWFEY-NRPADANISA-N 0.000 description 1
- ZVFVBBGVOILKPO-WHFBIAKZSA-N Ala-Gly-Ala Chemical compound C[C@H](N)C(=O)NCC(=O)N[C@@H](C)C(O)=O ZVFVBBGVOILKPO-WHFBIAKZSA-N 0.000 description 1
- BTBUEVAGZCKULD-XPUUQOCRSA-N Ala-Gly-His Chemical compound C[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CC1=CN=CN1 BTBUEVAGZCKULD-XPUUQOCRSA-N 0.000 description 1
- OBVSBEYOMDWLRJ-BFHQHQDPSA-N Ala-Gly-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@H](C)N OBVSBEYOMDWLRJ-BFHQHQDPSA-N 0.000 description 1
- HQJKCXHQNUCKMY-GHCJXIJMSA-N Ala-Ile-Asp Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)O)NC(=O)[C@H](C)N HQJKCXHQNUCKMY-GHCJXIJMSA-N 0.000 description 1
- QJABSQFUHKHTNP-SYWGBEHUSA-N Ala-Ile-Trp Chemical compound [H]N[C@@H](C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O QJABSQFUHKHTNP-SYWGBEHUSA-N 0.000 description 1
- OYJCVIGKMXUVKB-GARJFASQSA-N Ala-Leu-Pro Chemical compound C[C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N1CCC[C@@H]1C(=O)O)N OYJCVIGKMXUVKB-GARJFASQSA-N 0.000 description 1
- XIWKVCDQMCNKOZ-UVBJJODRSA-N Ala-Met-Trp Chemical compound C[C@@H](C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)O)N XIWKVCDQMCNKOZ-UVBJJODRSA-N 0.000 description 1
- BFMIRJBURUXDRG-DLOVCJGASA-N Ala-Phe-Asp Chemical compound OC(=O)C[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)C)CC1=CC=CC=C1 BFMIRJBURUXDRG-DLOVCJGASA-N 0.000 description 1
- CNQAFFMNJIQYGX-DRZSPHRISA-N Ala-Phe-Glu Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)C)CC1=CC=CC=C1 CNQAFFMNJIQYGX-DRZSPHRISA-N 0.000 description 1
- JAQNUEWEJWBVAY-WBAXXEDZSA-N Ala-Phe-Phe Chemical compound C([C@H](NC(=O)[C@@H](N)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 JAQNUEWEJWBVAY-WBAXXEDZSA-N 0.000 description 1
- DYXOFPBJBAHWFY-JBDRJPRFSA-N Ala-Ser-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@H](C)N DYXOFPBJBAHWFY-JBDRJPRFSA-N 0.000 description 1
- NZGRHTKZFSVPAN-BIIVOSGPSA-N Ala-Ser-Pro Chemical compound C[C@@H](C(=O)N[C@@H](CO)C(=O)N1CCC[C@@H]1C(=O)O)N NZGRHTKZFSVPAN-BIIVOSGPSA-N 0.000 description 1
- WQKAQKZRDIZYNV-VZFHVOOUSA-N Ala-Ser-Thr Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(O)=O WQKAQKZRDIZYNV-VZFHVOOUSA-N 0.000 description 1
- SYIFFFHSXBNPMC-UWJYBYFXSA-N Ala-Ser-Tyr Chemical compound C[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)O)N SYIFFFHSXBNPMC-UWJYBYFXSA-N 0.000 description 1
- WNHNMKOFKCHKKD-BFHQHQDPSA-N Ala-Thr-Gly Chemical compound [H]N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O WNHNMKOFKCHKKD-BFHQHQDPSA-N 0.000 description 1
- AENHOIXXHKNIQL-AUTRQRHGSA-N Ala-Tyr-Ala Chemical compound [O-]C(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@@H]([NH3+])C)CC1=CC=C(O)C=C1 AENHOIXXHKNIQL-AUTRQRHGSA-N 0.000 description 1
- AOAKQKVICDWCLB-UWJYBYFXSA-N Ala-Tyr-Asn Chemical compound C[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CC(=O)N)C(=O)O)N AOAKQKVICDWCLB-UWJYBYFXSA-N 0.000 description 1
- YCTIYBUTCKNOTI-UWJYBYFXSA-N Ala-Tyr-Asp Chemical compound C[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CC(=O)O)C(=O)O)N YCTIYBUTCKNOTI-UWJYBYFXSA-N 0.000 description 1
- XAXMJQUMRJAFCH-CQDKDKBSSA-N Ala-Tyr-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)C)CC1=CC=C(O)C=C1 XAXMJQUMRJAFCH-CQDKDKBSSA-N 0.000 description 1
- NLYYHIKRBRMAJV-AEJSXWLSSA-N Ala-Val-Pro Chemical compound C[C@@H](C(=O)N[C@@H](C(C)C)C(=O)N1CCC[C@@H]1C(=O)O)N NLYYHIKRBRMAJV-AEJSXWLSSA-N 0.000 description 1
- SSQHYGLFYWZWDV-UVBJJODRSA-N Ala-Val-Trp Chemical compound CC(C)[C@H](NC(=O)[C@H](C)N)C(=O)N[C@@H](Cc1c[nH]c2ccccc12)C(O)=O SSQHYGLFYWZWDV-UVBJJODRSA-N 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- 241000234282 Allium Species 0.000 description 1
- 235000002732 Allium cepa var. cepa Nutrition 0.000 description 1
- 241000223602 Alternaria alternata Species 0.000 description 1
- 241000213004 Alternaria solani Species 0.000 description 1
- 239000004254 Ammonium phosphate Substances 0.000 description 1
- 244000099147 Ananas comosus Species 0.000 description 1
- 235000007119 Ananas comosus Nutrition 0.000 description 1
- 244000105975 Antidesma platyphyllum Species 0.000 description 1
- 241000219194 Arabidopsis Species 0.000 description 1
- NONSEUUPKITYQT-BQBZGAKWSA-N Arg-Asn-Gly Chemical compound C(C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)NCC(=O)O)N)CN=C(N)N NONSEUUPKITYQT-BQBZGAKWSA-N 0.000 description 1
- OTUQSEPIIVBYEM-IHRRRGAJSA-N Arg-Asn-Tyr Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O OTUQSEPIIVBYEM-IHRRRGAJSA-N 0.000 description 1
- SQKPKIJVWHAWNF-DCAQKATOSA-N Arg-Asp-Lys Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCCN)C(O)=O SQKPKIJVWHAWNF-DCAQKATOSA-N 0.000 description 1
- YSUVMPICYVWRBX-VEVYYDQMSA-N Arg-Asp-Thr Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O YSUVMPICYVWRBX-VEVYYDQMSA-N 0.000 description 1
- XLWSGICNBZGYTA-CIUDSAMLSA-N Arg-Glu-Asp Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O XLWSGICNBZGYTA-CIUDSAMLSA-N 0.000 description 1
- PPPXVIBMLFWNSK-BQBZGAKWSA-N Arg-Gly-Cys Chemical compound C(C[C@@H](C(=O)NCC(=O)N[C@@H](CS)C(=O)O)N)CN=C(N)N PPPXVIBMLFWNSK-BQBZGAKWSA-N 0.000 description 1
- YNSGXDWWPCGGQS-YUMQZZPRSA-N Arg-Gly-Gln Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CCC(N)=O)C(O)=O YNSGXDWWPCGGQS-YUMQZZPRSA-N 0.000 description 1
- RFXXUWGNVRJTNQ-QXEWZRGKSA-N Arg-Gly-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)CNC(=O)[C@H](CCCN=C(N)N)N RFXXUWGNVRJTNQ-QXEWZRGKSA-N 0.000 description 1
- JTZUZBADHGISJD-SRVKXCTJSA-N Arg-His-Glu Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CCC(O)=O)C(O)=O JTZUZBADHGISJD-SRVKXCTJSA-N 0.000 description 1
- GXXWTNKNFFKTJB-NAKRPEOUSA-N Arg-Ile-Ser Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(O)=O GXXWTNKNFFKTJB-NAKRPEOUSA-N 0.000 description 1
- UZGFHWIJWPUPOH-IHRRRGAJSA-N Arg-Leu-Lys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CCCN=C(N)N)N UZGFHWIJWPUPOH-IHRRRGAJSA-N 0.000 description 1
- PZBSKYJGKNNYNK-ULQDDVLXSA-N Arg-Leu-Tyr Chemical compound CC(C)C[C@H](NC(=O)[C@@H](N)CCCN=C(N)N)C(=O)N[C@@H](Cc1ccc(O)cc1)C(O)=O PZBSKYJGKNNYNK-ULQDDVLXSA-N 0.000 description 1
- ZEBDYGZVMMKZNB-SRVKXCTJSA-N Arg-Met-Val Chemical compound CC(C)[C@@H](C(=O)O)NC(=O)[C@H](CCSC)NC(=O)[C@H](CCCN=C(N)N)N ZEBDYGZVMMKZNB-SRVKXCTJSA-N 0.000 description 1
- OWSMKCJUBAPHED-JYJNAYRXSA-N Arg-Pro-Tyr Chemical compound NC(N)=NCCC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 OWSMKCJUBAPHED-JYJNAYRXSA-N 0.000 description 1
- DNLQVHBBMPZUGJ-BQBZGAKWSA-N Arg-Ser-Gly Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)NCC(O)=O DNLQVHBBMPZUGJ-BQBZGAKWSA-N 0.000 description 1
- JKRPBTQDPJSQIT-RCWTZXSCSA-N Arg-Thr-Met Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCSC)C(=O)O)NC(=O)[C@H](CCCN=C(N)N)N)O JKRPBTQDPJSQIT-RCWTZXSCSA-N 0.000 description 1
- JBQORRNSZGTLCV-WDSOQIARSA-N Arg-Trp-Lys Chemical compound C1=CC=C2C(C[C@@H](C(=O)N[C@@H](CCCCN)C(O)=O)NC(=O)[C@@H](N)CCCN=C(N)N)=CNC2=C1 JBQORRNSZGTLCV-WDSOQIARSA-N 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- QEYJFBMTSMLPKZ-ZKWXMUAHSA-N Asn-Ala-Val Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](C(C)C)C(O)=O QEYJFBMTSMLPKZ-ZKWXMUAHSA-N 0.000 description 1
- QHBMKQWOIYJYMI-BYULHYEWSA-N Asn-Asn-Val Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C(C)C)C(O)=O QHBMKQWOIYJYMI-BYULHYEWSA-N 0.000 description 1
- FANGHKQYFPYDNB-UBHSHLNASA-N Asn-Asp-Trp Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CC(=O)N)N FANGHKQYFPYDNB-UBHSHLNASA-N 0.000 description 1
- ULRPXVNMIIYDDJ-ACZMJKKPSA-N Asn-Glu-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CC(=O)N)N ULRPXVNMIIYDDJ-ACZMJKKPSA-N 0.000 description 1
- JQSWHKKUZMTOIH-QWRGUYRKSA-N Asn-Gly-Phe Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)O)NC(=O)CNC(=O)[C@H](CC(=O)N)N JQSWHKKUZMTOIH-QWRGUYRKSA-N 0.000 description 1
- ODBSSLHUFPJRED-CIUDSAMLSA-N Asn-His-Asn Chemical compound C1=C(NC=N1)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](CC(=O)N)N ODBSSLHUFPJRED-CIUDSAMLSA-N 0.000 description 1
- NLRJGXZWTKXRHP-DCAQKATOSA-N Asn-Leu-Arg Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O NLRJGXZWTKXRHP-DCAQKATOSA-N 0.000 description 1
- WIDVAWAQBRAKTI-YUMQZZPRSA-N Asn-Leu-Gly Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)NCC(O)=O WIDVAWAQBRAKTI-YUMQZZPRSA-N 0.000 description 1
- TZFQICWZWFNIKU-KKUMJFAQSA-N Asn-Leu-Tyr Chemical compound NC(=O)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 TZFQICWZWFNIKU-KKUMJFAQSA-N 0.000 description 1
- NYGILGUOUOXGMJ-YUMQZZPRSA-N Asn-Lys-Gly Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)NCC(O)=O NYGILGUOUOXGMJ-YUMQZZPRSA-N 0.000 description 1
- ZVUMKOMKQCANOM-AVGNSLFASA-N Asn-Phe-Gln Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCC(N)=O)C(O)=O ZVUMKOMKQCANOM-AVGNSLFASA-N 0.000 description 1
- XMHFCUKJRCQXGI-CIUDSAMLSA-N Asn-Pro-Gln Chemical compound C1C[C@H](N(C1)C(=O)[C@H](CC(=O)N)N)C(=O)N[C@@H](CCC(=O)N)C(=O)O XMHFCUKJRCQXGI-CIUDSAMLSA-N 0.000 description 1
- HPASIOLTWSNMFB-OLHMAJIHSA-N Asn-Thr-Asp Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(O)=O)C(O)=O HPASIOLTWSNMFB-OLHMAJIHSA-N 0.000 description 1
- LTDGPJKGJDIBQD-LAEOZQHASA-N Asn-Val-Gln Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O LTDGPJKGJDIBQD-LAEOZQHASA-N 0.000 description 1
- PBVLJOIPOGUQQP-CIUDSAMLSA-N Asp-Ala-Leu Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(O)=O PBVLJOIPOGUQQP-CIUDSAMLSA-N 0.000 description 1
- UGKZHCBLMLSANF-CIUDSAMLSA-N Asp-Asn-Leu Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(O)=O UGKZHCBLMLSANF-CIUDSAMLSA-N 0.000 description 1
- UGIBTKGQVWFTGX-BIIVOSGPSA-N Asp-Asn-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC(=O)N)NC(=O)[C@H](CC(=O)O)N)C(=O)O UGIBTKGQVWFTGX-BIIVOSGPSA-N 0.000 description 1
- NAPNAGZWHQHZLG-ZLUOBGJFSA-N Asp-Asp-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CC(=O)O)N NAPNAGZWHQHZLG-ZLUOBGJFSA-N 0.000 description 1
- ZRAOLTNMSCSCLN-ZLUOBGJFSA-N Asp-Cys-Asn Chemical compound C([C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(=O)N)C(=O)O)N)C(=O)O ZRAOLTNMSCSCLN-ZLUOBGJFSA-N 0.000 description 1
- QQXOYLWJQUPXJU-WHFBIAKZSA-N Asp-Cys-Gly Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](CS)C(=O)NCC(O)=O QQXOYLWJQUPXJU-WHFBIAKZSA-N 0.000 description 1
- KIJLEFNHWSXHRU-NUMRIWBASA-N Asp-Gln-Thr Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O KIJLEFNHWSXHRU-NUMRIWBASA-N 0.000 description 1
- UFAQGGZUXVLONR-AVGNSLFASA-N Asp-Gln-Tyr Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CC(=O)O)N)O UFAQGGZUXVLONR-AVGNSLFASA-N 0.000 description 1
- PSLSTUMPZILTAH-BYULHYEWSA-N Asp-Gly-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CC(O)=O PSLSTUMPZILTAH-BYULHYEWSA-N 0.000 description 1
- POTCZYQVVNXUIG-BQBZGAKWSA-N Asp-Gly-Pro Chemical compound OC(=O)C[C@H](N)C(=O)NCC(=O)N1CCC[C@H]1C(O)=O POTCZYQVVNXUIG-BQBZGAKWSA-N 0.000 description 1
- TZOZNVLBTAFJRW-UGYAYLCHSA-N Asp-Ile-Asp Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)O)NC(=O)[C@H](CC(=O)O)N TZOZNVLBTAFJRW-UGYAYLCHSA-N 0.000 description 1
- RQHLMGCXCZUOGT-ZPFDUUQYSA-N Asp-Leu-Ile Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O RQHLMGCXCZUOGT-ZPFDUUQYSA-N 0.000 description 1
- MYOHQBFRJQFIDZ-KKUMJFAQSA-N Asp-Leu-Tyr Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O MYOHQBFRJQFIDZ-KKUMJFAQSA-N 0.000 description 1
- GWIJZUVQVDJHDI-AVGNSLFASA-N Asp-Phe-Glu Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCC(O)=O)C(O)=O GWIJZUVQVDJHDI-AVGNSLFASA-N 0.000 description 1
- ZKAOJVJQGVUIIU-GUBZILKMSA-N Asp-Pro-Arg Chemical compound OC(=O)C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCNC(N)=N)C(O)=O ZKAOJVJQGVUIIU-GUBZILKMSA-N 0.000 description 1
- CUQDCPXNZPDYFQ-ZLUOBGJFSA-N Asp-Ser-Asp Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(O)=O CUQDCPXNZPDYFQ-ZLUOBGJFSA-N 0.000 description 1
- GXHDGYOXPNQCKM-XVSYOHENSA-N Asp-Thr-Phe Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)NC(=O)[C@H](CC(=O)O)N)O GXHDGYOXPNQCKM-XVSYOHENSA-N 0.000 description 1
- PDIYGFYAMZZFCW-JIOCBJNQSA-N Asp-Thr-Pro Chemical compound C[C@H]([C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CC(=O)O)N)O PDIYGFYAMZZFCW-JIOCBJNQSA-N 0.000 description 1
- KCOPOPKJRHVGPE-AQZXSJQPSA-N Asp-Thr-Trp Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O KCOPOPKJRHVGPE-AQZXSJQPSA-N 0.000 description 1
- RMFITHMDQGFSDC-UBHSHLNASA-N Asp-Trp-Cys Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CC(=O)O)N RMFITHMDQGFSDC-UBHSHLNASA-N 0.000 description 1
- MRYDJCIIVRXVGG-QEJZJMRPSA-N Asp-Trp-Glu Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CCC(O)=O)C(O)=O MRYDJCIIVRXVGG-QEJZJMRPSA-N 0.000 description 1
- FIRWLDUOFOULCA-XIRDDKMYSA-N Asp-Trp-Lys Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC(=O)O)N FIRWLDUOFOULCA-XIRDDKMYSA-N 0.000 description 1
- PLNJUJGNLDSFOP-UWJYBYFXSA-N Asp-Tyr-Ala Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](C)C(O)=O PLNJUJGNLDSFOP-UWJYBYFXSA-N 0.000 description 1
- BJDHEININLSZOT-KKUMJFAQSA-N Asp-Tyr-Lys Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCCCN)C(O)=O BJDHEININLSZOT-KKUMJFAQSA-N 0.000 description 1
- BPAUXFVCSYQDQX-JRQIVUDYSA-N Asp-Tyr-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)NC(=O)[C@H](CC(=O)O)N)O BPAUXFVCSYQDQX-JRQIVUDYSA-N 0.000 description 1
- JGLWFWXGOINXEA-YDHLFZDLSA-N Asp-Val-Tyr Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 JGLWFWXGOINXEA-YDHLFZDLSA-N 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- 244000003416 Asparagus officinalis Species 0.000 description 1
- 235000005340 Asparagus officinalis Nutrition 0.000 description 1
- 241000228212 Aspergillus Species 0.000 description 1
- 241000228197 Aspergillus flavus Species 0.000 description 1
- 241000972773 Aulopiformes Species 0.000 description 1
- 108090001008 Avidin Proteins 0.000 description 1
- 235000000832 Ayote Nutrition 0.000 description 1
- 101000588395 Bacillus subtilis (strain 168) Beta-hexosaminidase Proteins 0.000 description 1
- 208000035143 Bacterial infection Diseases 0.000 description 1
- 241000219310 Beta vulgaris subsp. vulgaris Species 0.000 description 1
- 229920002498 Beta-glucan Polymers 0.000 description 1
- 241001480061 Blumeria graminis Species 0.000 description 1
- 241000255789 Bombyx mori Species 0.000 description 1
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 description 1
- 235000011293 Brassica napus Nutrition 0.000 description 1
- 235000006008 Brassica napus var napus Nutrition 0.000 description 1
- 235000011299 Brassica oleracea var botrytis Nutrition 0.000 description 1
- 240000003259 Brassica oleracea var. botrytis Species 0.000 description 1
- 235000004977 Brassica sinapistrum Nutrition 0.000 description 1
- 241000219193 Brassicaceae Species 0.000 description 1
- 101150015099 CHIA1 gene Proteins 0.000 description 1
- 101100520142 Caenorhabditis elegans pin-2 gene Proteins 0.000 description 1
- 241000222122 Candida albicans Species 0.000 description 1
- 244000025254 Cannabis sativa Species 0.000 description 1
- 235000009467 Carica papaya Nutrition 0.000 description 1
- 240000006432 Carica papaya Species 0.000 description 1
- 241001157813 Cercospora Species 0.000 description 1
- 101710115641 Chitooligosaccharidolytic beta-N-acetylglucosaminidase Proteins 0.000 description 1
- 101000749287 Clitocybe nebularis Clitocypin Proteins 0.000 description 1
- 101000767029 Clitocybe nebularis Clitocypin-1 Proteins 0.000 description 1
- 235000013162 Cocos nucifera Nutrition 0.000 description 1
- 244000060011 Cocos nucifera Species 0.000 description 1
- 108091033380 Coding strand Proteins 0.000 description 1
- 108700010070 Codon Usage Proteins 0.000 description 1
- 240000004270 Colocasia esculenta var. antiquorum Species 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 241000219112 Cucumis Species 0.000 description 1
- 235000015510 Cucumis melo subsp melo Nutrition 0.000 description 1
- 240000008067 Cucumis sativus Species 0.000 description 1
- 235000010799 Cucumis sativus var sativus Nutrition 0.000 description 1
- 241000219122 Cucurbita Species 0.000 description 1
- 235000009852 Cucurbita pepo Nutrition 0.000 description 1
- 235000009804 Cucurbita pepo subsp pepo Nutrition 0.000 description 1
- 241000219130 Cucurbita pepo subsp. pepo Species 0.000 description 1
- 235000003954 Cucurbita pepo var melopepo Nutrition 0.000 description 1
- XMTDCXXLDZKAGI-ACZMJKKPSA-N Cys-Ala-Gln Chemical compound C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)O)NC(=O)[C@H](CS)N XMTDCXXLDZKAGI-ACZMJKKPSA-N 0.000 description 1
- PRXCTTWKGJAPMT-ZLUOBGJFSA-N Cys-Ala-Ser Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O PRXCTTWKGJAPMT-ZLUOBGJFSA-N 0.000 description 1
- SMEYEQDCCBHTEF-FXQIFTODSA-N Cys-Pro-Ala Chemical compound [H]N[C@@H](CS)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C)C(O)=O SMEYEQDCCBHTEF-FXQIFTODSA-N 0.000 description 1
- IWVNIQXKTIQXCT-SRVKXCTJSA-N Cys-Tyr-Asn Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](CS)N)O IWVNIQXKTIQXCT-SRVKXCTJSA-N 0.000 description 1
- DGQJGBDBFVGLGL-ZKWXMUAHSA-N Cys-Val-Asp Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)O)NC(=O)[C@H](CS)N DGQJGBDBFVGLGL-ZKWXMUAHSA-N 0.000 description 1
- 229940094664 Cysteine protease inhibitor Drugs 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 239000003298 DNA probe Substances 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 240000007175 Datura inoxia Species 0.000 description 1
- 108010054576 Deoxyribonuclease EcoRI Proteins 0.000 description 1
- 235000002723 Dioscorea alata Nutrition 0.000 description 1
- 235000007056 Dioscorea composita Nutrition 0.000 description 1
- 235000009723 Dioscorea convolvulacea Nutrition 0.000 description 1
- 235000005362 Dioscorea floribunda Nutrition 0.000 description 1
- 235000004868 Dioscorea macrostachya Nutrition 0.000 description 1
- 235000005361 Dioscorea nummularia Nutrition 0.000 description 1
- 235000005360 Dioscorea spiculiflora Nutrition 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 101710107327 Endochitinase 1 Proteins 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 102000004533 Endonucleases Human genes 0.000 description 1
- 235000014755 Eruca sativa Nutrition 0.000 description 1
- 244000024675 Eruca sativa Species 0.000 description 1
- 241000221785 Erysiphales Species 0.000 description 1
- 240000006890 Erythroxylum coca Species 0.000 description 1
- 101710154757 Exo-beta-1,3-glucanase Proteins 0.000 description 1
- 108060002716 Exonuclease Proteins 0.000 description 1
- 208000004770 Fusariosis Diseases 0.000 description 1
- 241000145614 Fusarium bactridioides Species 0.000 description 1
- 241000146406 Fusarium heterosporum Species 0.000 description 1
- 206010051919 Fusarium infection Diseases 0.000 description 1
- 241001112697 Fusarium reticulatum Species 0.000 description 1
- 241001014439 Fusarium sarcochroum Species 0.000 description 1
- 241000145502 Fusarium subglutinans Species 0.000 description 1
- 241001465753 Fusarium torulosum Species 0.000 description 1
- 206010071602 Genetic polymorphism Diseases 0.000 description 1
- 108010061711 Gliadin Proteins 0.000 description 1
- LKUWAWGNJYJODH-KBIXCLLPSA-N Gln-Ala-Ile Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O LKUWAWGNJYJODH-KBIXCLLPSA-N 0.000 description 1
- CYTSBCIIEHUPDU-ACZMJKKPSA-N Gln-Asp-Ala Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(O)=O CYTSBCIIEHUPDU-ACZMJKKPSA-N 0.000 description 1
- BTSPOOHJBYJRKO-CIUDSAMLSA-N Gln-Asp-Arg Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O BTSPOOHJBYJRKO-CIUDSAMLSA-N 0.000 description 1
- XEYMBRRKIFYQMF-GUBZILKMSA-N Gln-Asp-Leu Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O XEYMBRRKIFYQMF-GUBZILKMSA-N 0.000 description 1
- AJDMYLOISOCHHC-YVNDNENWSA-N Gln-Gln-Ile Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O AJDMYLOISOCHHC-YVNDNENWSA-N 0.000 description 1
- VSXBYIJUAXPAAL-WDSKDSINSA-N Gln-Gly-Ala Chemical compound OC(=O)[C@H](C)NC(=O)CNC(=O)[C@@H](N)CCC(N)=O VSXBYIJUAXPAAL-WDSKDSINSA-N 0.000 description 1
- IKFZXRLDMYWNBU-YUMQZZPRSA-N Gln-Gly-Arg Chemical compound NC(=O)CC[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCCN=C(N)N IKFZXRLDMYWNBU-YUMQZZPRSA-N 0.000 description 1
- MFJAPSYJQJCQDN-BQBZGAKWSA-N Gln-Gly-Glu Chemical compound NC(=O)CC[C@H](N)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(O)=O MFJAPSYJQJCQDN-BQBZGAKWSA-N 0.000 description 1
- IHSGESFHTMFHRB-GUBZILKMSA-N Gln-Lys-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CCC(N)=O IHSGESFHTMFHRB-GUBZILKMSA-N 0.000 description 1
- FKXCBKCOSVIGCT-AVGNSLFASA-N Gln-Lys-Leu Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(O)=O FKXCBKCOSVIGCT-AVGNSLFASA-N 0.000 description 1
- ILKYYKRAULNYMS-JYJNAYRXSA-N Gln-Lys-Phe Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O ILKYYKRAULNYMS-JYJNAYRXSA-N 0.000 description 1
- ZVQZXPADLZIQFF-FHWLQOOXSA-N Gln-Phe-Tyr Chemical compound C([C@H](NC(=O)[C@H](CCC(N)=O)N)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)C1=CC=CC=C1 ZVQZXPADLZIQFF-FHWLQOOXSA-N 0.000 description 1
- DCWNCMRZIZSZBL-KKUMJFAQSA-N Gln-Pro-Tyr Chemical compound C1C[C@H](N(C1)C(=O)[C@H](CCC(=O)N)N)C(=O)N[C@@H](CC2=CC=C(C=C2)O)C(=O)O DCWNCMRZIZSZBL-KKUMJFAQSA-N 0.000 description 1
- LPIKVBWNNVFHCQ-GUBZILKMSA-N Gln-Ser-Leu Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(O)=O LPIKVBWNNVFHCQ-GUBZILKMSA-N 0.000 description 1
- PAOHIZNRJNIXQY-XQXXSGGOSA-N Gln-Thr-Ala Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(O)=O PAOHIZNRJNIXQY-XQXXSGGOSA-N 0.000 description 1
- UEILCTONAMOGBR-RWRJDSDZSA-N Gln-Thr-Ile Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O UEILCTONAMOGBR-RWRJDSDZSA-N 0.000 description 1
- NHMRJKKAVMENKJ-WDCWCFNPSA-N Gln-Thr-Leu Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(O)=O NHMRJKKAVMENKJ-WDCWCFNPSA-N 0.000 description 1
- CGYFDYFOAWDTPI-VJBMBRPKSA-N Gln-Trp-Trp Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CC3=CNC4=CC=CC=C43)C(=O)O)NC(=O)[C@H](CCC(=O)N)N CGYFDYFOAWDTPI-VJBMBRPKSA-N 0.000 description 1
- 108010044091 Globulins Proteins 0.000 description 1
- 102000006395 Globulins Human genes 0.000 description 1
- RUFHOVYUYSNDNY-ACZMJKKPSA-N Glu-Ala-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCC(O)=O RUFHOVYUYSNDNY-ACZMJKKPSA-N 0.000 description 1
- ITYRYNUZHPNCIK-GUBZILKMSA-N Glu-Ala-Leu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(O)=O ITYRYNUZHPNCIK-GUBZILKMSA-N 0.000 description 1
- KEBACWCLVOXFNC-DCAQKATOSA-N Glu-Arg-Met Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCSC)C(O)=O KEBACWCLVOXFNC-DCAQKATOSA-N 0.000 description 1
- WPLGNDORMXTMQS-FXQIFTODSA-N Glu-Gln-Ser Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(O)=O WPLGNDORMXTMQS-FXQIFTODSA-N 0.000 description 1
- LSPKYLAFTPBWIL-BYPYZUCNSA-N Glu-Gly Chemical compound OC(=O)CC[C@H](N)C(=O)NCC(O)=O LSPKYLAFTPBWIL-BYPYZUCNSA-N 0.000 description 1
- HVYWQYLBVXMXSV-GUBZILKMSA-N Glu-Leu-Ala Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(O)=O HVYWQYLBVXMXSV-GUBZILKMSA-N 0.000 description 1
- FMBWLLMUPXTXFC-SDDRHHMPSA-N Glu-Lys-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCCCN)NC(=O)[C@H](CCC(=O)O)N)C(=O)O FMBWLLMUPXTXFC-SDDRHHMPSA-N 0.000 description 1
- AOCARQDSFTWWFT-DCAQKATOSA-N Glu-Met-Arg Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O AOCARQDSFTWWFT-DCAQKATOSA-N 0.000 description 1
- ZWMYUDZLXAQHCK-CIUDSAMLSA-N Glu-Met-Asp Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(O)=O ZWMYUDZLXAQHCK-CIUDSAMLSA-N 0.000 description 1
- RZMXBFUSQNLEQF-QEJZJMRPSA-N Glu-Trp-Asn Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](CCC(=O)O)N RZMXBFUSQNLEQF-QEJZJMRPSA-N 0.000 description 1
- HBMRTXJZQDVRFT-DZKIICNBSA-N Glu-Tyr-Val Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](C(C)C)C(O)=O HBMRTXJZQDVRFT-DZKIICNBSA-N 0.000 description 1
- FGGKGJHCVMYGCD-UKJIMTQDSA-N Glu-Val-Ile Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O FGGKGJHCVMYGCD-UKJIMTQDSA-N 0.000 description 1
- 108010068370 Glutens Proteins 0.000 description 1
- BRFJMRSRMOMIMU-WHFBIAKZSA-N Gly-Ala-Asn Chemical compound NCC(=O)N[C@@H](C)C(=O)N[C@@H](CC(N)=O)C(O)=O BRFJMRSRMOMIMU-WHFBIAKZSA-N 0.000 description 1
- GZUKEVBTYNNUQF-WDSKDSINSA-N Gly-Ala-Gln Chemical compound NCC(=O)N[C@@H](C)C(=O)N[C@@H](CCC(N)=O)C(O)=O GZUKEVBTYNNUQF-WDSKDSINSA-N 0.000 description 1
- PHONXOACARQMPM-BQBZGAKWSA-N Gly-Ala-Met Chemical compound [H]NCC(=O)N[C@@H](C)C(=O)N[C@@H](CCSC)C(O)=O PHONXOACARQMPM-BQBZGAKWSA-N 0.000 description 1
- QSDKBRMVXSWAQE-BFHQHQDPSA-N Gly-Ala-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)CN QSDKBRMVXSWAQE-BFHQHQDPSA-N 0.000 description 1
- JXYMPBCYRKWJEE-BQBZGAKWSA-N Gly-Arg-Ala Chemical compound [H]NCC(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(O)=O JXYMPBCYRKWJEE-BQBZGAKWSA-N 0.000 description 1
- RJIVPOXLQFJRTG-LURJTMIESA-N Gly-Arg-Gly Chemical compound OC(=O)CNC(=O)[C@@H](NC(=O)CN)CCCN=C(N)N RJIVPOXLQFJRTG-LURJTMIESA-N 0.000 description 1
- VXKCPBPQEKKERH-IUCAKERBSA-N Gly-Arg-Pro Chemical compound NC(N)=NCCC[C@H](NC(=O)CN)C(=O)N1CCC[C@H]1C(O)=O VXKCPBPQEKKERH-IUCAKERBSA-N 0.000 description 1
- IWAXHBCACVWNHT-BQBZGAKWSA-N Gly-Asp-Arg Chemical compound NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CCCN=C(N)N IWAXHBCACVWNHT-BQBZGAKWSA-N 0.000 description 1
- NMROINAYXCACKF-WHFBIAKZSA-N Gly-Cys-Cys Chemical compound NCC(=O)N[C@@H](CS)C(=O)N[C@@H](CS)C(O)=O NMROINAYXCACKF-WHFBIAKZSA-N 0.000 description 1
- GHHAMXVMWXMGSV-STQMWFEESA-N Gly-Cys-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@H](CS)NC(=O)CN)C(O)=O)=CNC2=C1 GHHAMXVMWXMGSV-STQMWFEESA-N 0.000 description 1
- MOJKRXIRAZPZLW-WDSKDSINSA-N Gly-Glu-Ala Chemical compound [H]NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(O)=O MOJKRXIRAZPZLW-WDSKDSINSA-N 0.000 description 1
- KAJAOGBVWCYGHZ-JTQLQIEISA-N Gly-Gly-Phe Chemical compound [NH3+]CC(=O)NCC(=O)N[C@H](C([O-])=O)CC1=CC=CC=C1 KAJAOGBVWCYGHZ-JTQLQIEISA-N 0.000 description 1
- ZKLYPEGLWFVRGF-IUCAKERBSA-N Gly-His-Gln Chemical compound [H]NCC(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CCC(N)=O)C(O)=O ZKLYPEGLWFVRGF-IUCAKERBSA-N 0.000 description 1
- QSVMIMFAAZPCAQ-PMVVWTBXSA-N Gly-His-Thr Chemical compound [H]NCC(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H]([C@@H](C)O)C(O)=O QSVMIMFAAZPCAQ-PMVVWTBXSA-N 0.000 description 1
- SWQALSGKVLYKDT-UHFFFAOYSA-N Gly-Ile-Ala Natural products NCC(=O)NC(C(C)CC)C(=O)NC(C)C(O)=O SWQALSGKVLYKDT-UHFFFAOYSA-N 0.000 description 1
- LXTRSHQLGYINON-DTWKUNHWSA-N Gly-Met-Pro Chemical compound CSCC[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)CN LXTRSHQLGYINON-DTWKUNHWSA-N 0.000 description 1
- IGOYNRWLWHWAQO-JTQLQIEISA-N Gly-Phe-Gly Chemical compound OC(=O)CNC(=O)[C@@H](NC(=O)CN)CC1=CC=CC=C1 IGOYNRWLWHWAQO-JTQLQIEISA-N 0.000 description 1
- NSVOVKWEKGEOQB-LURJTMIESA-N Gly-Pro-Gly Chemical compound NCC(=O)N1CCC[C@H]1C(=O)NCC(O)=O NSVOVKWEKGEOQB-LURJTMIESA-N 0.000 description 1
- FGPLUIQCSKGLTI-WDSKDSINSA-N Gly-Ser-Glu Chemical compound NCC(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCC(O)=O FGPLUIQCSKGLTI-WDSKDSINSA-N 0.000 description 1
- JQFILXICXLDTRR-FBCQKBJTSA-N Gly-Thr-Gly Chemical compound NCC(=O)N[C@@H]([C@H](O)C)C(=O)NCC(O)=O JQFILXICXLDTRR-FBCQKBJTSA-N 0.000 description 1
- YDIDLLVFCYSXNY-RCOVLWMOSA-N Gly-Val-Asn Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)CN YDIDLLVFCYSXNY-RCOVLWMOSA-N 0.000 description 1
- IZVICCORZOSGPT-JSGCOSHPSA-N Gly-Val-Tyr Chemical compound [H]NCC(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O IZVICCORZOSGPT-JSGCOSHPSA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 101100161918 Glycine max SAC1 gene Proteins 0.000 description 1
- RVKIPWVMZANZLI-UHFFFAOYSA-N H-Lys-Trp-OH Natural products C1=CC=C2C(CC(NC(=O)C(N)CCCCN)C(O)=O)=CNC2=C1 RVKIPWVMZANZLI-UHFFFAOYSA-N 0.000 description 1
- PROLDOGUBQJNPG-RWMBFGLXSA-N His-Arg-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC2=CN=CN2)N)C(=O)O PROLDOGUBQJNPG-RWMBFGLXSA-N 0.000 description 1
- OMNVOTCFQQLEQU-CIUDSAMLSA-N His-Asn-Asp Chemical compound C1=C(NC=N1)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CC(=O)O)C(=O)O)N OMNVOTCFQQLEQU-CIUDSAMLSA-N 0.000 description 1
- WZOGEMJIZBNFBK-CIUDSAMLSA-N His-Asp-Asn Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O WZOGEMJIZBNFBK-CIUDSAMLSA-N 0.000 description 1
- NQKRILCJYCASDV-QWRGUYRKSA-N His-Gly-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CC1=CN=CN1 NQKRILCJYCASDV-QWRGUYRKSA-N 0.000 description 1
- BPOHQCZZSFBSON-KKUMJFAQSA-N His-Leu-His Chemical compound CC(C)C[C@H](NC(=O)[C@@H](N)Cc1cnc[nH]1)C(=O)N[C@@H](Cc1cnc[nH]1)C(O)=O BPOHQCZZSFBSON-KKUMJFAQSA-N 0.000 description 1
- YAEKRYQASVCDLK-JYJNAYRXSA-N His-Phe-Glu Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)O)NC(=O)[C@H](CC2=CN=CN2)N YAEKRYQASVCDLK-JYJNAYRXSA-N 0.000 description 1
- BSVLMPMIXPQNKC-KBPBESRZSA-N His-Phe-Gly Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)NCC(O)=O BSVLMPMIXPQNKC-KBPBESRZSA-N 0.000 description 1
- 101001018097 Homo sapiens L-selectin Proteins 0.000 description 1
- 108700025438 Hordeum vulgare ribosome-inactivating Proteins 0.000 description 1
- 235000008694 Humulus lupulus Nutrition 0.000 description 1
- 241000256023 Hyalophora cecropia Species 0.000 description 1
- LQSBBHNVAVNZSX-GHCJXIJMSA-N Ile-Ala-Asn Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C)C(=O)N[C@@H](CC(=O)N)C(=O)O)N LQSBBHNVAVNZSX-GHCJXIJMSA-N 0.000 description 1
- QICVAHODWHIWIS-HTFCKZLJSA-N Ile-Ala-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)O)N QICVAHODWHIWIS-HTFCKZLJSA-N 0.000 description 1
- QLRMMMQNCWBNPQ-QXEWZRGKSA-N Ile-Arg-Gly Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)NCC(=O)O)N QLRMMMQNCWBNPQ-QXEWZRGKSA-N 0.000 description 1
- BGZIJZJBXRVBGJ-SXTJYALSSA-N Ile-Asp-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)O)N BGZIJZJBXRVBGJ-SXTJYALSSA-N 0.000 description 1
- HGNUKGZQASSBKQ-PCBIJLKTSA-N Ile-Asp-Phe Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)N HGNUKGZQASSBKQ-PCBIJLKTSA-N 0.000 description 1
- DCQMJRSOGCYKTR-GHCJXIJMSA-N Ile-Asp-Ser Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(O)=O DCQMJRSOGCYKTR-GHCJXIJMSA-N 0.000 description 1
- ZDNORQNHCJUVOV-KBIXCLLPSA-N Ile-Gln-Ala Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C)C(O)=O ZDNORQNHCJUVOV-KBIXCLLPSA-N 0.000 description 1
- NZGTYCMLUGYMCV-XUXIUFHCSA-N Ile-Lys-Arg Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)N NZGTYCMLUGYMCV-XUXIUFHCSA-N 0.000 description 1
- OVDKXUDMKXAZIV-ZPFDUUQYSA-N Ile-Lys-Asn Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(=O)N)C(=O)O)N OVDKXUDMKXAZIV-ZPFDUUQYSA-N 0.000 description 1
- VGSPNSSCMOHRRR-BJDJZHNGSA-N Ile-Ser-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)O)N VGSPNSSCMOHRRR-BJDJZHNGSA-N 0.000 description 1
- JNLSTRPWUXOORL-MMWGEVLESA-N Ile-Ser-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)N1CCC[C@@H]1C(=O)O)N JNLSTRPWUXOORL-MMWGEVLESA-N 0.000 description 1
- NURNJECQNNCRBK-FLBSBUHZSA-N Ile-Thr-Thr Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O NURNJECQNNCRBK-FLBSBUHZSA-N 0.000 description 1
- DGTOKVBDZXJHNZ-WZLNRYEVSA-N Ile-Thr-Tyr Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)O)N DGTOKVBDZXJHNZ-WZLNRYEVSA-N 0.000 description 1
- YJRSIJZUIUANHO-NAKRPEOUSA-N Ile-Val-Ala Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C)C(=O)O)N YJRSIJZUIUANHO-NAKRPEOUSA-N 0.000 description 1
- AUIYHFRUOOKTGX-UKJIMTQDSA-N Ile-Val-Gln Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N AUIYHFRUOOKTGX-UKJIMTQDSA-N 0.000 description 1
- UYODHPPSCXBNCS-XUXIUFHCSA-N Ile-Val-Leu Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CC(C)C UYODHPPSCXBNCS-XUXIUFHCSA-N 0.000 description 1
- SWNRZNLXMXRCJC-VKOGCVSHSA-N Ile-Val-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)[C@@H](C)CC)C(O)=O)=CNC2=C1 SWNRZNLXMXRCJC-VKOGCVSHSA-N 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 240000000917 Impatiens balsamina Species 0.000 description 1
- 235000015912 Impatiens biflora Nutrition 0.000 description 1
- 238000012404 In vitro experiment Methods 0.000 description 1
- 244000017020 Ipomoea batatas Species 0.000 description 1
- 235000002678 Ipomoea batatas Nutrition 0.000 description 1
- 235000006350 Ipomoea batatas var. batatas Nutrition 0.000 description 1
- 101100288095 Klebsiella pneumoniae neo gene Proteins 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 1
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 1
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 1
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 1
- SENJXOPIZNYLHU-UHFFFAOYSA-N L-leucyl-L-arginine Natural products CC(C)CC(N)C(=O)NC(C(O)=O)CCCN=C(N)N SENJXOPIZNYLHU-UHFFFAOYSA-N 0.000 description 1
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 1
- 125000001176 L-lysyl group Chemical group [H]N([H])[C@]([H])(C(=O)[*])C([H])([H])C([H])([H])C([H])([H])C(N([H])[H])([H])[H] 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 1
- 102100033467 L-selectin Human genes 0.000 description 1
- 125000000769 L-threonyl group Chemical group [H]N([H])[C@]([H])(C(=O)[*])[C@](O[H])(C([H])([H])[H])[H] 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- 125000003798 L-tyrosyl group Chemical group [H]N([H])[C@]([H])(C(=O)[*])C([H])([H])C1=C([H])C([H])=C(O[H])C([H])=C1[H] 0.000 description 1
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 1
- 125000003580 L-valyl group Chemical group [H]N([H])[C@]([H])(C(=O)[*])C(C([H])([H])[H])(C([H])([H])[H])[H] 0.000 description 1
- 229920001543 Laminarin Polymers 0.000 description 1
- 239000005717 Laminarin Substances 0.000 description 1
- CQQGCWPXDHTTNF-GUBZILKMSA-N Leu-Ala-Glu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCC(O)=O CQQGCWPXDHTTNF-GUBZILKMSA-N 0.000 description 1
- WSGXUIQTEZDVHJ-GARJFASQSA-N Leu-Ala-Pro Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C)C(=O)N1CCC[C@@H]1C(O)=O WSGXUIQTEZDVHJ-GARJFASQSA-N 0.000 description 1
- MDVZJYGNAGLPGJ-KKUMJFAQSA-N Leu-Asn-Phe Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 MDVZJYGNAGLPGJ-KKUMJFAQSA-N 0.000 description 1
- KTFHTMHHKXUYPW-ZPFDUUQYSA-N Leu-Asp-Ile Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O KTFHTMHHKXUYPW-ZPFDUUQYSA-N 0.000 description 1
- JQSXWJXBASFONF-KKUMJFAQSA-N Leu-Asp-Phe Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O JQSXWJXBASFONF-KKUMJFAQSA-N 0.000 description 1
- NEEOBPIXKWSBRF-IUCAKERBSA-N Leu-Glu-Gly Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(O)=O NEEOBPIXKWSBRF-IUCAKERBSA-N 0.000 description 1
- OXRLYTYUXAQTHP-YUMQZZPRSA-N Leu-Gly-Ala Chemical compound [H]N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](C)C(O)=O OXRLYTYUXAQTHP-YUMQZZPRSA-N 0.000 description 1
- QPXBPQUGXHURGP-UWVGGRQHSA-N Leu-Gly-Met Chemical compound CC(C)C[C@@H](C(=O)NCC(=O)N[C@@H](CCSC)C(=O)O)N QPXBPQUGXHURGP-UWVGGRQHSA-N 0.000 description 1
- KEVYYIMVELOXCT-KBPBESRZSA-N Leu-Gly-Phe Chemical compound CC(C)C[C@H]([NH3+])C(=O)NCC(=O)N[C@H](C([O-])=O)CC1=CC=CC=C1 KEVYYIMVELOXCT-KBPBESRZSA-N 0.000 description 1
- HNDWYLYAYNBWMP-AJNGGQMLSA-N Leu-Ile-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC(C)C)N HNDWYLYAYNBWMP-AJNGGQMLSA-N 0.000 description 1
- LIINDKYIGYTDLG-PPCPHDFISA-N Leu-Ile-Thr Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(O)=O LIINDKYIGYTDLG-PPCPHDFISA-N 0.000 description 1
- JNDYEOUZBLOVOF-AVGNSLFASA-N Leu-Leu-Gln Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O JNDYEOUZBLOVOF-AVGNSLFASA-N 0.000 description 1
- YOKVEHGYYQEQOP-QWRGUYRKSA-N Leu-Leu-Gly Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)NCC(O)=O YOKVEHGYYQEQOP-QWRGUYRKSA-N 0.000 description 1
- RXGLHDWAZQECBI-SRVKXCTJSA-N Leu-Leu-Ser Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O RXGLHDWAZQECBI-SRVKXCTJSA-N 0.000 description 1
- SYRTUBLKWNDSDK-DKIMLUQUSA-N Leu-Phe-Ile Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O SYRTUBLKWNDSDK-DKIMLUQUSA-N 0.000 description 1
- YRRCOJOXAJNSAX-IHRRRGAJSA-N Leu-Pro-Lys Chemical compound CC(C)C[C@@H](C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCCN)C(=O)O)N YRRCOJOXAJNSAX-IHRRRGAJSA-N 0.000 description 1
- XGDCYUQSFDQISZ-BQBZGAKWSA-N Leu-Ser Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CO)C(O)=O XGDCYUQSFDQISZ-BQBZGAKWSA-N 0.000 description 1
- AIQWYVFNBNNOLU-RHYQMDGZSA-N Leu-Thr-Val Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(O)=O AIQWYVFNBNNOLU-RHYQMDGZSA-N 0.000 description 1
- SNOUHRPNNCAOPI-SZMVWBNQSA-N Leu-Trp-Gln Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N SNOUHRPNNCAOPI-SZMVWBNQSA-N 0.000 description 1
- AAKRWBIIGKPOKQ-ONGXEEELSA-N Leu-Val-Gly Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)NCC(O)=O AAKRWBIIGKPOKQ-ONGXEEELSA-N 0.000 description 1
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 1
- NFLFJGGKOHYZJF-BJDJZHNGSA-N Lys-Ala-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCCCN NFLFJGGKOHYZJF-BJDJZHNGSA-N 0.000 description 1
- NLOZZWJNIKKYSC-WDSOQIARSA-N Lys-Arg-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@@H](N)CCCCN)C(O)=O)=CNC2=C1 NLOZZWJNIKKYSC-WDSOQIARSA-N 0.000 description 1
- IWWMPCPLFXFBAF-SRVKXCTJSA-N Lys-Asp-Leu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O IWWMPCPLFXFBAF-SRVKXCTJSA-N 0.000 description 1
- QIJVAFLRMVBHMU-KKUMJFAQSA-N Lys-Asp-Phe Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O QIJVAFLRMVBHMU-KKUMJFAQSA-N 0.000 description 1
- VSRXPEHZMHSFKU-IUCAKERBSA-N Lys-Gln-Gly Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)NCC(O)=O VSRXPEHZMHSFKU-IUCAKERBSA-N 0.000 description 1
- LXNPMPIQDNSMTA-AVGNSLFASA-N Lys-Gln-His Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H](C(O)=O)CC1=CN=CN1 LXNPMPIQDNSMTA-AVGNSLFASA-N 0.000 description 1
- YVMQJGWLHRWMDF-MNXVOIDGSA-N Lys-Gln-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CCCCN)N YVMQJGWLHRWMDF-MNXVOIDGSA-N 0.000 description 1
- HAUUXTXKJNVIFY-ONGXEEELSA-N Lys-Gly-Val Chemical compound [H]N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](C(C)C)C(O)=O HAUUXTXKJNVIFY-ONGXEEELSA-N 0.000 description 1
- SPCHLZUWJTYZFC-IHRRRGAJSA-N Lys-His-Val Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](C(C)C)C(O)=O SPCHLZUWJTYZFC-IHRRRGAJSA-N 0.000 description 1
- ZXFRGTAIIZHNHG-AJNGGQMLSA-N Lys-Ile-Leu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(C)C)C(=O)O)NC(=O)[C@H](CCCCN)N ZXFRGTAIIZHNHG-AJNGGQMLSA-N 0.000 description 1
- WAIHHELKYSFIQN-XUXIUFHCSA-N Lys-Ile-Val Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C(C)C)C(O)=O WAIHHELKYSFIQN-XUXIUFHCSA-N 0.000 description 1
- MUXNCRWTWBMNHX-SRVKXCTJSA-N Lys-Leu-Asp Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O MUXNCRWTWBMNHX-SRVKXCTJSA-N 0.000 description 1
- VMTYLUGCXIEDMV-QWRGUYRKSA-N Lys-Leu-Gly Chemical compound OC(=O)CNC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCCCN VMTYLUGCXIEDMV-QWRGUYRKSA-N 0.000 description 1
- GAHJXEMYXKLZRQ-AJNGGQMLSA-N Lys-Lys-Ile Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O GAHJXEMYXKLZRQ-AJNGGQMLSA-N 0.000 description 1
- WLXGMVVHTIUPHE-ULQDDVLXSA-N Lys-Phe-Val Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](C(C)C)C(O)=O WLXGMVVHTIUPHE-ULQDDVLXSA-N 0.000 description 1
- MIROMRNASYKZNL-ULQDDVLXSA-N Lys-Pro-Tyr Chemical compound NCCCC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 MIROMRNASYKZNL-ULQDDVLXSA-N 0.000 description 1
- RMKJOQSYLQQRFN-KKUMJFAQSA-N Lys-Tyr-Asp Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(O)=O)C(O)=O RMKJOQSYLQQRFN-KKUMJFAQSA-N 0.000 description 1
- OHXUUQDOBQKSNB-AVGNSLFASA-N Lys-Val-Arg Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O OHXUUQDOBQKSNB-AVGNSLFASA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 241001344133 Magnaporthe Species 0.000 description 1
- 240000004658 Medicago sativa Species 0.000 description 1
- 235000017587 Medicago sativa ssp. sativa Nutrition 0.000 description 1
- BVXXDMUMHMXFER-BPNCWPANSA-N Met-Ala-Tyr Chemical compound [H]N[C@@H](CCSC)C(=O)N[C@@H](C)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O BVXXDMUMHMXFER-BPNCWPANSA-N 0.000 description 1
- CTVJSFRHUOSCQQ-DCAQKATOSA-N Met-Arg-Glu Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(O)=O CTVJSFRHUOSCQQ-DCAQKATOSA-N 0.000 description 1
- UZVWDRPUTHXQAM-FXQIFTODSA-N Met-Asp-Ala Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(O)=O UZVWDRPUTHXQAM-FXQIFTODSA-N 0.000 description 1
- JQECLVNLAZGHRQ-CIUDSAMLSA-N Met-Asp-Gln Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CCC(N)=O JQECLVNLAZGHRQ-CIUDSAMLSA-N 0.000 description 1
- HOZNVKDCKZPRER-XUXIUFHCSA-N Met-Lys-Ile Chemical compound [H]N[C@@H](CCSC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O HOZNVKDCKZPRER-XUXIUFHCSA-N 0.000 description 1
- LXCSZPUQKMTXNW-BQBZGAKWSA-N Met-Ser-Gly Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CO)C(=O)NCC(O)=O LXCSZPUQKMTXNW-BQBZGAKWSA-N 0.000 description 1
- 108700005443 Microbial Genes Proteins 0.000 description 1
- 108700005084 Multigene Family Proteins 0.000 description 1
- 108010086093 Mung Bean Nuclease Proteins 0.000 description 1
- 240000005561 Musa balbisiana Species 0.000 description 1
- 235000018290 Musa x paradisiaca Nutrition 0.000 description 1
- 108010079364 N-glycylalanine Proteins 0.000 description 1
- 108010066427 N-valyltryptophan Proteins 0.000 description 1
- 108010087066 N2-tryptophyllysine Proteins 0.000 description 1
- 238000005481 NMR spectroscopy Methods 0.000 description 1
- 241000244206 Nematoda Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 239000000020 Nitrocellulose Substances 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 108020005187 Oligonucleotide Probes Proteins 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 241001668545 Pascopyrum Species 0.000 description 1
- 244000115721 Pennisetum typhoides Species 0.000 description 1
- 235000007195 Pennisetum typhoides Nutrition 0.000 description 1
- 241000364057 Peoria Species 0.000 description 1
- ULECEJGNDHWSKD-QEJZJMRPSA-N Phe-Ala-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC1=CC=CC=C1 ULECEJGNDHWSKD-QEJZJMRPSA-N 0.000 description 1
- CSYVXYQDIVCQNU-QWRGUYRKSA-N Phe-Asp-Gly Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(O)=O CSYVXYQDIVCQNU-QWRGUYRKSA-N 0.000 description 1
- UMKYAYXCMYYNHI-AVGNSLFASA-N Phe-Gln-Asn Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CC(=O)N)C(=O)O)N UMKYAYXCMYYNHI-AVGNSLFASA-N 0.000 description 1
- FMMIYCMOVGXZIP-AVGNSLFASA-N Phe-Glu-Asn Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O FMMIYCMOVGXZIP-AVGNSLFASA-N 0.000 description 1
- KJJROSNFBRWPHS-JYJNAYRXSA-N Phe-Glu-Leu Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O KJJROSNFBRWPHS-JYJNAYRXSA-N 0.000 description 1
- NAXPHWZXEXNDIW-JTQLQIEISA-N Phe-Gly-Gly Chemical compound OC(=O)CNC(=O)CNC(=O)[C@@H](N)CC1=CC=CC=C1 NAXPHWZXEXNDIW-JTQLQIEISA-N 0.000 description 1
- AAERWTUHZKLDLC-IHRRRGAJSA-N Phe-Pro-Asp Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(O)=O)C(O)=O AAERWTUHZKLDLC-IHRRRGAJSA-N 0.000 description 1
- JXQVYPWVGUOIDV-MXAVVETBSA-N Phe-Ser-Ile Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O JXQVYPWVGUOIDV-MXAVVETBSA-N 0.000 description 1
- MCIXMYKSPQUMJG-SRVKXCTJSA-N Phe-Ser-Ser Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O MCIXMYKSPQUMJG-SRVKXCTJSA-N 0.000 description 1
- NJONQBYLTANINY-IHPCNDPISA-N Phe-Trp-Asn Chemical compound N[C@@H](Cc1ccccc1)C(=O)N[C@@H](Cc1c[nH]c2ccccc12)C(=O)N[C@@H](CC(N)=O)C(O)=O NJONQBYLTANINY-IHPCNDPISA-N 0.000 description 1
- ZOGICTVLQDWPER-UFYCRDLUSA-N Phe-Tyr-Val Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](C(C)C)C(O)=O ZOGICTVLQDWPER-UFYCRDLUSA-N 0.000 description 1
- IAJOBQBIJHVGMQ-UHFFFAOYSA-N Phosphinothricin Natural products CP(O)(=O)CCC(N)C(O)=O IAJOBQBIJHVGMQ-UHFFFAOYSA-N 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 241000235401 Phycomyces blakesleeanus Species 0.000 description 1
- 241000233629 Phytophthora parasitica Species 0.000 description 1
- 241000209049 Poa pratensis Species 0.000 description 1
- 241000209504 Poaceae Species 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 239000004743 Polypropylene Substances 0.000 description 1
- DZZCICYRSZASNF-FXQIFTODSA-N Pro-Ala-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1 DZZCICYRSZASNF-FXQIFTODSA-N 0.000 description 1
- HFZNNDWPHBRNPV-KZVJFYERSA-N Pro-Ala-Thr Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O HFZNNDWPHBRNPV-KZVJFYERSA-N 0.000 description 1
- VCYJKOLZYPYGJV-AVGNSLFASA-N Pro-Arg-Leu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(O)=O VCYJKOLZYPYGJV-AVGNSLFASA-N 0.000 description 1
- MLQVJYMFASXBGZ-IHRRRGAJSA-N Pro-Asn-Tyr Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CC2=CC=C(C=C2)O)C(=O)O MLQVJYMFASXBGZ-IHRRRGAJSA-N 0.000 description 1
- CJZTUKSFZUSNCC-FXQIFTODSA-N Pro-Asp-Asn Chemical compound NC(=O)C[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H]1CCCN1 CJZTUKSFZUSNCC-FXQIFTODSA-N 0.000 description 1
- PZSCUPVOJGKHEP-CIUDSAMLSA-N Pro-Gln-Asp Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O PZSCUPVOJGKHEP-CIUDSAMLSA-N 0.000 description 1
- ZPPVJIJMIKTERM-YUMQZZPRSA-N Pro-Gln-Gly Chemical compound OC(=O)CNC(=O)[C@H](CCC(=O)N)NC(=O)[C@@H]1CCCN1 ZPPVJIJMIKTERM-YUMQZZPRSA-N 0.000 description 1
- DIFXZGPHVCIVSQ-CIUDSAMLSA-N Pro-Gln-Ser Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(O)=O DIFXZGPHVCIVSQ-CIUDSAMLSA-N 0.000 description 1
- MGDFPGCFVJFITQ-CIUDSAMLSA-N Pro-Glu-Asp Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O MGDFPGCFVJFITQ-CIUDSAMLSA-N 0.000 description 1
- LXLFEIHKWGHJJB-XUXIUFHCSA-N Pro-Ile-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@@H]1CCCN1 LXLFEIHKWGHJJB-XUXIUFHCSA-N 0.000 description 1
- CPRLKHJUFAXVTD-ULQDDVLXSA-N Pro-Leu-Tyr Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O CPRLKHJUFAXVTD-ULQDDVLXSA-N 0.000 description 1
- GFHXZNVJIKMAGO-IHRRRGAJSA-N Pro-Phe-Ser Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(O)=O GFHXZNVJIKMAGO-IHRRRGAJSA-N 0.000 description 1
- RNEFESSBTOQSAC-DCAQKATOSA-N Pro-Ser-His Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC2=CN=CN2)C(=O)O RNEFESSBTOQSAC-DCAQKATOSA-N 0.000 description 1
- SXJOPONICMGFCR-DCAQKATOSA-N Pro-Ser-Lys Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)O SXJOPONICMGFCR-DCAQKATOSA-N 0.000 description 1
- AJJDPGVVNPUZCR-RHYQMDGZSA-N Pro-Thr-Lys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@@H]1CCCN1)O AJJDPGVVNPUZCR-RHYQMDGZSA-N 0.000 description 1
- QMABBZHZMDXHKU-FKBYEOEOSA-N Pro-Tyr-Trp Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O QMABBZHZMDXHKU-FKBYEOEOSA-N 0.000 description 1
- IMNVAOPEMFDAQD-NHCYSSNCSA-N Pro-Val-Glu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O IMNVAOPEMFDAQD-NHCYSSNCSA-N 0.000 description 1
- ZMLRZBWCXPQADC-TUAOUCFPSA-N Pro-Val-Pro Chemical compound CC(C)[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@@H]2CCCN2 ZMLRZBWCXPQADC-TUAOUCFPSA-N 0.000 description 1
- 108010029485 Protein Isoforms Proteins 0.000 description 1
- 102000001708 Protein Isoforms Human genes 0.000 description 1
- 102100030122 Protein O-GlcNAcase Human genes 0.000 description 1
- 108091034057 RNA (poly(A)) Proteins 0.000 description 1
- 108020004518 RNA Probes Proteins 0.000 description 1
- 239000003391 RNA probe Substances 0.000 description 1
- 244000088415 Raphanus sativus Species 0.000 description 1
- 235000006140 Raphanus sativus var sativus Nutrition 0.000 description 1
- 244000205939 Rhizopus oligosporus Species 0.000 description 1
- 235000000471 Rhizopus oligosporus Nutrition 0.000 description 1
- 108010083644 Ribonucleases Proteins 0.000 description 1
- 102000006382 Ribonucleases Human genes 0.000 description 1
- 241000193448 Ruminiclostridium thermocellum Species 0.000 description 1
- 101100244535 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) POP6 gene Proteins 0.000 description 1
- 240000000111 Saccharum officinarum Species 0.000 description 1
- 235000007201 Saccharum officinarum Nutrition 0.000 description 1
- 101100244540 Schizosaccharomyces pombe (strain 972 / ATCC 24843) pop7 gene Proteins 0.000 description 1
- 241000221696 Sclerotinia sclerotiorum Species 0.000 description 1
- 108091081021 Sense strand Proteins 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- ZUGXSSFMTXKHJS-ZLUOBGJFSA-N Ser-Ala-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(O)=O ZUGXSSFMTXKHJS-ZLUOBGJFSA-N 0.000 description 1
- LVVBAKCGXXUHFO-ZLUOBGJFSA-N Ser-Ala-Asp Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(O)=O)C(O)=O LVVBAKCGXXUHFO-ZLUOBGJFSA-N 0.000 description 1
- YQHZVYJAGWMHES-ZLUOBGJFSA-N Ser-Ala-Ser Chemical compound OC[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O YQHZVYJAGWMHES-ZLUOBGJFSA-N 0.000 description 1
- SWSRFJZZMNLMLY-ZKWXMUAHSA-N Ser-Asp-Val Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O SWSRFJZZMNLMLY-ZKWXMUAHSA-N 0.000 description 1
- SWIQQMYVHIXPEK-FXQIFTODSA-N Ser-Cys-Val Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CS)C(=O)N[C@@H](C(C)C)C(O)=O SWIQQMYVHIXPEK-FXQIFTODSA-N 0.000 description 1
- BQWCDDAISCPDQV-XHNCKOQMSA-N Ser-Gln-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CO)N)C(=O)O BQWCDDAISCPDQV-XHNCKOQMSA-N 0.000 description 1
- GZSZPKSBVAOGIE-CIUDSAMLSA-N Ser-Lys-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(O)=O GZSZPKSBVAOGIE-CIUDSAMLSA-N 0.000 description 1
- MHVXPTAMDHLTHB-IHPCNDPISA-N Ser-Phe-Trp Chemical compound C([C@H](NC(=O)[C@H](CO)N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(O)=O)C1=CC=CC=C1 MHVXPTAMDHLTHB-IHPCNDPISA-N 0.000 description 1
- BSXKBOUZDAZXHE-CIUDSAMLSA-N Ser-Pro-Glu Chemical compound [H]N[C@@H](CO)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(O)=O BSXKBOUZDAZXHE-CIUDSAMLSA-N 0.000 description 1
- GZGFSPWOMUKKCV-NAKRPEOUSA-N Ser-Pro-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)CO GZGFSPWOMUKKCV-NAKRPEOUSA-N 0.000 description 1
- OZPDGESCTGGNAD-CIUDSAMLSA-N Ser-Ser-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CO OZPDGESCTGGNAD-CIUDSAMLSA-N 0.000 description 1
- ZSDXEKUKQAKZFE-XAVMHZPKSA-N Ser-Thr-Pro Chemical compound C[C@H]([C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CO)N)O ZSDXEKUKQAKZFE-XAVMHZPKSA-N 0.000 description 1
- UQGAAZXSCGWMFU-UBHSHLNASA-N Ser-Trp-Asp Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)O)NC(=O)[C@H](CO)N UQGAAZXSCGWMFU-UBHSHLNASA-N 0.000 description 1
- SDFUZKIAHWRUCS-QEJZJMRPSA-N Ser-Trp-Glu Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)O)NC(=O)[C@H](CO)N SDFUZKIAHWRUCS-QEJZJMRPSA-N 0.000 description 1
- 241000607715 Serratia marcescens Species 0.000 description 1
- 235000008515 Setaria glauca Nutrition 0.000 description 1
- 235000000208 Solanum incanum Nutrition 0.000 description 1
- 101000966595 Solanum lycopersicum Glucan endo-1,3-beta-glucosidase B Proteins 0.000 description 1
- 102100021588 Sterol carrier protein 2 Human genes 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 235000021536 Sugar beet Nutrition 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- 108010006785 Taq Polymerase Proteins 0.000 description 1
- 108020005038 Terminator Codon Proteins 0.000 description 1
- 101710203193 Thaumatin-like protein Proteins 0.000 description 1
- NFMPFBCXABPALN-OWLDWWDNSA-N Thr-Ala-Trp Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](C)C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)O)N)O NFMPFBCXABPALN-OWLDWWDNSA-N 0.000 description 1
- TZKPNGDGUVREEB-FOHZUACHSA-N Thr-Asn-Gly Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(O)=O TZKPNGDGUVREEB-FOHZUACHSA-N 0.000 description 1
- JBHMLZSKIXMVFS-XVSYOHENSA-N Thr-Asn-Phe Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O JBHMLZSKIXMVFS-XVSYOHENSA-N 0.000 description 1
- VULNJDORNLBPNG-SWRJLBSHSA-N Thr-Glu-Trp Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)O)N)O VULNJDORNLBPNG-SWRJLBSHSA-N 0.000 description 1
- YSXYEJWDHBCTDJ-DVJZZOLTSA-N Thr-Gly-Trp Chemical compound C[C@H]([C@@H](C(=O)NCC(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)O)N)O YSXYEJWDHBCTDJ-DVJZZOLTSA-N 0.000 description 1
- FDALPRWYVKJCLL-PMVVWTBXSA-N Thr-His-Gly Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CNC=N1)C(=O)NCC(O)=O FDALPRWYVKJCLL-PMVVWTBXSA-N 0.000 description 1
- XYFISNXATOERFZ-OSUNSFLBSA-N Thr-Ile-Val Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C(C)C)C(=O)O)NC(=O)[C@H]([C@@H](C)O)N XYFISNXATOERFZ-OSUNSFLBSA-N 0.000 description 1
- YOOAQCZYZHGUAZ-KATARQTJSA-N Thr-Leu-Ser Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O YOOAQCZYZHGUAZ-KATARQTJSA-N 0.000 description 1
- KKPOGALELPLJTL-MEYUZBJRSA-N Thr-Lys-Tyr Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 KKPOGALELPLJTL-MEYUZBJRSA-N 0.000 description 1
- GUHLYMZJVXUIPO-RCWTZXSCSA-N Thr-Met-Val Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](C(C)C)C(O)=O GUHLYMZJVXUIPO-RCWTZXSCSA-N 0.000 description 1
- NWECYMJLJGCBOD-UNQGMJICSA-N Thr-Phe-Val Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](C(C)C)C(O)=O NWECYMJLJGCBOD-UNQGMJICSA-N 0.000 description 1
- WTMPKZWHRCMMMT-KZVJFYERSA-N Thr-Pro-Ala Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C)C(O)=O WTMPKZWHRCMMMT-KZVJFYERSA-N 0.000 description 1
- MUAFDCVOHYAFNG-RCWTZXSCSA-N Thr-Pro-Arg Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCNC(N)=N)C(O)=O MUAFDCVOHYAFNG-RCWTZXSCSA-N 0.000 description 1
- JAJOFWABAUKAEJ-QTKMDUPCSA-N Thr-Pro-His Chemical compound C[C@H]([C@@H](C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)N)O JAJOFWABAUKAEJ-QTKMDUPCSA-N 0.000 description 1
- FWTFAZKJORVTIR-VZFHVOOUSA-N Thr-Ser-Ala Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(O)=O FWTFAZKJORVTIR-VZFHVOOUSA-N 0.000 description 1
- BZTSQFWJNJYZSX-JRQIVUDYSA-N Thr-Tyr-Asp Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(O)=O)C(O)=O BZTSQFWJNJYZSX-JRQIVUDYSA-N 0.000 description 1
- VYVBSMCZNHOZGD-RCWTZXSCSA-N Thr-Val-Val Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C(C)C)C(O)=O VYVBSMCZNHOZGD-RCWTZXSCSA-N 0.000 description 1
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 1
- 239000004473 Threonine Substances 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- 241000223259 Trichoderma Species 0.000 description 1
- 241000223261 Trichoderma viride Species 0.000 description 1
- 244000098345 Triticum durum Species 0.000 description 1
- 235000007264 Triticum durum Nutrition 0.000 description 1
- OHGNSVACHBZKSS-KWQFWETISA-N Trp-Ala Chemical compound C1=CC=C2C(C[C@H]([NH3+])C(=O)N[C@@H](C)C([O-])=O)=CNC2=C1 OHGNSVACHBZKSS-KWQFWETISA-N 0.000 description 1
- MJBBMTOGSOSAKJ-HJXMPXNTSA-N Trp-Ala-Ile Chemical compound [H]N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O MJBBMTOGSOSAKJ-HJXMPXNTSA-N 0.000 description 1
- ADBFWLXCCKIXBQ-XIRDDKMYSA-N Trp-Asn-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)N ADBFWLXCCKIXBQ-XIRDDKMYSA-N 0.000 description 1
- NXJZCPKZIKTYLX-XEGUGMAKSA-N Trp-Glu-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)N NXJZCPKZIKTYLX-XEGUGMAKSA-N 0.000 description 1
- UDCHKDYNMRJYMI-QEJZJMRPSA-N Trp-Glu-Ser Chemical compound [H]N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(O)=O UDCHKDYNMRJYMI-QEJZJMRPSA-N 0.000 description 1
- HXNVJPQADLRHGR-JBACZVJFSA-N Trp-Glu-Tyr Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CC3=CC=C(C=C3)O)C(=O)O)N HXNVJPQADLRHGR-JBACZVJFSA-N 0.000 description 1
- YYXIWHBHTARPOG-HJXMPXNTSA-N Trp-Ile-Ala Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C)C(=O)O)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)N YYXIWHBHTARPOG-HJXMPXNTSA-N 0.000 description 1
- XGFGVFMXDXALEV-XIRDDKMYSA-N Trp-Leu-Asn Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)N XGFGVFMXDXALEV-XIRDDKMYSA-N 0.000 description 1
- AGSYHLPWNXGVSG-NYVOZVTQSA-N Trp-Trp-Cys Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CC3=CNC4=CC=CC=C43)C(=O)N[C@@H](CS)C(=O)O)N AGSYHLPWNXGVSG-NYVOZVTQSA-N 0.000 description 1
- IYHRKILQAQWODS-VJBMBRPKSA-N Trp-Trp-Glu Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CC3=CNC4=CC=CC=C43)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N IYHRKILQAQWODS-VJBMBRPKSA-N 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- XLMDWQNAOKLKCP-XDTLVQLUSA-N Tyr-Ala-Gln Chemical compound C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)N XLMDWQNAOKLKCP-XDTLVQLUSA-N 0.000 description 1
- DLZKEQQWXODGGZ-KWQFWETISA-N Tyr-Ala-Gly Chemical compound OC(=O)CNC(=O)[C@H](C)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 DLZKEQQWXODGGZ-KWQFWETISA-N 0.000 description 1
- PEVVXUGSAKEPEN-AVGNSLFASA-N Tyr-Asn-Glu Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O PEVVXUGSAKEPEN-AVGNSLFASA-N 0.000 description 1
- AYPAIRCDLARHLM-KKUMJFAQSA-N Tyr-Asn-Lys Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CCCCN)C(=O)O)N)O AYPAIRCDLARHLM-KKUMJFAQSA-N 0.000 description 1
- TZXFLDNBYYGLKA-BZSNNMDCSA-N Tyr-Asp-Tyr Chemical compound C([C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)C1=CC=C(O)C=C1 TZXFLDNBYYGLKA-BZSNNMDCSA-N 0.000 description 1
- UABYBEBXFFNCIR-YDHLFZDLSA-N Tyr-Asp-Val Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O UABYBEBXFFNCIR-YDHLFZDLSA-N 0.000 description 1
- BODHJXJNRVRKFA-BZSNNMDCSA-N Tyr-Cys-Tyr Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CS)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O BODHJXJNRVRKFA-BZSNNMDCSA-N 0.000 description 1
- RYSNTWVRSLCAJZ-RYUDHWBXSA-N Tyr-Gln-Gly Chemical compound OC(=O)CNC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 RYSNTWVRSLCAJZ-RYUDHWBXSA-N 0.000 description 1
- AKLNEFNQWLHIGY-QWRGUYRKSA-N Tyr-Gly-Asp Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)NCC(=O)N[C@@H](CC(=O)O)C(=O)O)N)O AKLNEFNQWLHIGY-QWRGUYRKSA-N 0.000 description 1
- BSCBBPKDVOZICB-KKUMJFAQSA-N Tyr-Leu-Asp Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O BSCBBPKDVOZICB-KKUMJFAQSA-N 0.000 description 1
- VTCKHZJKWQENKX-KBPBESRZSA-N Tyr-Lys-Gly Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCCCN)C(=O)NCC(O)=O VTCKHZJKWQENKX-KBPBESRZSA-N 0.000 description 1
- CDBXVDXSLPLFMD-BPNCWPANSA-N Tyr-Pro-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)CC1=CC=C(O)C=C1 CDBXVDXSLPLFMD-BPNCWPANSA-N 0.000 description 1
- YYLHVUCSTXXKBS-IHRRRGAJSA-N Tyr-Pro-Ser Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(O)=O YYLHVUCSTXXKBS-IHRRRGAJSA-N 0.000 description 1
- LVILBTSHPTWDGE-PMVMPFDFSA-N Tyr-Trp-Lys Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCCCN)C(O)=O)C1=CC=C(O)C=C1 LVILBTSHPTWDGE-PMVMPFDFSA-N 0.000 description 1
- PQPWEALFTLKSEB-DZKIICNBSA-N Tyr-Val-Glu Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O PQPWEALFTLKSEB-DZKIICNBSA-N 0.000 description 1
- GOPQNCQSXBJAII-ULQDDVLXSA-N Tyr-Val-Lys Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)N GOPQNCQSXBJAII-ULQDDVLXSA-N 0.000 description 1
- DDRBQONWVBDQOY-GUBZILKMSA-N Val-Ala-Arg Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O DDRBQONWVBDQOY-GUBZILKMSA-N 0.000 description 1
- NMANTMWGQZASQN-QXEWZRGKSA-N Val-Arg-Asp Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CC(=O)O)C(=O)O)N NMANTMWGQZASQN-QXEWZRGKSA-N 0.000 description 1
- HNWQUBBOBKSFQV-AVGNSLFASA-N Val-Arg-His Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N HNWQUBBOBKSFQV-AVGNSLFASA-N 0.000 description 1
- IDKGBVZGNTYYCC-QXEWZRGKSA-N Val-Asn-Pro Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)N1CCC[C@H]1C(O)=O IDKGBVZGNTYYCC-QXEWZRGKSA-N 0.000 description 1
- QHDXUYOYTPWCSK-RCOVLWMOSA-N Val-Asp-Gly Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)NCC(=O)O)N QHDXUYOYTPWCSK-RCOVLWMOSA-N 0.000 description 1
- COSLEEOIYRPTHD-YDHLFZDLSA-N Val-Asp-Tyr Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 COSLEEOIYRPTHD-YDHLFZDLSA-N 0.000 description 1
- NYTKXWLZSNRILS-IFFSRLJSSA-N Val-Gln-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](C(C)C)N)O NYTKXWLZSNRILS-IFFSRLJSSA-N 0.000 description 1
- ROLGIBMFNMZANA-GVXVVHGQSA-N Val-Glu-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](C(C)C)N ROLGIBMFNMZANA-GVXVVHGQSA-N 0.000 description 1
- UEHRGZCNLSWGHK-DLOVCJGASA-N Val-Glu-Val Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O UEHRGZCNLSWGHK-DLOVCJGASA-N 0.000 description 1
- BEGDZYNDCNEGJZ-XVKPBYJWSA-N Val-Gly-Gln Chemical compound CC(C)[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCC(N)=O BEGDZYNDCNEGJZ-XVKPBYJWSA-N 0.000 description 1
- WFENBJPLZMPVAX-XVKPBYJWSA-N Val-Gly-Glu Chemical compound CC(C)[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCC(O)=O WFENBJPLZMPVAX-XVKPBYJWSA-N 0.000 description 1
- XXROXFHCMVXETG-UWVGGRQHSA-N Val-Gly-Val Chemical compound CC(C)[C@H](N)C(=O)NCC(=O)N[C@@H](C(C)C)C(O)=O XXROXFHCMVXETG-UWVGGRQHSA-N 0.000 description 1
- SDSCOOZQQGUQFC-GVXVVHGQSA-N Val-His-Gln Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N SDSCOOZQQGUQFC-GVXVVHGQSA-N 0.000 description 1
- UZFNHAXYMICTBU-DZKIICNBSA-N Val-Phe-Gln Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N UZFNHAXYMICTBU-DZKIICNBSA-N 0.000 description 1
- DEGUERSKQBRZMZ-FXQIFTODSA-N Val-Ser-Ala Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(O)=O DEGUERSKQBRZMZ-FXQIFTODSA-N 0.000 description 1
- UJMCYJKPDFQLHX-XGEHTFHBSA-N Val-Ser-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](C(C)C)N)O UJMCYJKPDFQLHX-XGEHTFHBSA-N 0.000 description 1
- GVNLOVJNNDZUHS-RHYQMDGZSA-N Val-Thr-Lys Chemical compound [H]N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCCN)C(O)=O GVNLOVJNNDZUHS-RHYQMDGZSA-N 0.000 description 1
- YLBNZCJFSVJDRJ-KJEVXHAQSA-N Val-Thr-Tyr Chemical compound CC(C)[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](Cc1ccc(O)cc1)C(O)=O YLBNZCJFSVJDRJ-KJEVXHAQSA-N 0.000 description 1
- DFQZDQPLWBSFEJ-LSJOCFKGSA-N Val-Val-Asn Chemical compound CC(C)[C@@H](C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(=O)N)C(=O)O)N DFQZDQPLWBSFEJ-LSJOCFKGSA-N 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 241000219094 Vitaceae Species 0.000 description 1
- QWXOJIDBSHLIFI-UHFFFAOYSA-N [3-(1-chloro-3'-methoxyspiro[adamantane-4,4'-dioxetane]-3'-yl)phenyl] dihydrogen phosphate Chemical compound O1OC2(C3CC4CC2CC(Cl)(C4)C3)C1(OC)C1=CC=CC(OP(O)(O)=O)=C1 QWXOJIDBSHLIFI-UHFFFAOYSA-N 0.000 description 1
- FJJCIZWZNKZHII-UHFFFAOYSA-N [4,6-bis(cyanoamino)-1,3,5-triazin-2-yl]cyanamide Chemical compound N#CNC1=NC(NC#N)=NC(NC#N)=N1 FJJCIZWZNKZHII-UHFFFAOYSA-N 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 238000005273 aeration Methods 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- 238000012867 alanine scanning Methods 0.000 description 1
- 108010028939 alanyl-alanyl-lysyl-alanine Proteins 0.000 description 1
- 108010050181 aleurone Proteins 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 229910000148 ammonium phosphate Inorganic materials 0.000 description 1
- 235000019289 ammonium phosphates Nutrition 0.000 description 1
- 238000012870 ammonium sulfate precipitation Methods 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000000845 anti-microbial effect Effects 0.000 description 1
- 239000004599 antimicrobial Substances 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 108010062796 arginyllysine Proteins 0.000 description 1
- 210000004507 artificial chromosome Anatomy 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000010165 autogamy Effects 0.000 description 1
- 238000000376 autoradiography Methods 0.000 description 1
- QLTSDROPCWIKKY-PMCTYKHCSA-N beta-D-glucosaminyl-(1->4)-beta-D-glucosamine Chemical compound O[C@@H]1[C@@H](N)[C@H](O)O[C@H](CO)[C@H]1O[C@H]1[C@H](N)[C@@H](O)[C@H](O)[C@@H](CO)O1 QLTSDROPCWIKKY-PMCTYKHCSA-N 0.000 description 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- 239000012681 biocontrol agent Substances 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 239000007844 bleaching agent Substances 0.000 description 1
- 239000001055 blue pigment Substances 0.000 description 1
- UDSAIICHUKSCKT-UHFFFAOYSA-N bromophenol blue Chemical compound C1=C(Br)C(O)=C(Br)C=C1C1(C=2C=C(Br)C(O)=C(Br)C=2)C2=CC=CC=C2S(=O)(=O)O1 UDSAIICHUKSCKT-UHFFFAOYSA-N 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 229940095731 candida albicans Drugs 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000036978 cell physiology Effects 0.000 description 1
- 108091092328 cellular RNA Proteins 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 101150014959 chi1 gene Proteins 0.000 description 1
- 210000003763 chloroplast Anatomy 0.000 description 1
- VJYIFXVZLXQVHO-UHFFFAOYSA-N chlorsulfuron Chemical compound COC1=NC(C)=NC(NC(=O)NS(=O)(=O)C=2C(=CC=CC=2)Cl)=N1 VJYIFXVZLXQVHO-UHFFFAOYSA-N 0.000 description 1
- 238000011098 chromatofocusing Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 230000002759 chromosomal effect Effects 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 235000008957 cocaer Nutrition 0.000 description 1
- ZPUCINDJVBIVPJ-LJISPDSOSA-N cocaine Chemical compound O([C@H]1C[C@@H]2CC[C@@H](N2C)[C@H]1C(=O)OC)C(=O)C1=CC=CC=C1 ZPUCINDJVBIVPJ-LJISPDSOSA-N 0.000 description 1
- 239000002361 compost Substances 0.000 description 1
- 230000001010 compromised effect Effects 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 238000012864 cross contamination Methods 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 108010004073 cysteinylcysteine Proteins 0.000 description 1
- 108010016616 cysteinylglycine Proteins 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000007123 defense Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 239000005547 deoxyribonucleotide Substances 0.000 description 1
- 125000002637 deoxyribonucleotide group Chemical group 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 230000001627 detrimental effect Effects 0.000 description 1
- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical compound [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 0.000 description 1
- 239000005546 dideoxynucleotide Substances 0.000 description 1
- FFYPMLJYZAEMQB-UHFFFAOYSA-N diethyl pyrocarbonate Chemical compound CCOC(=O)OC(=O)OCC FFYPMLJYZAEMQB-UHFFFAOYSA-N 0.000 description 1
- 230000009699 differential effect Effects 0.000 description 1
- 238000002050 diffraction method Methods 0.000 description 1
- 235000004879 dioscorea Nutrition 0.000 description 1
- FSXRLASFHBWESK-UHFFFAOYSA-N dipeptide phenylalanyl-tyrosine Natural products C=1C=C(O)C=CC=1CC(C(O)=O)NC(=O)C(N)CC1=CC=CC=C1 FSXRLASFHBWESK-UHFFFAOYSA-N 0.000 description 1
- 230000008034 disappearance Effects 0.000 description 1
- 238000011037 discontinuous sequential dilution Methods 0.000 description 1
- 235000021186 dishes Nutrition 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 210000003278 egg shell Anatomy 0.000 description 1
- 239000005712 elicitor Substances 0.000 description 1
- 230000013020 embryo development Effects 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 102000013165 exonuclease Human genes 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 230000004720 fertilization Effects 0.000 description 1
- 230000008124 floral development Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000004459 forage Substances 0.000 description 1
- 230000000855 fungicidal effect Effects 0.000 description 1
- 108010063718 gamma-glutamylaspartic acid Proteins 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 238000003209 gene knockout Methods 0.000 description 1
- MSWZFWKMSRAUBD-IVMDWMLBSA-N glucosamine group Chemical group OC1[C@H](N)[C@@H](O)[C@H](O)[C@H](O1)CO MSWZFWKMSRAUBD-IVMDWMLBSA-N 0.000 description 1
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 1
- IAJOBQBIJHVGMQ-BYPYZUCNSA-N glufosinate-P Chemical compound CP(O)(=O)CC[C@H](N)C(O)=O IAJOBQBIJHVGMQ-BYPYZUCNSA-N 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 108010040856 glutamyl-cysteinyl-alanine Proteins 0.000 description 1
- 108010079547 glutamylmethionine Proteins 0.000 description 1
- 108010050792 glutenin Proteins 0.000 description 1
- 108010000434 glycyl-alanyl-leucine Proteins 0.000 description 1
- 108010077435 glycyl-phenylalanyl-glycine Proteins 0.000 description 1
- 108010010147 glycylglutamine Proteins 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 235000011868 grain product Nutrition 0.000 description 1
- 235000021021 grapes Nutrition 0.000 description 1
- 101150054900 gus gene Proteins 0.000 description 1
- 235000009424 haa Nutrition 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 239000001307 helium Substances 0.000 description 1
- 229910052734 helium Inorganic materials 0.000 description 1
- SWQJXJOGLNCZEY-UHFFFAOYSA-N helium atom Chemical compound [He] SWQJXJOGLNCZEY-UHFFFAOYSA-N 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 108010040030 histidinoalanine Proteins 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 230000014726 immortalization of host cell Effects 0.000 description 1
- 230000001976 improved effect Effects 0.000 description 1
- 238000007901 in situ hybridization Methods 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 238000001155 isoelectric focusing Methods 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 1
- 108010078274 isoleucylvaline Proteins 0.000 description 1
- 108010000761 leucylarginine Proteins 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 108010003700 lysyl aspartic acid Proteins 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 108010034507 methionyltryptophan Proteins 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 244000000010 microbial pathogen Species 0.000 description 1
- 210000001724 microfibril Anatomy 0.000 description 1
- 210000003470 mitochondria Anatomy 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000006919 modified lb Substances 0.000 description 1
- 238000001823 molecular biology technique Methods 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 238000000329 molecular dynamics simulation Methods 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- 108091027963 non-coding RNA Proteins 0.000 description 1
- 102000042567 non-coding RNA Human genes 0.000 description 1
- 108010058731 nopaline synthase Proteins 0.000 description 1
- 230000031787 nutrient reservoir activity Effects 0.000 description 1
- 239000002751 oligonucleotide probe Substances 0.000 description 1
- 229920001542 oligosaccharide Polymers 0.000 description 1
- 150000002482 oligosaccharides Chemical class 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 230000003071 parasitic effect Effects 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 210000002824 peroxisome Anatomy 0.000 description 1
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- 108010070409 phenylalanyl-glycyl-glycine Proteins 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 229930195732 phytohormone Natural products 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 230000008119 pollen development Effects 0.000 description 1
- 230000010152 pollination Effects 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 229920001155 polypropylene Polymers 0.000 description 1
- 230000004481 post-translational protein modification Effects 0.000 description 1
- 230000001124 posttranscriptional effect Effects 0.000 description 1
- 235000011056 potassium acetate Nutrition 0.000 description 1
- 239000000276 potassium ferrocyanide Substances 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 108060006613 prolamin Proteins 0.000 description 1
- 108010089416 prolyl-leucyl-leucyl-glutaminyl-lysine Proteins 0.000 description 1
- 108010004914 prolylarginine Proteins 0.000 description 1
- 108010090894 prolylleucine Proteins 0.000 description 1
- 230000001902 propagating effect Effects 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 235000021251 pulses Nutrition 0.000 description 1
- 235000015136 pumpkin Nutrition 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 108010026134 purothionin Proteins 0.000 description 1
- 150000003856 quaternary ammonium compounds Chemical class 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
- 238000010188 recombinant method Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000003362 replicative effect Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000001177 retroviral effect Effects 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 239000012882 rooting medium Substances 0.000 description 1
- 239000012146 running buffer Substances 0.000 description 1
- 235000019515 salmon Nutrition 0.000 description 1
- 239000012723 sample buffer Substances 0.000 description 1
- 235000014102 seafood Nutrition 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 108010048818 seryl-histidine Proteins 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 239000002002 slurry Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- FQENQNTWSFEDLI-UHFFFAOYSA-J sodium diphosphate Chemical compound [Na+].[Na+].[Na+].[Na+].[O-]P([O-])(=O)OP([O-])([O-])=O FQENQNTWSFEDLI-UHFFFAOYSA-J 0.000 description 1
- 229940048086 sodium pyrophosphate Drugs 0.000 description 1
- ADWNFGORSPBALY-UHFFFAOYSA-M sodium;2-[dodecyl(methyl)amino]acetate Chemical compound [Na+].CCCCCCCCCCCCN(C)CC([O-])=O ADWNFGORSPBALY-UHFFFAOYSA-M 0.000 description 1
- 210000001082 somatic cell Anatomy 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 229940063673 spermidine Drugs 0.000 description 1
- 229940063675 spermine Drugs 0.000 description 1
- 238000001694 spray drying Methods 0.000 description 1
- 235000020354 squash Nutrition 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 108010058363 sterol carrier proteins Proteins 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 239000004575 stone Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 230000004960 subcellular localization Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 230000000153 supplemental effect Effects 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 230000002459 sustained effect Effects 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- XOGGUFAVLNCTRS-UHFFFAOYSA-N tetrapotassium;iron(2+);hexacyanide Chemical compound [K+].[K+].[K+].[K+].[Fe+2].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-] XOGGUFAVLNCTRS-UHFFFAOYSA-N 0.000 description 1
- 235000019818 tetrasodium diphosphate Nutrition 0.000 description 1
- 239000001577 tetrasodium phosphonato phosphate Substances 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 238000000844 transformation Methods 0.000 description 1
- 230000010474 transient expression Effects 0.000 description 1
- 229930013292 trichothecene Natural products 0.000 description 1
- LZAJKCZTKKKZNT-PMNGPLLRSA-N trichothecene Chemical compound C12([C@@]3(CC[C@H]2OC2C=C(CCC23C)C)C)CO1 LZAJKCZTKKKZNT-PMNGPLLRSA-N 0.000 description 1
- PIEPQKCYPFFYMG-UHFFFAOYSA-N tris acetate Chemical compound CC(O)=O.OCC(N)(CO)CO PIEPQKCYPFFYMG-UHFFFAOYSA-N 0.000 description 1
- 108010080629 tryptophan-leucine Proteins 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
- IBIDRSSEHFLGSD-UHFFFAOYSA-N valinyl-arginine Natural products CC(C)C(N)C(=O)NC(C(O)=O)CCCN=C(N)N IBIDRSSEHFLGSD-UHFFFAOYSA-N 0.000 description 1
- 108010009962 valyltyrosine Proteins 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01014—Chitinase (3.2.1.14)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
- C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
- C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
- C12N15/823—Reproductive tissue-specific promoters
- C12N15/8231—Male-specific, e.g. anther, tapetum, pollen
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
- C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
- C12N15/823—Reproductive tissue-specific promoters
- C12N15/8234—Seed-specific, e.g. embryo, endosperm
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
- C12N15/8282—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for fungal resistance
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
- C12N9/2442—Chitinase (3.2.1.14)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01052—Beta-N-acetylhexosaminidase (3.2.1.52)
Definitions
- the present invention relates to nucleic acid sequences derived from fungal genes which encode polypeptides having cell wall-degrading activity and isolated polypeptides having cell wall-degrading activity.
- the invention also relates to recombinant nucleic acid molecules, vectors, and host cells comprising the nucleic acid sequences as well as methods for producing and using the polypeptides, including expression in plant cells to confer or enhance a plant's resistance to Fusarium and other pathogens.
- Cereal crops including wheat, maize, barley, oats, and rye, are susceptible to infection by many types of fungi and by many species of the pathogenic fungus Fusarium.
- Fusarium graminearum (Schwabe) and Fusarium culmorum are the primary causal agents of a disease known as Fusarium head blight, head blight, or scab, of wheat, barley, oats and rye (reviewed in Bai and Shaner 1994; Parry et al. 1995).
- the life cycle of the pathogen alternates between two hosts, wheat and maize.
- F. graminearum causes death of the floral organs (florets) that harbor the developing grain, giving the head a bleached and “scabby” appearance, and resulting in moderate to severe reductions in grain yield.
- F. graminearum and other Fusarium species produce trichothecene mycotoxins, such as deoxynivalenol (DON), that exacerbate disease severity and pose a health threat to humans and livestock that ingest contaminated cereal products.
- DON deoxynivalenol
- Glycinebetaine an osmoprotectant, accumulates over the normal course of pollen development during desiccation (reviewed in McCue and Hanson 1990). Along with choline, it is postulated to serve as a fortuitous source of carbon and nitrogen for F. graminearum and for other fungal pathogens. Whether fungal hyphae readily access these compounds at the pollen surface or must invade the pollen cytoplasm is not known at this time.
- the outer wall layer of Fusarium is comprised of polymers of glucose (1,3/1,6- ⁇ -D-glucan) with ⁇ -1,3 and ⁇ -1,6 linkages.
- the basal-most inner layer consists primarily of chitin microfibrils, linear polymers of ⁇ -1,4-N-acetylglucosamine that account for about one-third of the mass of the hyphal wall (Barbosa and Kemmelmeier 1993).
- Fusarium cell walls appear to be more refractory to the action of hydrolytic enzymes, possibly because of high levels of protein and chitin (Sivan and Chet 1989a), and clustering of acetylated glucosamine residues (Fukamizo et al. 1992).
- Chitinases and glucanases are produced by naturally occurring bacteria, fungi and plants. In the fungi, these proteins have roles in the self-hydrolysis of cell wall chitin and glucan, respectively, and in the hydrolysis of cell wall components of other microorganisms (Srivastava et al. 1985; Sivan and Chet 1989b, Chérif and Benhamou 1990, Vázquez-Garciduefias et al. 1998). This latter feature has been applied to the discovery of anti-microbial biocontrol agents. Chitin is also found in the cuticles of insects and in nematode egg shells.
- chitinase might have a role in fungal metabolism (Flach et al. 1992). Endochitinases cleave randomly between C1 and C4 linkages within the chitin polymer (Flach et al. 1992, Graham and Sticklen 1994), whereas exochitinases cleave sequentially between each linkage, releasing chitobiose from the terminus of the chitin polymer. N-acetyl-glucosaminidase also cleaves terminally, releasing N-acetylglucosamine (Flach et al. 1992).
- endoglucanases randomly cleave ⁇ -linkages within the glucan polymer, generating short oligosaccharides, whereas the exoglucanases cleave single glucose residues from the nonreducing end of the polymer (Vázquez-Garciduefias et al. 1998). Hydrolysis of the glucan layer of fungal cell walls is attributed to the combined action of both endo- and exo-glucanases.
- glucan and chitin composition of the fungal cell wall has led to extensive engineering of chitinases and glucanases as antifungal proteins.
- Chitinases and glucanases produced by plants have been widely characterized as pathogenesis-related (Pr) proteins involved in defense against pathogens and insect pests. Pr protein genes in plants are induced upon exposure to microbial pathogens and insect pests.
- Cell wall-degrading proteins have been grouped into classes on the basis of their biochemical and structural properties. Both endo- and exochitinases appear to be effective in the hydrolysis of fungal cell wall chitin.
- Class I chitinases have chitinolytic activity against bacterial cell walls and are known to bind chitin directly.
- class II chitinases do not act on bacterial cell walls and lack chitin binding activity; they are postulated to play a role in production of fungal elicitors that trigger the host defense response (Graham and Sticklen 1994, Fritig et al. 1998).
- the basic chitinases and glucanases (class I) accumulate intracellularly in the vacuole, and the acidic isoforms (class II) are extracellular, with some exceptions (e.g., Wu et al. 1994; Graham and Sticklen 1994).
- Class I chitinases are encoded by small gene families in most plants and in the fungi. The structure of various chitinase genes and proteins has been reviewed (Graham and Sticklen 1994).
- ⁇ -1,3-glucanases are members of small multigene families in plants (Payne et al. 1990, Xu et al. 1992, Beffa and Meins 1996, Simmons 1994), but are single-copy genes in Fusarium sporotrichioides (FIG. 27).
- Class I glucanases accumulate in the vacuoles, whereas Class II and III glucanases are acidic and extracellular (see Beffa and Meins 1996).
- Class I glucanases have been widely studied in the context of pathogen defense and stress responses in tobacco (Linthorst et al. 1990; Linthorst 1991; Payne et al. 1990), barley (Jutidamrongphan et al. 1991; Xu et al. 1992; Malehorn et al. 1993), wheat (Jutidamrongphan et al. 1991; Cruz-Ortega et al. 1997), and other species (Krishnaveni et al. 1999a, reviewed in Simmons 1994). Chitinases and glucanases act preferentially on the tips of growing fungal hypha (for instance, Broekaert et al. 1988, Collinge et al.
- chitinases and ⁇ -1,3-glucanases are differentially expressed during plant development (Lotan et al. 1989) as well as in response to pathogen attack.
- Glucanases appear to have roles in cell division and flower development (Beffa and Meins 1996) and the stress response (Simmons 1994) in plants.
- the role of chitinases in plant development is not as well-characterized; however, they have been implicated in embryogenesis and cell division (for review, see Collinge et al. 1993)
- chitinases and ⁇ -1,3-glucanases produced by naturally occurring bacteria and fungi have anti-Fusarium properties (Mitchell and Alexander 1961; Michael and Nelson 1972, Chérif and Benhamou 1990).
- Glucanases and chitinases from plants can degrade isolated cell walls of Fusarium solani (Mauch et al. 1988).
- Chitinases from tobacco were inhibitory to the growth of F. oxysporum (Yun et al. 1996) and F. solani (Sela-Buurlage et al. 1993) in culture.
- Krishnaveni et al. (1999b) have described three chitinases from sorghum seeds that inhibit the growth of F. moniliforme.
- the enzymes were active against a wide range of other fungi.
- a tobacco chitinase with activity against Fusarium and Trichoderma were inactive against Aspergillus flavus, Phytophthora parasitica and other pathogens (Yun et al. (1996).
- the differential activities of the chitinases are attributed to inherent properties of the enzymes (Sela-Buurlage et al. 1993, Brunner et al. 1998), to differences in cell wall architecture (Sivan and Chet 1989a, Van Loon 1997) among the fungi, or to other factors.
- Hu and Reddy (1997) isolated a thaumatin-like protein from Arabidopsis thaliana with activity against F. oxysporum.
- the present invention is directed to nucleic acid sequences derived from Fusarium fungal genes which encode polypeptides having cell wall-degrading activity as well as isolated polypeptides having cell wall-degrading activity.
- the invention is also directed to recombinant nucleic acid molecules, vectors, and host cells comprising the nucleic acid sequences and methods for producing and using the polypeptides, including expression in plant cells to confer or enhance a plant's resistance to Fusarium and other pathogens.
- the invention provides isolated nucleic acid molecules that encode polypeptides having cell wall-degrading activity comprising glucanase, endochitinase or exochitinase activity.
- Genomic sequences encoding glucanase and exochitinase and cDNA sequences encoding glucanase, endochitinase and exochitinase are specifically exemplified herein.
- nucleic acid sequences encoding a polypeptide having the glucanase, endochitinase or exochitinase polypeptide sequences exemplified below and nucleic acid molecules encoding polypeptides having glucanase, endochitinase or exochitinase activity.
- Nucleic acid sequences which hybridize specifically to an enzyme coding sequence or its complement under medium or high stringency conditions and which encode a polypeptide having glucanase, endochitinase or exochitinase activity are also encompassed by the present invention.
- Nucleic acid sequences having at least 70% sequence identity with the exemplified glucanase sequences as described in detail, below, and which encode a polypeptide having glucanase activity are also encompassed by the present invention.
- Nucleic acid sequences encoding a polypeptide having at least 80% sequence identity with the exemplified glucanase polypeptide sequences as described in detail, below, and which encode a polypeptide having glucanase activity are also encompassed by the present invention.
- Nucleic acid sequences having at least 75% sequence identity with the exemplified endochitinase or exochitinase sequences as described in detail, below, and which encode a polypeptide having endochitinase or exochitinase activity are also encompassed by the present invention.
- Nucleic acid sequences encoding a polypeptide having at least 85% sequence identity with the exemplified endochitinase or exochitinase polypeptide sequences as described in detail, below, and which encode a polypeptide having endochitinase or exochitinase activity are also encompassed by the present invention.
- the present invention is also directed to isolated polypeptides having glucanase, endochitinase or exochitinase activity.
- Polypeptides encoded by a nucleic acid sequence which hybridizes under medium or high stringency conditions with exemplified nucleic acid sequences as discussed in detail, below, are also encompassed by the invention. Variants of the polypeptides are encompassed by the invention as well as fragments having glucanase, endochitinase or exochitinase activity.
- the invention is also directed to methods of producing and using the polypeptides of the invention.
- a further aspect of the invention is the provision of recombinant nucleic acid molecules containing the sequences encoding polypeptides having the fungal cell wall-degrading activity, including glucanase, endochitinase or exochitinase activity.
- Such molecules include, for example, recombinant vectors, such as cloning, expression or transformation vectors, which contain a DNA sequence encoding a glucanase, endochitinase or exochitinase.
- Another aspect of the invention is the provision of cells which are transformed by the above vectors or DNA sequences.
- a particular use of the invention is the provision of cells transformed with one or more nucleic acid sequences of the invention.
- a more particular use of the invention is the provision of plants, plant seeds or plant cells transformed with one or more nucleic acid sequences encoding a polypeptide having glucanase, endochitinase or exochitinase coding activity to provide plants having resistance to plant pathogens, including fungi, particularly, Fusarium species or to provide plants having enhanced resistance to plant pathogens.
- a further aspect of the invention is the provision of oligonucleotide probes capable of detecting a glucanase, endochitinase or exochitinase gene or functional equivalents thereof in fungi of the genus Fusarium and the use of the probes to isolate nucleic acid sequences encoding a glucanase, endochitinase or exochitinase gene or functional equivalent thereof.
- the nucleic acid sequences which specifically hybridize to the probes and which encode a functional glucanase, endochitinase or exochitinase are encompassed by the present invention.
- nucleic acid sequences of the invention facilitates the isolation of homologous genes from fungi to obtain genes which protect host cells, including fungi, bacteria, and plants against related fungal pathogens.
- the invention also includes the application of transgene constructs in combination with each other, that is, glucanase and endochitinase; glucanase and exochitinase; endochitinase and exochitinase; and glucanase, endochitinase and exochitinase. Also included are transgenic monocot or dicot lines, plant cells, and progeny obtained by sexual or asexual propagation, that carry any combination of the transgene constructs.
- nucleic acid sequences encoding fungal cell wall-degrading enzymes selected from the group consisting of glucanase, exochitinase, and endochitinase; isolated polypeptides having glucanase, endochitinase or exochitinase activity; recombinant nucleic acid molecules including expression vectors encoding polypeptides having cell wall-degrading activity; and cells harboring the recombinant nucleic acid molecules or expression vectors.
- transformation vectors comprising a cell wall-degrading recombinant molecule, which vectors are effective for stably introducing the recombinant molecule into a plant.
- a further object of the invention is to provide fungal genes which generate cell wall-degrading enzymes, including proteins having the capability of degrading the glucan and chitin cell wall components of F. venenatum and other Fusarium species, including F. graminearum and F. culmorum, the principle causal agents of head blight (scab) in the U.S.
- Another object of the invention is expression of the cell wall-degrading enzymes in transgenic monocots, including wheat, barley or oats, to confer partial or complete resistance to Fusarium species and/or to other fungal pathogens of wheat and other cereal crops.
- transgenic lines will be useful genetic stocks for generating improved crops.
- a still further object of the invention is the provision of novel wheat germplasms that express genes designed to limit the spread of the pathogenic fungus Fusarium and indirectly to curtail the accumulation of DON in infected heads.
- FIG. 1 is a comparison of glucanases encoded by F. venenatum cDNA (upper) and F. sporotrichioides genomic DNA (lower) (SEQ ID NOS:5 and 4, respectively).
- FIG. 2 shows the F. venenatum endochitinase 5′ cDNA PCR product and the deduced polypeptide sequence (SEQ ID NOS:6 and 7, respectively).
- Bold type indicates new sequence obtained in 5′ PCR product.
- Non-bold type shows the portion of the F. venenatum endochitinase 5′ cDNA sequence that matches the F. venenatum endochitinase partial cDNA sequence.
- FIG. 3 shows the F. sporotrichioides exochitinase 5′ genomic PCR product and a portion of the deduced polypeptide sequence (SEQ ID NOS:8 and 9, respectively).
- the underlined segment indicates an intron.
- the nucleotide segment before the ATG (first M) is the promoter and 5′ untranslated region.
- Bold type indicates new sequence obtained in 5′ PCR product.
- Non-bold type shows the portion of the F. sporotrichioides genomic sequence that matches the F. venenalum exochitinase cDNA sequence.
- FIG. 4 shows the comparison of exochitinases encoded by F. venenatum cDNA (upper) and F. sporotrichioides genomic DNA (lower) (SEQ ID NOS:14 and 15, respectively).
- FIG. 5 illustrates the modification to the Ubiquitin-1 promoter.
- FIG. 6 shows the map of plasmid FvGluS; monocot expression vector having the modified full-length glucanase cDNA in the sense orientation.
- FIG. 7 shows the map of plasmid FvGluAS; monocot expression vector having the modified full-length glucanase cDNA in the antisense orientation.
- FIG. 8 shows the map of plasmid FvEndoS; monocot expression vector having the modified full-length endochitinase cDNA in the sense orientation.
- FIG. 9 shows the map of plasmid FvEndoAS;.monocot expression vector having the modified full-length endochitinase cDNA in the antisense orientation.
- FIG. 10 shows the map of plasmid FvExoS; monocot expression vector having the modified full-length exochitinase cDNA in the sense orientation.
- FIG. 11 shows the map of plasmid FvExoAS; monocot expression vector having the modified full-length exochitinase cDNA in the antisense orientation.
- FIG. 12 shows the PCR analyses of transgenic lines for stably integrated transgene DNA.
- UT indicates untransformed plant; T1-T3 indicates transformed plants; C indicates plasmid (construct) control; mw indicates 100 bp ladder.
- FIG. 13 shows the Northern blot of FvEndo mRNA in Triticum aestivum line AB8-108.
- FIG. 14 shows detection of 5 ′portion of glucanase transcripts in endosperm and glume of Triticum aestivum using RT-PCR.
- FIG. 15 shows detection of 5′ and 3′ portions of endo- and exo-chitinase transcripts in endosperm and glume of Triticum aestivum using RT-PCR.
- FIG. 16 shows authentication of endochitinase 5′ RT-PCR product using restriction endonucleases.
- FIG. 17 shows authentication of exochitinase 5′ RT-PCR product using restriction exonucleases.
- FIG. 18 shows FvEndo 5′ and 3′ RT-PCR products are cDNA-dependent.
- FIG. 19 shows FvExo 5′ and 3′ RT-PCR products are cDNA-dependent.
- FIG. 20 shows Southern blots of F. venenatum genomic DNA hybridized with FvGlu, FvEndo, FvExo cDNAs.
- SEQ ID NO:1 is a F. venenatum glucanase full-length unmodified cDNA sequence.
- SEQ ID NO:2 is the glucanase encoded by SEQ ID NO:1.
- SEQ ID NO:3 is the genomic DNA segment containing the F. sporotrichioides glucanase gene.
- SEQ ID NO:4 is the glucanase sequence encoded by SEQ ID. NO:3.
- SEQ ID NO:5 is a glucanase encoded by a F. venenatum cDNA sequence.
- SEQ ID NO:6 is a F. venenatum endochitinase 5′ cDNA PCR product.
- SEQ ID NO:7 is the polypeptide encoded by SEQ ID NO:6.
- SEQ ID NO:8 is a F. sporotrichioides exochitinase 5′ genomic PCR product.
- SEQ ID NO:9 is a portion of the polypeptide sequence encoded by SEQ ID NO:8.
- SEQ ID NO:10 is a F. venenatum endochitinase unmodified full-length cDNA sequence.
- SEQ ID NO:11 is the endochitinase encoded by SEQ ID NO:10.
- SEQ ID NO:12 is a F. sporotrichioides - F. venenatum chimeric sequence.
- SEQ ID NO:13 is the exochitinase encoded by SEQ ID NO:12.
- SEQ ID NO:14 comprises amino acids 1 to 249 of an exochitinase encoded by a F. venenatum cDNA.
- SEQ ID NO:15 comprises amino acids 1 to 249 of a F. sporotrichioides genomic DNA.
- SEQ ID NO:16 is a F. venenatum glucanase modified full-length cDNA sequence.
- SEQ ID NO:17 is the glucanase encoded by SEQ ID NO:16.
- SEQ ID NO:18 is a F. venenatum endochitinase modified full-length cDNA sequence.
- SEQ ID NO:19 is the endochitinase encoded by SEQ ID NO:18.
- SEQ ID NO:20 is a F. venenatum exochitinase modified full-length cDNA sequence.
- SEQ ID NO:21 is the exochitinase encoded by SEQ ID NO:20.
- SEQ ID NOS:22-24 are the native Ubiquitin-1 and modified Ubiquitin-1 sequences shown in FIG. 5 (upper, middle, and lower, respectively).
- SEQ ID NO:25 is a leader sequence.
- SEQ ID NO:26 is primer M13F.
- SEQ ID NO:27 is primer M13R.
- SEQ ID NOS:28-74 are the primers shown in Table 2.
- SEQ ID NOS:75-82 are the PCR primers shown in Table 3.
- Glucanase refers to a protein or polypeptide with hydrolytic activity on glucan, a component of fungal cell walls.
- glucanase refers to a polypeptide having the enzymatic ability to degrade the ⁇ -1,3 linkages of glucan.
- glucanase activity is defined herein as hydrolytic activity which catalyzes the degradation of the ⁇ -1,3 linkages of glucan.
- Glucanase activity can be measured by adding a suitable substrate (e.g., glucan, laminarin) to a cellular extract, e.g., wheat endosperm tissue, leaf tissue, according to published methods. Assays are described in Keen and Yoshikawa (1983) and Fontaine et al. (1997).
- Chitinase refers to a protein or polypeptide with hydrolytic activity on chitin, a macromolecule composed primarily of N-acetyl-glucosamine and found in cell walls of fungal hyphae and spores.
- Endochitinase refers to a protein or polypeptide that enzymatically degrades chitin by cleaving randomly between C1 and C4 linkage with the chitin polymer.
- endochitinase activity is defined herein to mean the ability to degrade chitin by cleaving randomly between C1 and C4 linkage with the chitin polymer.
- Exochitinase refers to a protein or polypeptide that enzymatically degrades chitin by cleaving sequentially between each C1 and C4 linkages from the terminus of the chitin polymer.
- exochitinase activity is defined herein to mean ability to degrade chitin by cleaving sequentially between each C1 and C4 linkages from the terminus of the chitin polymer.
- Chitinase (endochitinase or exochitinase) activity can be measured by adding a suitable substrate to a cellular extract, e.g., wheat endosperm tissue, leaf tissue, according to published methods. Assays are described in Harman et al. (1993); McCreath and Gooday (1992); Tronsmo and Harman (1993), Bolar et al. (2000), and U.S. Pat. No. 5,378,821.
- transgenic when used in reference to a cell refers to a cell which contains a transgene, or whose genome has been altered by the introduction of a transgene.
- transgenic when used in reference to a tissue or to a plant refers to a tissue or plant, respectively, which comprises one or more cells that contain a transgene, or whose genome has been altered by the introduction of a transgene.
- Transgenic cells, tissues and plants may be produced by several methods including the introduction of a “transgene” comprising nucleic acid (usually DNA) into a target cell or integration of the transgene into a chromosome of a target cell by way of human intervention, such as by the methods described herein.
- transgene refers to any nucleic acid sequence which is introduced into the genome of a cell by experimental manipulations.
- a transgene may be a “native DNA sequence,” or a “heterologous DNA sequence” (i.e., “foreign DNA”).
- native DNA sequence refers to a nucleotide sequence which is naturally found in the cell into which it is introduced so long as it does not contain some modification (e.g., a point mutation, the presence of a selectable marker gene, etc.) relative to the naturally-occurring sequence.
- heterologous DNA sequence refers to a nucleotide sequence which is ligated to, or is manipulated to become ligated to, a nucleic acid sequence to which it is not ligated in nature, or to which it is ligated at a different location in nature.
- Heterologous DNA is not endogenous to the cell into which it is introduced, but has been obtained from another cell.
- Heterologous DNA also includes a native DNA sequence which contains some modification.
- heterologous DNA encodes RNA and proteins that are not normally produced by the cell into which it is expressed. Examples of heterologous DNA include reporter genes, transcriptional and translational regulatory sequences, selectable marker proteins (e.g., proteins which confer drug resistance), etc.
- transformation refers to the introduction of a transgene into a cell. Transformation of a cell may be stable or transient.
- stable transformation refers to the introduction and integration of one or more transgenes into the genome of a cell. Stable transformation of a cell may be detected by Southern blot hybridization of genomic DNA of the cell with nucleic acid sequences which are capable of binding to one or more of the transgenes. Stable transformation of a plant may also be detected by using the polymerase chain reaction to amplify transgene sequences from genomic DNA from cells of the progeny of that plant.
- stable transformant refers to a cell which has stably integrated one or more transgenes into the genomic DNA. Thus, a stable transformant is distinguished from a transient transformant in that, whereas genomic DNA from the stable transformant contains one or more transgenes, genomic DNA from the transient transformant does not contain a transgene.
- isolated when used in relation to a nucleic acid molecule, as in “an isolated nucleic acid sequence” refers to a nucleic acid sequence that is identified and separated from at least one contaminant nucleic acid with which it is ordinarily associated in its natural source. Isolated nucleic acid is nucleic acid present in a form or setting that is different from that in which it is found in nature. The isolated nucleic acid sequence may be present in single-stranded or double-stranded form.
- nucleic acid sequence When an isolated nucleic acid sequence is to be utilized to express a protein, the nucleic acid sequence will contain at a minimum at least a portion of the sense or coding strand (i.e., the nucleic acid sequence may be single-stranded). Alternatively, it may contain both the sense and anti-sense strands (i.e., the nucleic acid sequence may be double-stranded).
- the techniques used to isolate or clone a nucleic acid sequence encoding a polypeptide include isolation from genomic DNA, preparation from cDNA, or a combination thereof.
- the cloning of the nucleic acid sequences of the present invention from such genomic DNA can be effected, e.g., by using the well known polymerase chain reaction (PCR) or antibody screening of expression libraries to detect cloned DNA fragments with shared structural features. See, e.g., Innis et al., 1990, PCR: A Guide to Methods and Application, Academic Press, New York.
- nucleic acid amplification procedures such as ligase chain reaction (LCR), ligated activated transcription (LAT) and nucleic acid sequence-based amplification (NASBA) may be used.
- LCR ligase chain reaction
- LAT ligated activated transcription
- NASBA nucleic acid sequence-based amplification
- the nucleic acid sequence may be cloned from a strain of Fusarium, or another or related organism and thus, for example, may be an allelic or species variant of the polypeptide encoding region of the nucleic acid sequence.
- identity is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by a comparison of the sequences. In the art, “identity” also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as determined by the match between strings of such sequences. “Identity” can be readily calculated by known methods, including but not limited to those described in Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H.
- Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA (Altschul, S. F. et al., J. Molec. Biol. 215: 403-410 (1990).
- BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990) and Altschul et al., Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res.
- Percentage of sequence identity is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. It is preferred that the comparison window is at least 50% of the coding sequence, preferably 60%, more preferably 75% or 85%, and even more preferably 95% to 100%.
- hybridization includes “any process by which a strand of nucleic acid joins with a complementary strand through base pairing.” [Coombs J (1994) Dictionary of Biotechnology, Stockton Press, New York N.Y.]. Hybridization and the strength of hybridization (i. e., the strength of the association between the nucleic acids) is impacted by such factors as the degree of complementarity between the nucleic acids, stringency of the conditions involved, the T m of the formed hybrid, and the G:C ratio within the nucleic acids.
- hybridizes under stringent conditions refers to the formation of a double-stranded duplex from two single-stranded nucleic acids.
- the region of double-strandedness can include the full-length of one or both of the single-stranded nucleic acids, or all of one single stranded nucleic acid and a subsequence of the other single stranded nucleic acid, or the region of double-strandedness can include a subsequence of each nucleic acid.
- Nucleic acid probes to identify and clone DNA encoding polypeptides having the desired enzyme activity from strains of different genera or species can be prepared according to methods well known in the art. Such probes can be used for hybridization with the genomic or cDNA of the genus or species of interest, following standard Southern blotting procedures, in order to identify and isolate the corresponding gene therein. Both DNA and RNA probes can be used. The probes are typically labeled for detecting the corresponding gene (for example, with 32 P, 3 H, 35 S, biotin, or avidin).
- High stringency conditions when used in reference to nucleic acid hybridization comprise conditions equivalent to binding or hybridization at 68° C. in a solution consisting of 5 ⁇ SSPE, 1% SDS, 5 ⁇ Denhardt's reagent and 100 ⁇ g/ml denatured salmon sperm DNA followed by washing in a solution comprising 0.1 ⁇ SSPE, and 0.1% SDS at 68° C. when a probe of about 100 to about 1000 nucleotides in length is employed, or the above-mentioned conditions with 50% formamide at 42° C.
- High stringency washes can include 0.1 ⁇ SSC to 0.2 ⁇ SSC, 1% SDS, 65° C., 15-20 min.
- An example of stringent wash conditions for a Southern blot of such nucleic acids is a 0.2 ⁇ SSC wash at 65° C. for 15 minutes (see, Sambrook et al., Molecular Cloning—A Laboratory Manual (2 nd ed.) Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY, 1989, for a description of SSC buffer).
- Other exemplary high stringency hybridization conditions include, for example, 7% SDS, 0.25 M sodium phosphate buffer, pH 7.0-7.2, 0.25 M sodium chloride at 65° C.-68° C. or the above-mentioned conditions with 50% formamide at 42° C.
- Exemplary medium stringency conditions are as described above for high stringency except that 35% formamide at 42° C. is used, and the washes are carried out at 55° C.
- stringency conditions are defined as prehybridization, hybridization, and washing post-hybridization at about 5° C. to about 10° C. below the calculated T m using the calculation according to Bolton and McCarthy (1962, Proceedings of the National Academy of Sciences USA 48:1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA, 0.5% NP-40, 1 ⁇ Denhardt's solution, 1 mM sodium pyrophosphate, 1 mM sodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per ml following standard Southern blotting procedures.
- a genomic DNA or cDNA library prepared from other organisms may be screened for DNA which hybridizes with the probes described above and which encodes a polypeptide having the desired enzyme activity.
- Genomic or other DNA from such other organisms may be separated by agarose or polyacrylamide gel electrophoresis, or other separation techniques.
- DNA from the libraries or the separated DNA may be transferred to and immobilized on nitrocellulose or other suitable material.
- the material with immobilized DNA is used in a Southern blot.
- hybridization indicates that the nucleic acid sequence hybridizes to a labeled nucleic acid probe corresponding to the selected nucleic acid sequence, its complementary strand, or a subsequence thereof, under medium to high stringency conditions. Molecules to which the nucleic acid probe hybridizes under these conditions are detected using X-ray film.
- nucleic acid construct refers to a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or which has been modified to contain segments of nucleic acid which are combined and juxtaposed in a manner which would not otherwise exist in nature.
- the nucleic acid construct can include, for example, a coding sequence of the invention, and control sequences such as a promoter, and transcriptional and translation stop signals.
- control sequences such as a promoter, and transcriptional and translation stop signals.
- nucleic acid construct is synonymous with the term expression cassette when the nucleic acid construct contains all the control sequences required for expression of a coding sequence of the present invention.
- Exemplary constructs include plasmids and vectors, including cloning vectors, recombinant expression vectors.
- a “vector” is a nucleic acid composition which can transduce, transform or infect a cell and generally be replicated in the cell, thereby causing the cell to express vector-encoded nucleic acids and, optionally, proteins other than those native to the cell, or in a manner not native to the cell.
- a vector includes a nucleic acid (ordinarily RNA or DNA) to be expressed by the cell.
- a vector optionally includes materials to aid in achieving entry of the nucleic acid into the cell, such as a retroviral particle, liposome, protein coating or the like. Vectors contain nucleic acid sequences which allow their propagation and selection in bacteria or other non-plant organisms.
- sense orientation refers to the orientation of a cDNA sequence or coding sequence with respect to the promoter in a construct, such that the 5′ end of the cDNA sequence or coding sequence is adjoined to the promoter.
- antisense refers to the orientation of a nucleic acid sequence such as the cDNA or coding sequence with respect to the promoter in a construct, such that the 3′ end of the sequence is adjoined to the promoter.
- control sequences is defined to include all components which are necessary or advantageous for the expression of a polypeptide.
- control sequences include, but are not limited to, a leader, a polypeptide sequence, a promoter, a signal peptide sequence or a targeting sequence, an enhancer, and a transcription terminator.
- the control sequences include a promoter, and a transcriptional terminator sequence.
- the portion of a gene or nucleic acid construct containing the 5′ leader sequence generally 5 to 15 nucleotides in length immediately upstream of the ATG start codon, can also be considered a control sequence as it can affect the efficiency of translation.
- control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the nucleic acid sequence encoding a polypeptide.
- linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the nucleic acid sequence encoding a polypeptide.
- operably linked is defined here as a configuration in which a control sequence is placed at a position relative to the coding sequence of the nucleic acid sequence such that the control sequence directs the production of a messenger RNA and/or a polypeptide.
- control sequence may also be a suitable transcription terminator sequence, a sequence recognized by a host cell to terminate transcription.
- the terminator sequence is operably linked to the 3′ terminus of the nucleic acid sequence encoding the polypeptide.
- the control sequence may also be a suitable leader sequence, a nontranslated region of an mRNA which is important for translation by the host cell.
- the leader sequence is operably linked to the 5′ terminus of the nucleic acid sequence encoding the polypeptide.
- control sequence may also be a polyadenylation sequence, a sequence operably linked to the 3′ terminus of the nucleic acid sequence and which, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA.
- the control sequence may also be a signal peptide coding region that codes for an amino acid sequence linked to the amino terminus of a polypeptide and directs the encoded polypeptide into the cell's secretory pathway.
- the 5′ end of the coding sequence of the nucleic acid sequence may inherently contain a signal peptide coding region naturally linked in translation reading frame with the segment of the coding region which encodes the secreted polypeptide.
- the 5′ end of the coding sequence may contain a signal peptide coding region which is foreign to the coding sequence.
- the foreign signal peptide coding region may be required where the coding sequence does not naturally contain a signal peptide coding region.
- the foreign signal peptide coding region may simply replace the natural signal peptide coding region in order to enhance secretion of the polypeptide.
- Antisense technology comprises cloning a nucleic acid segment and operatively linking it to a promoter such that the antisense (or complementary) strand of RNA will be transcribed. The construct is then transformed into the host cell and the antisense strand of RNA is produced.
- expression vector refers to a vector comprising a nucleic acid construct and sequences for delivery into and autonomous replication in microbial host cells.
- the various nucleic acid and control sequences described above may be joined together to produce a recombinant expression vector which may include one or more convenient restriction sites to allow for insertion or substitution of the nucleic acid sequence encoding the polypeptide at such sites.
- the nucleic acid sequence of the present invention may be expressed by inserting the nucleic acid sequence or a nucleic acid construct comprising the sequence into an appropriate vector for expression.
- the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.
- Modification of a nucleic acid sequence encoding a polypeptide of the present invention may be necessary for the synthesis of polypeptides substantially similar to the polypeptide.
- the term “substantially similar” to the polypeptide refers to non-naturally occurring forms of the polypeptide.
- These polypeptides may differ in some engineered way from the polypeptide isolated from its native source, e.g., variants that differ in specific activity, therrnostability, pH optimum, subcellular localization or the like.
- the variant sequence may be constructed on the basis of the nucleic acid sequence presented as the polypeptide encoding part of an exemplified sequence, a subsequence thereof, and/or by introduction of nucleotide substitutions which do not give rise to another amino acid sequence of the polypeptide encoded by the nucleic acid sequence, but which correspond to the codon usage of the host organism intended for production of the enzyme, or by introduction of nucleotide substitutions which may give rise to a different amino acid sequence.
- nucleotide substitution see, e.g., Ford et al., 1991, Protein Expression and Purification 2: 95-107.
- amino acid residues essential to the activity of the polypeptide encoded by the isolated nucleic acid sequence of the invention may be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (see, e.g., Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, mutations are introduced at every positively charged residue in the molecule, and the resultant mutant molecules are tested for enzyme activity to identify amino acid residues that are critical to the activity of the molecule.
- Sites of substrate-enzyme interaction can also be determined by analysis of the three-dimensional structure as determined by such techniques as nuclear magnetic resonance analysis, crystallography or photoaffinity labelling (see, e.g., de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, Journal of Molecular Biology 224: 899-904; Wlodaver et al., 1992, FEBS Letters 309: 59-64).
- Plant host cells include but are not limited to, somatic cells, gametes or embryos.
- Embryo refers to a sporophytic plant before the start of germination. Embryos can be formed by fertilization of gametes by sexual crossing or by selfing.
- a “sexual cross” is pollination of one plant by another.
- Selfing is the production of seed by self-pollenization, i.e., pollen and ovule are from the same plant.
- breeding refers to crossing a F 1 hybrid plant to one of its parents. Typically, backcrossing is used to transfer genes which confer a simply inherited, highly heritable trait into an inbred line. The inbred line is termed the recurrent parent.
- the source of the desired trait is the donor parent. After the donor and the recurrent parents have been sexually crossed, F 1 hybrid plants which possess the desired trait of the donor parent are selected and repeatedly crossed (i.e., backcrossed) to the recurrent parent or inbred line.
- progeny refers to the descendants of a particular plant or regenerant (self-cross) or pair of plants (crossed or backcrossed).
- the descendants by self-fertilization can be of the T 1 , the T 2 , or any subsequent generation, and descendants by crossing can be of the F 1 , the F 2 , or any subsequent generation.
- the parents are the pollen donor and the ovule donor which are crossed to make the progeny plant of this invention.
- Parents also refer to F 1 parents of a hybrid plant of this invention (the F 2 plants).
- parents refer to a recurrent parent which is backcrossed to hybrid plants of this invention to produce another hybrid plant of this invention.
- plant parts are stem, callus, leaves, root, fruits, seeds, and tubers. Also specific plant tissues, such as chloroplast, apoplast, mitochondria, vacuole, peroxisomes, and cytoplasm are considered to be a plant part. Furthermore, any plant cell, whatever the tissue origin, is considered to be a plant part.
- Line pertains to a plant or its germplasm, a primary regenerant from transformation, (T o ) plant or its progeny, resulting from genetic transformation, in which all or a portion of transgene has been stably integrated.
- the term “monocot” refers to a plant species having a single cotyledon, including wheat, oat, barley, rice, rye, triticale, maize (corn), and other cereals, as well as sugarcane, sorghum, pineapple, yam, onion, banana, coconut, date, hops, and grasses such as meadow grass and forage grass.
- the common names of cereal crop plants used throughout this disclosure refer to varieties of plants of the following genera: Common Name Genera Wheat (soft, hard and durum varieties) Triticum Sorghum Sorghum Rice Oryza Barley Hordeum Maize or corn Zea Rye Secale Triticale Triticale Oat Avena
- plants susceptible to fungal or bacterial diseases are also encompassed, including, for example, grapes, coca beans, and nuts.
- EST refers to Expressed sequence tag; nucleotide sequence of a complete or partial cDNA, representing a transcript or messenger RNA that is expressed in an organ, tissue or cell type; usually 100 or more nucleotides in length.
- the present invention is directed to isolated nucleic acid sequences derived from Fusarium fungal genes which encode polypeptides having cell wall-degrading activity comprising glucanase, endochitinase or exochitinase activity as well as isolated polypeptides having cell wall-degrading activity.
- nucleic acid sequence having at least 70% nucleotide sequence identity with SEQ ID NO:1 from nucleotide 37 through nucleotide 942, SEQ ID NO:3 from nucleotide 1801 through nucleotide 2761 or SEQ ID NO:16 from nucleotide 18 to nucleotide 923;
- the isolated nucleic acid molecules which encode a polypeptide having glucanase activity include genomic sequences which encode a glucanase and which direct and regulate the transcriptional and translational expression of the glucanase coding sequences, and cDNA sequences which encode a polypeptide having glucanase activity.
- nucleotide sequences which encode polypeptides having glucanase activity are given in SEQ ID NOS:1, 3, and 16.
- An exemplified glucanase gene product has a predicted amino acid sequence as given in SEQ ID NOS:2, 4, 5, and 17.
- a genomic DNA sequence containing the full-length glucanase gene is presented in SEQ ID NO:3.
- the genomic DNA sequence is 3622 bp in length, and nucleotide sequence analysis reveals 2 exons and 1 intron.
- the coding region comprises nucleotide 1801 to 2014 and 2070 to 2761 which encodes a protein 301 amino acids in length (SEQ ID NO:4).
- the nucleic acid molecule is the sequence contained in plasmid GLU2 (SEQ ID NO:16 which is a F. venenatum glucanase modified full-length cDNA sequence) that is contained in Escherichia coli NRRL B-30201.
- the nucleic acid molecule is the sequence contained in plasmids FvGluS or FvGluAS that are contained in Escherichia coli NRRL B-30204 and NRRL B-30205, respectively.
- the invention also encompasses nucleic acid sequences which have a degree of sequence identity to the coding region of SEQ ID NO:1, 3 or 16 or coding region plus intron of SEQ ID NO:3 of at least 70%, preferably at least about 75%, preferably at least about 80%, more preferably at least about 85%, more preferably at least about 90%, and even more preferably about 95% and which encode a polypeptide effective to degrade ⁇ -1,3-linkages of glucan.
- the degree of identity between two nucleic acid sequences is determined by the Clustal method (Thompson et al.
- the invention further encompasses a complementary strand of a nucleic acid sequence or subsequence of at least 100 contiguous nucleotides and preferably at least 200 contiguous nucleotides of a nucleic acid sequence which hybridizes under medium stringency or high stringency conditions, as defined above, with the coding region of the DNA sequence of SEQ ID NO:1, 3 or 16 or coding region plus intron of SEQ ID NO:3.
- the invention is further directed to a subsequence of the aforenamed nucleic acid sequences wherein the subsequence encodes a polypeptide fragment which has glucanase activity.
- the present invention is also directed to isolated polypeptides having glucanase activity which are encoded by the nucleic acid molecules described above.
- isolated polypeptides having glucanase activity comprise:
- FIG. 1 is a comparison of full-length glucanases encoded by F. venenatum cDNA (upper) and F. sporotrichioides genomic DNA (lower) (SEQ ID NOS:5 and 4, respectively).
- Isolated nucleic acid sequences encoding polypeptides comprising an amino acid sequence which has a degree of identity to the amino acid sequence of SEQ ID NO:2, 4, 5 or 17 with amino acids 1 to 301 of at least about 80%, preferably at least 85%, more preferably at least about 90%, further more preferably at least about 95%, and most preferably at least about 97%, and which have glucanase activity (homologous polypeptides), are encompassed by the invention.
- a fragment of SEQ ID NO:2, 4, 5 or 17 is a polypeptide having one or more amino acids deleted from the amino and/or carboxyl terminus of this amino acid sequence.
- a fragment contains at least 256 amino acid residues, more preferably at least 271 amino acid residues, and most preferably at least 286 amino acid residues.
- the present invention relates to isolated polypeptides having glucanase activity which are encoded by nucleic acid sequences which hybridize under medium stringency conditions or high stringency conditions, as described in detail above, with (i) SEQ ID NO:1 from nucleotide 37 through nucleotide 942, SEQ ID NO:3 from nucleotide 1801 through nucleotide 2761 or SEQ ID NO:16 from nucleotide 18 to nucleotide 923; (ii) a subsequence of (i) of at least 100 nucleotides, or (iii) a complementary strand of (i) or (ii).
- the subsequence of SEQ ID NO:1, 3 or 16 may be at least 100 contiguous nucleotides or preferably at least 200 contiguous nucleotides. Moreover, the subsequence may encode a polypeptide fragment which has glucanase activity.
- Examples of conservative substitutions are within the groups of basic amino acids (such as arginine, lysine and histidine), acidic amino acids (such as glutamic acid and aspartic acid), polar amino acids (such as glutamine and asparagine), hydrophobic amino acids (such as leucine isoleucine and valine), aromatic amino acids (such as phenylalanine, tryptophan and tyrosine), and small amino acids (such as glycine, alanine, serine, threonine and methionine).
- Amino acid substitutions which do not generally alter the specific activity are known in the art as described, for example, by H. Neurath and R. L.
- polypeptides of the present invention have at least 20%, preferably at least 40%, more preferably at least 60%, even more preferably at least 80%, even more preferably at least 90%, and most preferably at least 100% of the glucanase activity of the polypeptide of SEQ ID NO:2, 4, 5 or 17.
- a polypeptide of the present invention may be obtained from microorganisms of any genus.
- the term “obtained from” as used herein in connection with a given source shall mean that the polypeptide encoded by the nucleic acid sequence is produced by the source or by a cell in which the nucleic acid sequence from the source has been inserted.
- a polypeptide of the present invention may be a fungal polypeptide, for example, Aspergillus, Fusarium, Magnaporthe, or Phytophthora.
- the polypeptide is a Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium helerosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, or Fusarium venenatum polypeptide.
- the Fusarium venenatum cell is Fusarium venenatum A3/5, which was originally deposited as Fusarium graminearum ATCC 20334 and recently reclassified as Fusarium venenatum by Yoder and Christianson, 1998, Fungal Genetics and Biology 23: 62-80 and O'Donnell et al., 1998, Fungal Genetics and Biology 23: 57-67; as well as taxonomic equivalents of Fusarium venenatum regardless of the species name by which they are currently known.
- the Fusarium venenatum cell is a morphological mutant of Fusarium venenatum A3/5 or Fusarium venenatum ATCC 20334, as disclosed in WO 97/26330.
- taxonomic equivalents of Fusarium are defined by D. L. Hawksworth, P. M. Kirk, B. C. Sutton, and D. N. Pegler (editors), 1995, In Ainsworth & Bisby's Dictionary of the Fungi, Eighth Edition, CAB International, University Press, Cambridge, England, pp. 173- 174.
- polypeptides may be identified and obtained from other sources including microorganisms isolated from nature (e.g., soil, composts, water, etc.) using the above-mentioned probes. Techniques for isolating microorganisms from natural habitats are well known in the art.
- the nucleic acid sequence may then be derived by similarly screening a genomic or cDNA library of another microorganism. Once a nucleic acid sequence encoding a polypeptide has been detected with the probe(s), the sequence may be isolated or cloned by utilizing techniques which are known to those of ordinary skill in the art (see, e.g., Sambrook et al., 1989, supra).
- an “isolated” polypeptide is a polypeptide which is essentially free of other non-glucanase polypeptides, e.g., at least about 20% pure, preferably at least about 40% pure, more preferably about 60% pure, even more preferably about 80% pure, most preferably about 90% pure, and even most preferably about 95% pure, as determined by SDS-PAGE.
- Polypeptides encoded by nucleic acid sequences of the present invention also include fused polypeptides or cleavable fusion polypeptides in which another polypeptide is fused at the N-terminus or the C-terminus of the polypeptide or fragment thereof.
- a fused polypeptide is produced by fusing a nucleic acid sequence (or a portion thereof) encoding another polypeptide to a nucleic acid sequence (or a portion thereof) of the present invention.
- Techniques for producing fusion polypeptides are known in the art, and include ligating the coding sequences encoding the polypeptides so that they are in frame and that expression of the fused polypeptide is under control of the same promoter(s) and terminator.
- Endochitinase refers to a polypeptide that degrades chitin by cleaving randomly between C1 and C4 linkages within the chitin polymer.
- the present invention is directed to isolated nucleic acid molecules which encode a polypeptide having endochitinase activity, selected from the group consisting of:
- nucleic acid sequence having at least 75% nucleotide sequence identity with SEQ ID NO:10 from nucleotide 186 through nucleotide 1385 or SEQ ID NO:18 from nucleotide 18 to nucleotide 1217;
- nucleic acid sequence which hybridizes under medium stringency or high stringency conditions with (i) SEQ ID NO:10 from nucleotide 186 through nucleotide 1385 or SEQ ID NO:18 from nucleotide 18 to nucleotide 1217; (ii) a subsequence of (i) of at least 100 contiguous nucleotides, or (iii) a complementary strand of (i) or (ii); and
- the fungal endochitinase nucleic acid molecules include genomic sequences which encode an endochitinase and which direct and regulate the transcriptional and translational expression of the endochitinase coding sequences, and cDNA sequences which encode an endochitinase.
- nucleotide sequences which encode a polypeptide having endochitinase activity are given in SEQ ID NOS :10 and 18.
- An exemplified endochitinase gene product has a predicted amino acid sequence as given in SEQ ID NOS:11 AND 19.
- a F. venenatum endochitinase full-length unmodified cDNA sequence is given in SEQ ID NO:10.
- the cDNA sequence is 1494 bp in length.
- the resulting open reading frame (coding portion), initiating at base 186 and terminating at base 1385 encodes a protein 399 amino acids in length (SEQ ID NO:11).
- a modified full length cDNA sequence which encodes a polypeptide having endochitinase activity is given in SEQ ID NO:18.
- This cDNA is 1227 bp in length.
- the open reading frame initiating at base 18 and terminating at base 1217 encodes a protein of 399 amino acids (SEQ ID NO:19).
- One modification made to the unmodified endochitinase sequence is the substitution of a CA-rich 5′ leader sequence, GGATCCACCAACCAGCG, (bases #1-#17 of ENDO 5′ primer, Table 2).
- the modified 5′ leader sequence occurs immediately upstream of the ATG start codon of the endochitinase coding sequence.
- the modification renders the leader sequence rich in C and A nucleotides, and minimizes the number of T nucleotides. These characteristics are reported to enhance the translational efficiency of genes in plants.
- the DNA sequence containing the 5′ portion of the endochitinase cDNA (FIG. 2), from which the remainder of the primer was designed, is presented in SEQ ID NO:6.
- the partial cDNA is 467 bp in length.
- the nucleic acid molecule is the sequence contained in plasmid Endo 167 (SEQ ID NO:18 which is a F. venenatum endochitinase modified full-length cDNA sequence) that is contained in Escherichia coli NRRL B-30202.
- the nucleic acid molecule is the sequence contained in plasmids FvEndoS or FvEndoAS that are contained in Escherichia coli NRRL B-30206 and NRRL B-30207, respectively.
- the invention also encompasses nucleic acid sequences which have a degree of sequence identity to the coding region of SEQ ID NO:I0 or 18 of at least 75%, preferably at least about 80%, more preferably at least about 85%, more preferably at least about 90%, and even more preferably about 95% and which encode a polypeptide having endochitinase activity, that is, the ability to degrade chitin by cleavage of internal glycosidic linkages.
- the degree of identity between two nucleic acid sequences is determined by the Clustal method (Thompson et al.
- nucleic acid sequences which hybridize under medium stringency or high stringency conditions, as defined above, with the coding region of the DNA sequence of SEQ ID NO:10 or 18 and which encode a polypeptide having endochitinase activity are encompassed by this invention.
- the invention further encompasses a complementary strand of a nucleic acid sequence or subsequence of at least 100 contiguous nucleotides and preferably at least 200 contiguous nucleotides of a nucleic acid sequence which hybridizes under medium stringency or high stringency conditions, as defined above, with the coding region of the DNA sequence of SEQ ID NO:10 or 18.
- the invention is further directed to a subsequence of the aforenamed nucleic acid sequences wherein the subsequence encodes a polypeptide fragment which has endochitinase activity.
- the present invention is also directed to isolated polypeptides having endochitinase activity which are encoded by the nucleic acid molecules described above.
- isolated polypeptides having endochitinase activity comprise:
- Isolated nucleic acid sequences encoding polypeptides comprising an amino acid sequence which has a degree of identity to the amino acid sequence of SEQ ID NO:11 or 19 of at least about 85%, preferably at least about 90%, more preferably at last about 95%, and most preferably at least about 97%, and which have endochitinase activity, (homologous polypeptides), are encompassed by the invention.
- the polypeptides of the present invention comprise an amino acid sequence of SEQ ID NO:11 or 19 or a fragment thereof that has endochitinase activity.
- the polypeptide of the present invention comprises amino acids 1 to 399 of SEQ ID NO:11 or 19 or a fragment thereof that has endochitinase activity.
- a fragment of SEQ ID NO:11 or 19 is a polypeptide having one or more amino acids deleted from the amino and/or carboxyl terminus of this amino acid sequence.
- a fragment contains at least 339 amino acid residues, more preferably at least 359 amino acid residues, and most preferably at least 379 amino acid residues.
- the present invention relates to isolated polypeptides having endochitinase activity which are encoded by nucleic acid sequences which hybridize under medium stringency conditions or high stringency conditions, as described in detail above, with (i) SEQ ID NO:10 from nucleotide 186 through nucleotide 1385 or SEQ ID NO:18 from nucleotide 18 to nucleotide 1217; (ii) a subsequence of (i) of at least 100 contiguous nucleotides, or (iii) a complementary strand of (i) or (ii).
- the subsequence of SEQ ID NO:10 or 18 may be at least 100 contiguous nucleotides or preferably at least 200 contiguous nucleotides. Moreover, the subsequence may encode a polypeptide fragment which has endochitinase activity. Synonymous coding sequences and conservative amino acid substitutions, as described in detail, above, are incorporated herein by reference.
- polypeptides of the present invention have at least 20%, preferably at least 40%, more preferably at least 60%, even more preferably at least 80%, even more preferably at least 90%, and most preferably at least 100% of the endochitinase activity of the polypeptide of SEQ ID NO:11 or 19.
- a polypeptide of the present invention may be obtained from microorganisms of any genus as described in detail above and which is incorporated herein by reference.
- an “isolated” polypeptide is a polypeptide which is essentially free of other non-endochitinase polypeptides, e.g., at least about 20% pure, preferably at least about 40% pure, more preferably about 60% pure, even more preferably about 80% pure, most preferably about 90% pure, and even most preferably about 95% pure, as determined by SDS-PAGE.
- Polypeptides encoded by nucleic acid sequences of the present invention also include fused polypeptides or cleavable fusion polypeptides as described in detail above and which is incorporated herein by reference.
- Exochitinase refers to a polypeptide having the ability to degrade chitin by cleaving sequentially between each C1-C4 linkage initiating from the terminus of the chitin polymer.
- the present invention is directed to isolated nucleic acid molecules which encode a polypeptide having endochitinase activity, selected from the group consisting of:
- nucleic acid sequence having at least 75% nucleotide sequence identity with SEQ ID NO:12 from nucleotide 108 to 1853 or SEQ ID NO:20 from nucleotide 18 to 1763;
- nucleic acid sequence which hybridizes under medium stringency or high stringency conditions with (i) SEQ ID NO:12 from nucleotide 108 to 1853 or SEQ ID NO:20 from nucleotide 18 to 1763; (ii) a subsequence of (i) of at least 100 contiguous nucleotides, or (iii) a complementary strand of (i) or (ii); and
- the isolated nucleic acid molecules which encode a polypeptide having exochitinase activity include genomic sequences which encode an exochitinase and which direct and regulate the transcriptional and translational expression of the exochitinase coding sequences, and cDNA sequences which encode an exochitinase.
- nucleotide sequences which encode exochitinase are given in SEQ ID NOS:8, 12 and 20.
- An exemplified exochitinase gene product has a predicted amino acid sequence as given in SEQ ID NOS:13 and 21.
- a Fusarium exochitinase full-length unmodified cDNA sequence is given in SEQ ID NO:12.
- the cDNA sequence is 1949 bp in length.
- the resulting open reading frame (coding portion), initiating at base 108 and terminating at base 1853 encodes a protein 581 amino acids in length.
- the encoded protein is described in SEQ ID NO:13.
- venenatum modified full length cDNA sequence which encodes exochitinase is given in SEQ ID NO:20.
- This cDNA is 1781 bp in length.
- the open reading frame, initiating at base 18 and terminating at base 1763 encodes a protein of 581 amino acids (SEQ ID NO:21).
- One modification made to the unmodified exochitinase sequence is the substitution of a CA-rich 5′ leader sequence, GGATCCACCAACCAGCG, (bases #1-#17 of EXO 5′ primer, Table 2).
- the modified 5′ leader sequence occurs immediately upstream of the ATG start codon of the exochitinase coding sequence.
- the modification renders the leader sequence rich in C and A nucleotides, and minimizes the number of T nucleotides.
- the genomic DNA sequence containing a 5′ portion of the exochitinase gene of F. sporotrichioides is presented in SEQ ID NO:8 (FIG. 3).
- the deduced polypeptide sequence is given in SEQ ID NO:9.
- the genomic DNA sequence is 995 bp in length, and nucleotide sequence analysis reveals 2 exons and 1 intron.
- the nucleic acid molecule is the sequence contained in plasmid Exo9 (SEQ ID NO:20 which is a F. venenatum exochitinase modified full-length cDNA sequence) that is contained in Escherichia coli NRRL B-30203.
- the nucleic acid molecule is the sequence contained in plasmids FvExoS or FvExoAS that are contained in Escherichia coli NRRL B-30208 and NRRL B-30209, respectively.
- the invention also encompasses nucleic acid sequences which have a degree of sequence identity to the coding region of SEQ ID NO:12 or 20 of at least 75%, preferably at least about 80%, more preferably at least about 85%, more preferably about 90%, and even more preferably about 95% and which encode a polypeptide having exochitinase activity, that is, ability to degrade chitin by cleavage of terminal glycosidic linkages.
- the degree of identity between two nucleic acid sequences is determined by the Clustal method (Thompson et al.
- nucleic acid sequences which hybridize under medium stringency or high stringency conditions, as defined above, with the coding region of the DNA sequence of SEQ ID NO:12 or 20 and which encode a polypeptide having exochitinase activity are encompassed by this invention.
- the invention further encompasses a complementary strand of a nucleic acid sequence or subsequence of at least 100 contiguous nucleotides and preferably at least 200 contiguous nucleotides of a nucleic acid sequence which hybridizes under medium stringency or high stringency conditions, as defined above, with the coding region of the DNA sequence of SEQ ID NO:12 or 20.
- the invention is further directed to a subsequence of the aforenamed nucleic acid sequences wherein the subsequence encodes a polypeptide fragment which has exochitinase activity.
- the present invention is also directed to isolated polypeptides having exochitinase activity which are encoded by the nucleic acid molecules described above.
- Isolated polypeptides having exochitinase activity comprise:
- FIG. 4 shows the comparison of exochitinases encoded by F. venenatum cDNA (upper) and F. sporotrichioides genomic DNA (lower) (SEQ ID NOS:14 and 15, respectively).
- Isolated nucleic acid sequences encoding polypeptides comprising an amino acid sequence which has a degree of identity to the amino acid sequence of SEQ ID NO:13 or 21 of at least about 85%, preferably at least about 90%, more preferably at last about 95%, and most preferably at least about 97%, and which have exochitinase activity, (homologous polypeptides), are encompassed by the invention.
- polypeptides of the present invention comprise an amino acid sequence of SEQ ID NO:13 or 21 or a fragment thereof that has exochitinase activity.
- polypeptide of the present invention comprises amino acids 1 to 581 of SEQ ID NO:13 or 21 or a fragment thereof that has exochitinase activity.
- a fragment of SEQ ID NO:13 or 21 is a polypeptide having one or more amino acids deleted from the amino and/or carboxyl terminus of this amino acid sequence.
- a fragment contains at least 494 amino acid residues, more preferably at least 523 amino acid residues, and most preferably at least 552 amino acid residues.
- the present invention relates to isolated polypeptides having exochitinase activity which are encoded by nucleic acid sequences which hybridize under medium stringency conditions or high stringency conditions, as described in detail above, with (i) SEQ ID NO:12 from nucleotide 108 to 1853 or SEQ ID NO:20 from nucleotide 18 to 1763; (ii) a subsequence of (i) of at least 100 contiguous nucleotides, or (iii) a complementary strand of (i) or (ii).
- the subsequence of SEQ ID NO:12 or 20 may be at least 100 contiguous nucleotides or preferably at least 200 contiguous nucleotides. Moreover, the subsequence may encode a polypeptide fragment which has exochitinase activity. Synonymous coding sequences and conservative amino acid substitutions, as described in detail, above, are incorporated herein by reference.
- a polypeptide of the present invention may be obtained from microorganisms of any genus as described in detail above and which is incorporated herein by reference.
- an “isolated” polypeptide is a polypeptide which is essentially free of other non-exochitinase polypeptides, e.g., at least about 20% pure, preferably at least about 40% pure, more preferably about 60% pure, even more preferably about 80% pure, most preferably about 90% pure, and even most preferably about 95% pure, as determined by SDS-PAGE.
- Polypeptides encoded by nucleic acid sequences of the present invention also include fused polypeptides or cleavable fusion polypeptides as described in detail above and which is incorporated herein by reference.
- Any isolated cell wall-degrading nucleic acid molecule encoding a polypeptide having glucanase, endochitinase or exochitinase activity can be used in the present invention.
- the particular polynucleotide and amino acid sequences used are not critical features of the invention, so long as the desired cell wall-degrading function is achieved.
- the F. venenatum endochitinase and exochitinase are similar to chitinases from other fungi.
- the endochitinase shares 56% amino acid sequence identity with an Aspergillus nidulans chitinase (GenBank accession no. D87063), 53% identity with chitinase antigens from Coccidioides immitis (Yang et al. 1996; Zimmerman et al.
- the F. venenatum exochitinase had 66% and 58% amino acid sequence identity with two exochitinases from Trichoderma harzianum (Draborg et al. 1995), and 64% identity with a N-acetyl- ⁇ -D-glucosaminidase from T. harzianum (Peterbauer et al. 1996).
- nucleic acid probes based on the disclosed sequences can be used to isolate the desired gene from a cDNA or genomic DNA library, as described in detail, above.
- Nucleic acid probes can be either DNA or RNA.
- the desired gene can be isolated by hybridization of the probes to target sequences, including genomic library clones, cDNA library clones, or uncloned isolated DNA or cDNA fragments. Hybridization can be carried out under stringent, highly stringent, or low-stringency conditions either on membranes or in solution.
- Nucleic acid probes can be used as restriction fragment length polymorphic (RFLP) markers for the genetic mapping of loci, especially in fungi, or for the identification of homologous genes or loci in fungi.
- RFLP restriction fragment length polymorphic
- Primers based on the disclosed sequences can be used to isolate the desired genes from a cDNA or genomic library.
- the nucleic acids of interest can be amplified from nucleic acid samples using amplification techniques. For instance, polymerase chain reaction (PCR) technology can be used to amplify the sequences of the Fusarium cell wall-degrading enzymes and related genes directly from genomic DNA, from cDNA, from genomic libraries or cDNA libraries.
- PCR and other in vitro amplification methods may also be useful, for example, to clone nucleic acid sequences that code for proteins to be expressed, to make nucleic acids to use as probes for detecting the presence of the desired DNA or mRNA in samples, for nucleic acid sequencing, or for other purposes.
- PCR PROTOCOLS A GUIDE TO METHODS AND APPLICATIONS, Innis, et al. (eds.), Academic Press, San Diego (1990).
- nucleic acid molecules which contain isolated cell wall-degrading sequences and are suitable for transformation of host cells are prepared.
- a nucleic acid sequence coding for the desired polypeptide for example, a cDNA or a genomic sequence encoding a full length protein or, a nucleic acid sequence encoding a homologous polypeptide is conveniently used to construct a recombinant expression cassette which can be introduced into the desired host cell.
- An expression cassette will typically comprise the Fusarium cell wall-degrading nucleic acid sequence operably linked to a one or more control sequences which direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences, and may include other transcriptional and translational initiation regulatory sequences which will direct the transcription of the sequence from the sense or antisense gene in the intended tissues of the transformed host cell.
- Expression will be understood to include any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.
- An isolated nucleic acid sequence encoding a polypeptide of the present invention may be manipulated in a variety of ways to provide for expression of the polypeptide. Manipulation of the nucleic acid sequence prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying nucleic acid sequences utilizing recombinant DNA methods are well known in the art.
- control sequences include all components which are necessary or advantageous for the expression of a polypeptide of the present invention.
- control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator sequence, as described in detail above.
- Promoter sequence refers to a DNA sequence which when ligated to a nucleotide sequence of interest is capable of controlling the transcription of the nucleotide sequence of interest into mRNA. This is discussed in detail, above.
- Promoters may be constitutive.
- the term “constitutive” when made in reference to a promoter means that the promoter is capable of directing transcription of an operably linked nucleic acid sequence under most environmental and developmental conditions in the absence of a stimulus (e.g., heat shock, chemicals, light, etc.), and/or in several or many tissues, cell types, or organs.
- Promoters may be tissue specific or cell specific.
- tissue specific refers to a promoter that is capable of directing selective expression of a nucleotide sequence of interest to a specific type of tissue (e.g., petals) in the relative absence of expression of the same nucleotide sequence of interest in a different type of tissue (e.g., roots).
- Tissue specificity of a promoter may be evaluated by, for example, operably linking a reporter gene to the promoter sequence to generate a reporter construct, introducing the reporter construct into the genome of a plant such that the reporter construct is integrated into every tissue of the resulting transgenic plant, and detecting the expression of the reporter gene (e.g., detecting mRNA, protein, or the activity of a protein encoded by the reporter gene) in different tissues of the transgenic plant.
- the reporter gene e.g., detecting mRNA, protein, or the activity of a protein encoded by the reporter gene
- a constitutive plant promoter fragment may be employed which will direct expression of the Fusarium cell wall-degrading enzyme in some to many tissues of a plant.
- Such promoters are active under most environmental conditions and states of development or cell differentiation.
- Examples of constitutive promoters include the cauliflower mosaic virus (CaMV) 35S transcription initiation region, the 1′- or 2′- promoter derived from T-DNA of Agrobacterium tumefaciens, and other transcription initiation regions from various plant genes known to those of skill.
- Promoters for constitutive expression of anti-Fusarium genes in cereals and the tissues in which they have been shown to be active are the following: maize ubiquitin-1, young leaf, root, pollen, seed (cereals) (Christensen and Quail, 1996); maize Adh-1, embryo shoots, roots, anther, pollen, seed (rice) (Freeling and Bennett, 1985); rice ACT-1, shoot leaf, root, floral parts, including pollen (rice) (Zhang et al., 1991); CaMV 35S, leaf, root (rice) (Battraw et al.); ScBv, stem, palea, lemma, other floret organs, anther (oat) (Tzafrir et al.),1998.
- Organ-specific promoters may be, for example, a promoter from storage sink tissues such as seeds, potato tubers, and fruits (Edwards & Coruzzi, 1990, Ann. Rev. Genet. 24:275-303), or from metabolic sink tissues such as meristems (Ito et al., 1994, Plant Mol. Biol. 24: 863-878), a seed specific promoter such as the glutelin, prolamin, globulin, or albumin promoter from rice (Wu et al., 1998, Plant and Cell Physiology 39:885-889), the storage protein napA promoter from Brassica napus, or any other seed specific promoter known in the art.
- a promoter from storage sink tissues such as seeds, potato tubers, and fruits (Edwards & Coruzzi, 1990, Ann. Rev. Genet. 24:275-303), or from metabolic sink tissues such as meristems (Ito et al., 1994, Plant Mol. Biol. 24: 863-878)
- the promoter may be a leaf specific promoter such as the rbcS promoter from rice or tomato (Kyozuka et al., 1993, Plant Physiology 102:991 -1000, or the aldP gene promoter from rice (Kagaya et al., 1995, Molecular and General Genetics 248: 668-674), or a wound inducible promoter such as the potato pin2 promoter (Xu et al., 1993, Plant Molecular Biology 22:573-588).
- a leaf specific promoter such as the rbcS promoter from rice or tomato (Kyozuka et al., 1993, Plant Physiology 102:991 -1000, or the aldP gene promoter from rice (Kagaya et al., 1995, Molecular and General Genetics 248: 668-674), or a wound inducible promoter such as the potato pin2 promoter (Xu et al., 1993, Plant Molecular Biology 22:573-588).
- the plant promoter may be under environmental or developmental control. Such promoters are referred to here as “inducible” promoters. Examples of environmental conditions that may effect transcription by inducible promoters include pathogen attack, anaerobic conditions, or the presence of light.
- a promoter enhancer element may also be used to achieve higher expression of the gene in the plant.
- the promoter enhancer element may be an intron which is placed between the promoter and the nucleotide sequence encoding a polypeptide of the present invention.
- the first intron of the rice actin gene discloses the use of the first intron of the rice actin gene to enhance expression.
- regulatory sequences or promoters that are particularly suitable for expression of genes in monocots are used.
- the promoter of the maize Polyubiquitin-1 gene (Ubi-1, Christensen and Quail 1996) for regulating the expression of the glucanase and chitinase genes in wheat.
- the Ubi-1 promoter is active in a number of cereal species. It is termed a “constitutive” promoter because it functions in many different organs and tissues, including callus, young leaves, endosperm (A. Blechl, unpub.), mature pollen, and the floret organs of wheat (P. Okubara and A.
- Ubi-1 promoter is envisioned to extend the application of the constructs to pathogens that attack other organs of wheat and to pathogens that attack other cereal crops, such as rice, maize, oat and barley.
- the promoters can also be used to initiate transcription of mRNA molecules to inhibit expression of the gene.
- Means for inhibiting gene expression in host cells using recombinant DNA techniques are well known.
- antisense Technology can be conveniently used. See, e.g., Sheehy et al., Proc. Nat. Acad. Sci. USA, 85:8805-8809 (1988), and Hiatt et al., U.S. Pat. No. 4,801,340.
- Introduction of nucleic acid configured in the sense orientation has also been shown to be an effective means by which to block the transcription of target genes.
- the control sequence may also be a suitable leader sequence, a nontranslated region of an mRNA which is important for translation by the host cell.
- the leader sequence is operably linked to the 5′ terminus of the nucleic acid sequence encoding the polypeptide. Any leader sequence that is functional in the host cell of choice may be used in the present invention.
- control sequence may also be a polyadenylation sequence, a sequence operably linked to the 3′ terminus of the nucleic acid sequence and which, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence which is functional in the host cell of choice may be used in the present invention.
- the control sequence may also be a signal peptide coding region that codes for an amino acid sequence linked to the amino terminus of a polypeptide and directs the encoded polypeptide into the cell's secretory pathway.
- the 5′ end of the coding sequence of the nucleic acid sequence may inherently contain a signal peptide coding region naturally linked in translation reading frame with the segment of the coding region which encodes the secreted polypeptide.
- the 5′ end of the coding sequence may contain a signal peptide coding region which is foreign to the coding sequence.
- the foreign signal peptide coding region may be required where the coding sequence does not naturally contain a signal peptide coding region.
- the foreign signal peptide coding region may simply replace the natural signal peptide coding region in order to enhance secretion of the polypeptide.
- any signal peptide coding region which directs the expressed polypeptide into the secretory pathway of a host cell of choice may be used in the present invention.
- the vector comprising the sequences from the cell wall-degrading genes may comprise a marker gene which confers a selectable phenotype on plant cells.
- a marker gene may be co-transformed on a separate vector molecule.
- the marker may encode biocide resistance, particularly antibiotic resistance, such as resistance to kanamycin, carbenicillin, ampicillin or herbicide resistance, such as resistance to chlorosulfuron, or phosphinothricin (the active ingredient in bialaphos and Basta).
- glucanase and chitinase genes we constructed a vector derived from pAHC20 (Christensen and Quail 1996), in which the bar selectable marker gene is regulated by the maize Ubi-1 promoter (0.9 kb, including the first exon and first intron located in the 5′ untranslated region) and NOS 3′ terminator.
- the bar gene conferring resistance to the herbicide bialaphos (de Block et al. 1987), is successfully and routinely used in our laboratory as a selectable marker for transgenic wheat plants.
- pUBK engineered by Dr.
- Dr. McCue replaced the origin of replication and gene for ampicillin resistance (bla) of pAHC20 (Christensen and Quail 1996) with the corresponding portion of pBGS9 (Spratt et al. 1986), which encodes kanamycin resistance (nptII).
- pUBK is publicly available from Dr. McCue.
- Another factor in making recombinant constructs of transgenes is the efficiency of translation of the transgene mRNA.
- Computerized analyses of gene sequences and in vitro experiments indicate that the efficiency of translation initiation is mediated by three nucleotides ( ⁇ 3 to ⁇ 1) immediately preceding the ATG start (initiation) codon.
- the ⁇ 3 to ⁇ 1 start codon context consensus for eukaryotic genes was reported to be ACC (Kozak 1987); a survey of plant genes yielded a consensus of ACA (Fütterer and Hohn 1996); the start codon context for 85 maize genes was preferentially GGC or AAG (Luehrsen and Walbot 1994).
- the 15-20 nucleotides upstream of ATG, including ⁇ 3 to ⁇ 1, are AC-rich in many monocot genes. Enhancements of translational efficiency of heterologous genes, e.g., fungal genes in monocots have been engineered.
- the present invention also relates to recombinant expression vectors comprising a nucleic acid sequence of the present invention, a promoter, and transcriptional and translational stop signals.
- the various nucleic acid and control sequences described above may be joined together to produce a recombinant expression vector which may include one or more convenient restriction sites to allow for insertion or substitution of the nucleic acid sequence encoding the polypeptide at such sites.
- the nucleic acid sequence of the present invention may be expressed by inserting the nucleic acid sequence or a nucleic acid construct comprising the sequence into an appropriate vector for expression.
- the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.
- the recombinant expression vector may be any vector (e.g., a plasmid or virus) which can be conveniently subjected to recombinant DNA procedures and can bring about the expression of the nucleic acid sequence.
- the choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced.
- the vectors may be linear or closed circular plasmids.
- the vector may be an autonomously replicating vector, i.e., a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome.
- the vector may contain any means for assuring self-replication.
- the vector may be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated.
- a single vector or plasmid or two or more vectors or plasmids which together contain the total DNA to be introduced into the genome of the host cell, or a transposon may be used.
- the vector may also contain sequences that stabilize the integration and expression of the nucleic acid construct in a host cell. These include DNA sequences carrying matrix attachment regions also known as scaffold attachment regions, for example, for yeast or plant, e.g., Arabidopsis or wheat, sources.
- the vector may also contain one or more selectable markers which permit easy selection a transformed cells.
- the vector may also contain an element(s) that permits integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome.
- the vector may rely on the nucleic acid sequence encoding the polypeptide or any other element of the vector for integration of the vector into the genome by homologous or nonhomologous recombination.
- the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question.
- More than one copy of a nucleic acid sequence of the present invention may be inserted into the host cell to increase production of the gene product.
- An increase in the copy number of the nucleic acid sequence can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the nucleic acid sequence where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the nucleic acid sequence, can be selected for by cultivating the cells in the presence of the appropriate selectable agent.
- the present invention also relates to recombinant host cells, comprising a nucleic acid sequence of the invention, which are advantageously used in the recombinant production of the polypeptides.
- recombinant host cells comprising a nucleic acid sequence of the invention, which are advantageously used in the recombinant production of the polypeptides.
- Preparation of transformed host cells and cloning methods are described by U.S. Pat. No. 5,374,540, which is incorporated herein by reference.
- the host cell may be a unicellular microorganism, e.g., a prokaryote, or a non-unicellular microorganism, e.g., a eukaryote.
- Eukaryote cells can include any cell such as from an insect, fimgus or plant.
- plant includes whole plants, plant parts or organs, plant tissue, as described in detail, above.
- the present invention also relates to methods for producing a polypeptide comprising cultivating a host cell under conditions suitable for production of the polypeptide and recovering the polypeptide.
- the cells are cultivated in nutrient medium suitable for production of the polypeptide using methods know in the art.
- the polypeptides may be detected and recovered using methods known in the art.
- the present invention also relates to methods for producing a polypeptide of the present invention comprising (a) cultivating a transgenic plant or a plant cell comprising a nucleic acid sequence encoding a polypeptide having enzyme activity of the present invention under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide.
- polypeptides may be detected using methods known in the art that are specific for the polypeptides. These detection methods may include use of specific antibodies, formation of an enzyme product, or disappearance of an enzyme substrate. For example, an enzyme assay may be used to determine the activity of the polypeptide as described herein.
- the resulting polypeptide may be recovered by methods known in the art.
- the polypeptide may be recovered from the nutrient medium by conventional procedures including, but not limited to, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation.
- polypeptides of the present invention may be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g., Protein Purification, J.-C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989).
- chromatography e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion
- electrophoretic procedures e.g., preparative isoelectric focusing
- differential solubility e.g., ammonium sulfate precipitation
- SDS-PAGE or extraction
- the transgenic plant or plant cell expressing an RNA transcript or polypeptide of the present invention may be constructed in accordance with methods known in the art.
- the plant or plant cell is constructed by incorporating one or more expression constructs encoding a polypeptide of the present invention into the plant host genome and propagating the resulting modified plant or plant cell into a transgenic plant or plant cell.
- the transgenic plants of this invention are cereal crop plants, including but not limited to, wheat, rye, triticale, barley, maize, sorghum and rice.
- the transgenic plants are wheat, maize, barley, oats, and rye.
- the transgenic plants of this invention are dicotyledonous plants.
- DNA constructs described above may be introduced into the genome of the desired plant host by a variety of conventional techniques. Techniques for transforming a wide variety of higher plant species are well known and described in the technical and scientific literature. See, for example, Weising, et al., Ann. Rev. Genet. 22:421-477 (1988).
- the DNA construct may be introduced directly into the genomic DNA of the plant cell using techniques such as biolistic methods, electroporation, PEG poration, and microinjection of plant cell protoplasts or embryogenic callus.
- the DNA constructs may be combined with suitable T-DNA flanking regions and introduced using an Agrobacterium tumefaciens or A. rhizogenes vector.
- biolistic bombardment refers to the process of accelerating particles towards a target biological sample (e.g., cell, tissue, etc.) to effect wounding of the cell membrane of a cell in the target biological sample and/or entry of the particles into the target biological sample.
- a target biological sample e.g., cell, tissue, etc.
- Methods for biolistic bombardment are known in the art (e.g., U.S. Pat. No. 5,584,807, the contents of which are herein incorporated by reference), and are commercially available (e.g., the helium gas-driven microprojectile accelerator (PDS-1000/He) (BioRad).
- Particle bombardment techniques are described in Klein, et al., Nature 327:70-73 (1987).
- a particularly preferred method of transforming wheat and other cereals is the bombardment of calli derived from immature embryos as described by Weeks, et al., Plant Physiol. 102:1077-1084 (1993).
- Microinjection techniques are known in the art and well described in the scientific and patent literature.
- the introduction of DNA constructs using polyethylene glycol precipitation is described in Paszkowski, et al., EMBO J. 3:2717-2722 (1984).
- Electroporation techniques are described in Fromm, et al., Proc. Nat'l Acad. Sci. USA 82:5824 (1985).
- Agrobacterium tumefaciens -meditated transformation techniques are also well described in the scientific literature. See, for example Horsch, et al., Science 233:496-498 (1984), and Fraley, et al. Proc. Nat'l Acad. Sci. USA 80:4803 (1983). Although Agrobacterium is useful primarily in dicots, certain monocots can be transformed by Agrobacterium. For instance, Agrobacterium transformation of rice is described by Hiei, et al, Plant J. 6:271-282 (1994); U.S. Pat. No. 5,187, 073; U.S. Pat. No.
- the present invention is particularly useful in wheat and other cereals.
- a number of methods of transforming cereals have been described in the literature. For instance, reliable methods for stable transformation of wheat, including the highly-regenerable cultivars such as the hard white spring wheat Bobwhite, are described (Vasil, et al., 1992, 1993; Weeks, et al., 1993; Becker et al., 1994; Nehra et al. 1994, Blechl and Anderson, 1996).
- U.S. Pat. Nos. 5,650,558 and 5,914,450 to Blechl et al. describe transformation of wheat and non-wheat cereal plants, which patents are incorporated herein by reference. Chen et al.
- Transgenic maize regenerants have been described by Fromm, et al., Bio/Technology 8:833-839 (1990) and Gordon-Kamm, et al., Plant Cell 2:603-618 (1990)). Similarly, oats (Sommers, et al., Bio/Technology 10:1589-1594 (1992)), sorghum (Casas, et al., Proc. Nat'l Acad. Sci. USA 90:11212-11216 (1993)), rice (Li, et al., Plant Cell Rep. 12:250-255 (1993)), barley (Yuechun & Lemaux, Plant Physiol.
- the present invention further relates to plants, seeds, plant tissues, plant organs, and plant cells transiently expressing the claimed sequences.
- Transient expression refers to the generation of mRNA and/or protein from the claimed DNA sequences in plants without stable integration of the plasmid into host genomes. Such expression might be observed after 1 to 14 days following introduction of DNA by particle gun bombardment, Agrobacterium, and other means used for plant transformation.
- Transformed plant cells that are derived by any of the above transformation techniques can be cultured to regenerate a whole plant which possesses the transformed genotype and thus the desired phenotype.
- Such regeneration techniques rely on manipulation of certain phytohormones in a tissue culture growth medium, typically relying on a biocide and/or herbicide marker which has been introduced together with the cell wall-degrading polynucleotide sequences. Plant regeneration from cultured protoplasts is described in Evans, et al., PROTOPLASTS ISOLATION AND CULTURE, HANDBOOK OF PLANT CELL CULTURE, Macmillian Publishing Company, New York, pp.
- Regeneration can also be obtained from plant callus, explants, organs, or parts thereof. Such regeneration techniques are described generally in Klee, et al. Ann. Rev. of Plant Phys. 38:467-486 (1987).
- Transformed plants are evaluated for the presence of the desired properties and/or the extent to which the desired properties are expressed.
- a first evaluation may include the level of expression of the newly introduced genes, the level of fungal resistance of the transformed plants, and stable heritability of the desired properties.
- the expression cassette is stably incorporated in transgenic plants and confirmed to be operable, it can be introduced into other plants by sexual crossing.
- a technique used to transfer a desired phenotype to a breeding population of plants is through backcrossing.
- any of a number of standard breeding techniques can be used, depending upon the species to be crossed.
- Any plant species or variety that is subject to fungal attack may be transformed with one or more genetic constructs according to the invention in order to improve resistance to Fusarium or other pathogens, for example, to improve resistance to Fusarium head blight and other fungal diseases, including by (a) generation of cell wall-degrading enzymes, including proteins having the capability of degrading the glucan and chitin cell wall components of F. venenatum and other Fusarium species, including F. graminearum and F.
- cell wall-degrading enzymes including proteins having the capability of degrading the glucan and chitin cell wall components of F. venenatum and other Fusarium species, including F. graminearum and F.
- culmorum the principle causal agents of head blight (scab) in the U.S.
- scab head blight
- expression of the cell wall-degrading enzymes in transgenic monocots, including wheat, barley or oats, to confer partial or complete resistance to Fusarium species and to other fungal pathogens of wheat and other cereal crops (c) limiting the spread of the pathogenic fungus Fusarium and curtailing the accumulation of DON in infected heads, and (d) expression of the cell wall-degrading enzymes in dicots to confer resistance to plant pathogens.
- Analysis of transgenic lines may be done by northern blots, in situ hybridization, RT-PCR, S1 nuclease protection assays, RNase protection assays, Western blots, enzyme assays or other methods for detection of expressed mRNA or protein.
- a particular use of the invention is the provision of plants or plant cells transformed with a DNA sequence encoding a glucanase, endochitinase or exochitinase coding sequence to provide plants having resistance to plant pathogens, particularly, Fusarium species.
- Another use of the invention is as probes and primers capable of detecting a glucanase, endochitinase or exochitinase gene or functional equivalents thereof in fungi of the genus Fusarium and the use of the probes to isolate DNA sequences encoding a glucanase, endochitinase or exochitinase gene or functional equivalent thereof.
- nucleic acid sequences of the invention facilitates the isolation of homologous genes from hosts to obtain genes which protect host cells, including fungi and plants against related fungal pathogens.
- sequences are in the antisense orientation as a gene knock-out.
- Another use is to generate cell-wall degrading chitinases and glucanases for use in degrading seafood waste, such as shells that contain chitin, or for use for chemical modification of chitin or glucan.
- F. venenatum and F. sporotrichioides produce mycotoxins similar to that made by the head blight pathogen.
- Cell-wall degrading enzymes from a Fusarium species are required by the fungus for normal growth and development.
- glucanase cDNA clones The nucleotide sequences of two glucanase cDNA clones, an endochitinase cDNA clone, and 3 exochitinase cDNA clones from a collection of F. venenatum ESTs were examined. Both glucanase cDNA clones were determined to be full-length, whereas all the chitinase clones were partial cDNA clones that were missing the 5′ ends, including the ATG translational start codons. To restore the missing sequences, we performed a polymerase chain reaction (PCR) method known as 5′ anchored PCR on a genomic library of F. sporotrichioides.
- PCR polymerase chain reaction
- the PCR primers added a 5′ BamHI restriction site and a 3′ BglII restriction site to the termini of the amplified portions of the cDNAs. These sites were useful for cloning the cDNAs into our monocot expression vector.
- the monocot expression vector carried a promoter from the maize Polyubiquitin-1 (Ubi-1) gene, the bar gene for resistance to the herbicide bialaphos, a 3′ terminator segment from the nopaline synthase (nos) gene of Agrobacterium tumefaciens, and the nptll marker gene for resistance to the antibiotic kanamycin.
- a BglII restriction enzyme site located in the Ubi-1 promoter was destroyed by site-directed mutagenesis. This allowed the removal of the bar gene from pUBKBglII- with restriction enzymes BglII and BamHI, and substitution of the F. venenatum cDNAs. Both sense and antisense orientations of the three cDNAs (6 total) were obtained, as determined from nucleotide sequences of the promoter-transgene and transgene-terminator junctions.
- Embryos of Triticum aestivum cultivar Bobwhite were bombarded simultaneously with plasmids carrying the sense orientations of the cDNAs plus a selector marker plasmid carrying the bar gene. Calli and regenerated plants were cultured using methods developed in the laboratory. Candidate transgenic plants were selected on the basis of growth in the presence of the herbicide bialaphos (conferred by the bar gene). DNA from leaf sections of candidate tranformants were further analyzed by PCR with a primer designed from the maize Ubi-1 promoter sequence and a second primer specific for each of the transgene (glucanase, endochitinase, and exochitinase) sequences.
- transgene DNA Those plants carrying transgene DNA are being propagated for production of seed that are homozygous for the transgenes. Homozygous lines are tested for expression of the transgenes and tested for resistance to F. graminearum and other fungal pathogens.
- Fusarium venenatum CC1-3 a morphological mutant of Fusarium strain ATCC 20334 (Wiebe et al., 1991, Mycol. Research 95: 1284-1288), was grown in a two-liter lab-scale fermentor using a fed-batch fermentation scheme with NUTRIOSETM (Roquette Freres, S. A., Beinheim, France) as the carbon source and yeast extract. Ammonium phosphate was provided in the feed. The pH was maintained at 6 to 6.5, and the temperature was kept at 30° C. with positive dissolved oxygen.
- Double-stranded cDNA was synthesized using approximately 5 ⁇ g of poly(A)+ mRNA according to the method of Gubler and Hoffman (1983, Gene 25: 263-269) except a NotI-(dT) 18 primer (Pharmacia Biotech, Inc., Piscataway, N.J.) was used to initiate first strand synthesis.
- the cDNA was treated with mung bean nuclease (Boehringer Mannheim Corporation, Indianapolis, Ind.) and the ends were made blunt with T4 DNA polymerase (New England Biolabs, Beverly, Mass.).
- the cDNA was digested with NotI, size selected by agarose gel electrophoresis (ca. 0.7-4.5 kb), and ligated with pZErO-2.1 (Invitrogen Corporation, Carlsbad, Calif.) which had been cleaved with NotI plus EcoRV and dephosphorylated with calf-intestine alkaline phosphatase (Boehringer Mannheim Corporation, Indianapolis, Ind.).
- the ligation mixture was used to transform competent E. coli TOP 10 cells (Invitrogen Corporation, Carlsbad, Calif.). Transformants were selected on 2YT agar plates (Miller, 1992, A Short Course in Bacterial Genetics. A Laboratory Manual and Handbook for Escherichia coli and Related Bacteria, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.) which contained kanamycin at a final concentration of 50 ⁇ g/ml.
- Library A (4 day cells) consisted about 7.5 ⁇ 10 4 independent clones and Library B (6 day cells) consisted of roughly 1.2 ⁇ 10 5 clones.
- the primary microtiter plates were stored frozen at ⁇ 80° C.
- the secondary deep-dish plates were incubated at 37° C. overnight with vigorous agitation (300 rpm) on rotary shaker.
- each secondary culture plate was covered with a polypropylene pad (Advanced Genetic Technologies Corporation, Gaithersburg, Md.) and a plastic microtiter dish cover.
- Nucleotide sequence data were scrutinized for quality, and samples giving improper spacing or ambiguity levels exceeding 2% were discarded or re-run. Vector sequences were removed with assistance of FACTURATM software (Perkin-Elmer Applied Biosystems, Inc., Foster City, Calif.). In addition, sequences were truncated at the end of each sample when the number of ambiguous base calls increased. All sequences were compared to each other to determine multiplicity using AutoAssemblerTM software (Perkin-Elmer Applied Biosystems, Inc., Foster City, Calif.).
- NRDB non-redundant data base
- GeneAssistTM software Perkin-Elmer Applied Biosystems, Inc., Foster City, Calif.
- Smith-Waterman algorithm using the BLOSUM 62 matrix with a threshold score of 70.
- the NRDB was assembled from Genpept, Swiss-Prot, and PIR databases.
- EST clones encoding putative chitinolytic enzymes were identified by partial sequencing of random cDNA clones using an Applied Biosystems Model 377 XL Automated DNA Sequencer according to the manufacturer's instructions and comparison of the deduced amino acid sequence of each EST to the amino acid sequences in a NRDS made from publicly available protein and nucleic acid databases (e.g., GENBANK, EMBL, TREMBL, GENPEPT, SWISSPROT, PIR).
- Electroporation was performed on 50 ⁇ L aliquots of cells (3.5 ⁇ 10 7 / ⁇ g) using the Bio-Rad Gene Pulser (Bio-Rad, Hercules, Calif.), in 0.2 cm gap cuvettes at 2500 Volts. Following electroporation, the cells were suspended in 1 mL 2% (w:v) Bacto tryptone, 0.5% (w:v) yeast extract, 0.05% (w:v) sodium chloride, 20 mM glucose, 10 mM magnesium chloride, and 2.5 mM potassium chloride.
- the cells were maintained at 37° C. for 60 minutes with agitation at 200-300 rpm. Aliquots of 50 to 200 ⁇ L of cells were spread onto agar plates of Luria broth (LB) containing 50 ⁇ g/mL kanamycin sulfate. After culture at 37° C. for 16-20 hr, bacterial colonies showing good growth were transferred to a fresh LB-kanamycin plate. From this stock plate, additional cultures of individual colonies were grown for plasmid DNA isolation.
- LB Luria broth
- Plasmid DNA was isolated according to the protocol described in the Qiagen Plasmid Midi Kit (Qiagen, Inc., Valencia, Calif.). Plasmid DNA was isolated from 1.5 mL cultures using the QIAprep 8 kit and QIAvac Manifold-6S (Qiagen, Inc., Valencia, Calif.).
- EST Preliminary nucleotide sequence
- EST data were analyzed for the presence of restriction enzyme recognition sites using the Lasergene software programs EditSeq and MapDraw (DNASTAR, Inc, Madison, Wis.). Restriction enzymes that cleaved in one or more of the cDNA coding sequences or in the polylinker region of pZErO-2 were used. Plasmid DNAs ( ⁇ 0.5-1 ⁇ g), prepared as described in Example 7, were incubated with Apal, BamHI, EcoRI or Kpn I in a total volume of 15 ⁇ L as prescribed by the enzyme manufacturers.
- the cleavage products were partitioned on 1% agarose in 40 mM TRIS acetate, pH 8.2, 1 mM EDTA, stained with an ethidium bromide solution ( ⁇ 1 ⁇ g/mL), and visualized by irradiation with an ultraviolet light source (UVT 400-M transilluminator, IBI Kodak, Rochester, N.Y.).
- UVT 400-M transilluminator IBI Kodak, Rochester, N.Y.
- glucanase cDNA clone One glucanase cDNA clone, the endochitinase cDNA clone, and the longest exochitinase cDNA clone were selected for more rigorous, double strand nucleotide sequence determinations.
- Each cDNA was sequenced by a modified method of Sanger et al. (1977), using 250-500 ng of plasmid DNA in 2 ⁇ L, 8 ⁇ L of BigDye Terminator Mix and 1 ⁇ L of primer (4 uM) in a total volume of 20 ⁇ L, according to the ABI Prism BigDyeTM Terminator Cycle Sequencing Ready Reaction Kit (Perkin Elmer Applied Biosystems, Foster City, Calif.).
- the PCR protocol (25 cycles of 96° C., 30 sec, 50° C. for 15 sec, 60° C. for 4 min) was performed in a PTC-100 Programmable Thermal Controller (MJ Research, Watertown, Mass.). Fluorescently-labeled PCR products were enriched from unincorporated fluorescent dideoxynucleotides by passage through a column containing 50 mg (dry weight) of Sephadex G-50 Fine, DNA grade, (Amersham Pharmacia, Biotech AB, Uppsala, Sweden) imbibed in water. Sequence data of these clones and all other DNA templates were obtained with the ABI Prism 310 Genetic Analyzer.
- Nucleotide sequences of the 5′ and 3′ ends of the cDNA inserts in pZErO-2 were obtained with primers M13F (5′ GTAAAACGACGGCCAG) and M13R (5′ AGCGAATAACAATTTCACACAGGA). Additional sequences were obtained by primer walking. Primers synthesized for this purpose, shown in Table 2, were 902A and 902B for the glucanase cDNA; 958A-D for the endochitinase clone; and 1082 A-D for the exochitinase clone. The glucanase cDNA was 1023 bp.
- the full length unmodified glucanase cDNA is shown in SEQ ID NO:1.
- the unmodified partial cDNA endochitinase was 1290 bp
- the unmodified partial exochitinase cDNA was 1390 bp.
- venenatum endochitinase partial cDNA had 70-80% amino acid sequence identity to a chitinase antigen from Coccidioides immitis [Accession Number U33265, Yang et al., Infect. Immun., 64:1992-1997 (1996)]. Segments of the exochitinase partial cDNA sequence shared about 80% amino acid sequence identity with an exochitinase gene from Trichoderma harzianum [Accession Number S80069, Draborg et al., Biochem. Mol. Biol. Int. 36:781-791 (1995)].
- Glucanase The nucleotide sequence of a F. sporotrichioides genomic fragment encoding a glucanase was obtained as described in Example 9 from plasmid clone pFSC22-2. Primers used for sequencing were: 1014, 1015, 1016, 1017, 1018, 1021, 1023, 1038, 1040, 1042, Glucgene-1 and Glucgene-2 (Table 2). The nucleotide sequence was proofread and assembled as in Example 9.
- Endochitinase The 5′ end of the endochitinase cDNA (FIG. 2) was obtained by PCR amplification of 0.5 ⁇ g of the F. venenatum cDNA library using primers 1010 (from the endochitinase EST) and 720 (from the pZERO-2 sequence for M13 reverse primer). PCR conditions for the 1010/720 reaction were 94° C., 30 sec (1 min. first cycle); 52° C., 25 sec; 72° C., 60 sec, for 25 cycles. The PCR product was purified after partitioning on an agarose gel.
- Nucleotide sequence data of the endochitinase PCR product (5′ end) was obtained using primers 410, corresponding to the SP6 promoter of the cloning vector pZERO-2 (Invitrogen), and 1011, corresponding to the endochitinase cDNA.
- PCR conditions for the 1011/410 cycle sequencing reactions were 94° C., 30 sec (1 min. first cycle); 52° C., 25 sec; 72° C., 50 sec, for 25 cycles. Note: the reaction for 1011/410 took advantage of internal primers to reduce non-specific amplification of the initial PCR reaction.
- Exochitinase A genomic library of Fusarium sporotrichioides was constructed in phage Lambda ⁇ gt11 as described in Huynh et al. 1984. The 5′ portion of the exochitinase gene (FIG. 3) was amplified from the F. sporotrichioides library (0.5 ⁇ g of DNA in a 20 ⁇ L reaction) with primers 129, corresponding to sequence located upstream of the EcoRI site of the ⁇ gt11 cloning vector (Huynh et al. 1984), and 1003, corresponding to the exochitinase cDNA. PCR conditions for the 129/1003 reaction were 94° C., 30 sec (1 min.
- PCR product was purified from an agarose gel. Nucleotide sequence data of the PCR product (5′ portion of the exochitinase gene) was obtained with primers 128, corresponding to sequence located upstream of the EcoRI site of ⁇ gt11, and 1009, corresponding to the exochitinase cDNA. PCR conditions for the 128/1009 cycle sequencing reactions were 94° C., 30 sec (1 min. first cycle); 52° C., 25 sec; 72° C., 50 sec, for 25 cycles. Note: the second PCR reaction (128/1009) took advantage of internal primers to reduce background from the initial PCR reaction.
- a 930 bp, full-length glucanase cDNA with an AC-rich 5′ untranslated region was obtained by PCR amplification.
- the PCR amplification reaction consisted of: 50 ng of glucanase plasmid DNA cleaved with EcoRI, 0.4 uM GLUC 3′ and GLUC 5′ primers (Table 2), 0.2 uM of each deoxyribonucleotide (dATP, dCTP, dGTP, TTP), and 1 Unit of Elongase (Gibco BRL, Rockville, Md.) in a total volume of 50 ⁇ L buffer (60 mM TRIS sulfate, pH 9.1, 18 mM ammonium sulfate, 1 mM magnesium sulfate).
- PCR was carried out for 5 cycles at 92° C. for 30 sec, 57° C. for 30 sec, 68° C. for 60 sec followed by 25 cycles at 92° C. for 30 sec, 62° C. for 30 sec, 68° C. for 60 sec.
- Full-length cDNAs of the endochitinase and exochitinase were obtained by PCR amplification of genomic DNA fragments using ENDO 3′ plus ENDO 5′ primers or EXO 3′ plus EXO 5′ primers, respectively (Table 2).
- the final step in generating the full-length endochitinase cDNA was accomplished in a 100 ⁇ L reaction using 32 pmol each of primers ENDO 5′ and ENDO 3′. Amplification was performed with approximately 0.5 ⁇ g of F. venenatum cDNA library “A” and Pfu polymerase (Stratagene, La Jolla, Calif.) in Pfu buffer according to the manufacturer's protocol.
- Conditions for amplification were: 94° C., 30 sec (1 minute for the first cycle); 56° C., 25 sec; 72° C., 150 sec, for 25 cycles.
- the PE 2400 thermocycler Perkin Elmer was used.
- the fragments were cloned into bacterial and monocot expression vectors as described in Examples 6 and 17, respectively.
- PCR amplification of the exochitinase full-length cDNA was accomplished in a 100 ⁇ L reaction using 32 pmol each of primers EXO 5′ and EXO 3′.
- Amplification of F. venenatum cDNA library “A” (approx. 0.5 ⁇ g) was performed with Pfu (Stratagene, La Jolla, Calif.) in Pfu buffer according to the manufacturers' protocol, using a PE 2400 thermocycler (Perkin Elmer). PCR conditions were 94° C., 30 sec (1 min first cycle); 56° C. 25 sec; 72° C., 150 sec, for 25 cycles.
- the resulting PCR product was gel-purified and cloned into bacterial and monocot expression vectors, as described in Examples 6 and 18.
- cDNA fragments were separated from unincorporated primers and from other PCR products by partitioning on agarose gels (Example 8).
- PCR products of the desired size (930 bp for glucanase, 1240 bp for endochitinase, 1780 bp for exochitinase) were excised from gels with a clean razor blades, then recovered from the gel segments by adsorption onto silica beads (GENECLEAN Spin Kit, BIO 101, Inc., Vista, Calif.) as recommended.
- silica beads GEMLEAN Spin Kit, BIO 101, Inc., Vista, Calif.
- pUBK plasmid DNA 50 ng was suspended in a reaction mixture of: 125 ng primer UBI 1E (Table 2), 125 ng primer UBI 1F (Table 2), 10 mM of each deoxynucleotide, and 2.5 Units of Pfu polymerase in a total volume of 50 ⁇ L 20 mM TRIS chloride, pH 8.0, 10 mM potassium chloride, 6 mM ammonium sulfate, 2 mM magnesium sulfate, 0.1% Triton X-100, and 10 ⁇ g/mL bovine serum albumin.
- PCR was performed with a DNA Thermal Cycler (Perkin Elmer Cetus, currently Foster City, Calif.) and an amplification protocol of 16 cycles of 96° C. for 30 sec, 55° C. for 60 sec, 68° C. for 13 min.
- the resulting amplified products were treated with 10 Units of Dpn I restriction enzyme for 1 hour at 37° C.
- Competent E. coli XL 1-Blue host cells were incubated on ice with 1 ⁇ L of the Dpn-treated PCR mix for 30 minutes, immediately transferred to a water bath at 42° C. for 45 sec, then placed on ice for 2 minutes.
- Cells were suspended in 0.5 mL of liquid medium containing 1% (w:v) NZ amine, 0.5% (w:v) yeast extract, 0.5% (w:v) sodium chloride, 20 mM glucose, 12.5 mM magnesium chloride, and 12.5 mM magnesium sulfate. Cells were incubated at 37° C. for 1 hour with agitation at 225 rpm. Cells were concentrated by centrifugation at 4° C., 1700 ⁇ g for 3 minutes, then spread onto an LB agar plate containing 50 /g/mL kanamycin sulfate.
- the mutagenized plasmid was further treated with BamHI+BglII, PstI and PvuII restriction enzymes as recommended by the manufacturer, and the cleavage products visualized on an agarose gel as described in Example 8.
- the mutagenized plasmid lacked about 0.7 kb of DNA that was present in pUBK prior to site-directed mutagenesis.
- a 2.0 kb portion of the mutagenized plasmid was excised with HindIII and BamHI restriction enzymes, then ligated to pUBK from which the native HindIII-BamHI fragment had been removed.
- the ligation reaction contained approximately 50 ng of 2.0 kb fragment, 50 ng of pUBK vector, 0.5 Units of T4 DNA ligase in 10 ⁇ L of 50 mM TRIS chloride, pH 7.8, 10 mM magnesium chloride, 10 mM dithiothreitol, 2 mM ATP, and 50 ⁇ g/mL bovine serum albumin. The reaction was incubated at 15° C. for 17 hours. Electrocompetent E. coli NM522 cells were subjected to electroporation in the presence of 1 ⁇ L of the ligation mixture and further treated as described in Example 6.
- the floret organs were immediately immersed in a substrate solution containing 10 mM sodium phosphate buffer, pH 7, 0.5 mM potassium ferrocyanide, 0.5 mM potassium ferricyanide, 0.5% (w:v) Triton X-100, and 330 ⁇ g/mL 5-bromo-4-chloro-3-indoyl-beta-D-glucuronide (Jersey Lab and Glove Supply, Livingston, N.J.). After 16-20 hours of incubation in darkness, the substrate solution was withdrawn. The plant materials were rinsed twice with 0.1 M sodium phosphate buffer, pH 7.
- Chlorophyll and other pigments were removed from the green tissues by successive immersions in 70%, 80% and 90% ethanol solutions, 20-24 hours per solution. Treatments with 90% ethanol were repeated until the tissues appeared translucent and nearly white. Activity of the GUS gene was evident as a dark blue pigment within the developing seed and pollen of wheat, indicating that the modified Ubi-1 promoter was active in these organs. GUS activity was also detected in the chlorophyll-containing cells and tissues of the glume, lemma and palea. No GUS activity was seen in the brush or in the anther.
- PCR products representing full-length cDNA fragments of the glucanase, endochitinase, and exochitinase, with 15 to 16 bases of AC-rich 5′ untranslated sequence were mobilized into either pCR2.1 (Invitrogen, Carlsbad, Calif.; for glucanase) or Bluescript vector pBKS+(Stratagene, La Jolla, Calif.; for endochitinase and exochitinase). Ligations were carried out as described in Example 13 with approximately 50-100 ng of bacterial vector DNA that was previously treated with SmaI restriction enzyme, and 60 ng of the PCR product. Transformations of E.
- coli NM522 host cells for glucanase
- DH5-alpha host cells for endochitinase and exochitinase
- Nylon disks with colonies were placed on a sheet of 3MM (Whatman) paper that was saturated with 0.5 N sodium hydroxide, 1.5 M sodium chloride for 5 minutes, blotted briefly on clean paper, then transferred to paper saturated with 0.5 M TRIS-CL, pH 7.0, 3 M sodium chloride for 5 minutes.
- the colony disks were submerged in 2 ⁇ SSC (0.3 M sodium chloride, 0.03 M sodium citrate, pH 7.0) for 10 minutes with agitation at 60-70 rpm to loosen bacterial debris, rinsed in fresh 2 ⁇ SSC and dried in air.
- the colony disks were incubated in glass tubes in a Hybaid hybridization oven (National Labnet, Woodbridge, N.J.) at 65° C.
- Digoxygenin-labeled hybridization probes were made with ⁇ 100 ng of the endochitinase and exochitinase PCR products and 4 ⁇ L of DIG HighPrime mix in a final volume of 20 ⁇ L (Boehringer Mannheim), incubated at 37° C. for 16 to 20 hours. Unincorporated label was removed by precipitation of the probe DNA in 0.5 M lithium chloride and 2.5 volumes of 95% or 100% ethanol at ⁇ 80° C. for 20 minutes. Probe DNA was resuspended in water for use in hybridization solutions.
- Hybridized filters were washed in the hybridization tubes as follows: two washes of 50 mL 2 ⁇ SSC, 0.1% SDS at room temperature for 5 minutes and two washes of 50 mL 0.5 ⁇ SSC, 0.1% SDS, 65° C. for 25 minutes.
- Candidate glucanase cDNA clones were treated with BamHI+BglII, Eco RI or EcoRV; candidate endochitinase cDNA clones were treated with BamHI, Bgl II, and BamHI+BglII; candidate exochitinase cDNA clones were treated with BamHI, Bgl II, BamHI+BglII, Clal, and XhoI.
- the restriction enzyme digestion patterns indicated that all three cDNAs had been cloned in both orientations within the bacterial plasmid vectors, and that a BamHI and a BglII site had been added to each cDNA.
- nucleotide sequences of both strands of each cDNA were obtained by automated fluorescent sequencing as described in Example 9 (see SEQ ID NOS. 16, 18, and 20). Double-stranded sequences of the cDNAs were analyzed for the presence of restriction sites as described in Part 3, and for matches to entries in the GenBank database with BLASTX.
- PCR-modified cDNAs having nucleotide sequences identical to the original cDNAs were introduced into a monocot expression vector, pUBKBglII-, for use in wheat transformation experiments.
- the monocot expression plasmid vector was digested extensively with BamHI and BglII restriction enzymes.
- the pUBKBglII- plasmid vector DNA was purified by partitioning on agarose as described in Example 8.
- the modified glucanase cDNA was excised from pCR2.1 by partial digestion with BamHI restriction enzyme as follows: In a 1.5 mL conical bottom tube, 25 ⁇ g of glucanase plasmid DNA was suspended in 125 ⁇ L of 150 mM sodium chloride, 10 mM TRIS-Cl, pH 7.9, 1 mM magnesium chloride, 1 mM dithiothreitol, and 100 ⁇ g/mL bovine serum albumin. Thirty microliter aliquots of the mixture were dispensed to two other tubes. To the mix remaining in the first tube, 10 Units of BamHI enzyme was added and thoroughly mixed.
- Plasmids that showed slower migration in the agarose gel were further treated with BamHI+BglII or BamHI restriction enzymes. Insertion of the modified glucanase cDNA in the sense orientation with respect to the Ubi-1 promoter gave BamHI fragments of 0.55 kb in size, and were distinguishable from the antisense orientation of the cDNA, which resulted in a 0.39 kb BamHI fragment.
- the BamHI site at the Ubi-1-cDNA junction is followed by 47 bp of the pCR2.1 polylinker region (with an EcoRI site), then the BglII site that occurs at the 3′ end of the modified glucanase cDNA (FIG. 7).
- the antisense version contains a chimeric non-functional BamHI-BglII site at the cDNA-NOS terminator junction.
- the modified full-length endochitinase cDNA was obtained from a clone in which the 5′ end of the coding sequence was proximal to the T7 promoter site of pBKS+. About 4 ⁇ g of plasmid DNA was cleaved extensively with 30 Units of BamHI and 30 Units of BglII restriction enzymes in 50 ⁇ L of 150 mM sodium chloride, 10 mM TRIS-Cl, pH 7.9, 1 mM magnesium chloride, 1 mM dithiothreitol, and 100 ⁇ g/mL bovine serum albumin. The cDNA fragment was recovered from the agarose gel by partitioning on silica beads as described in Example 12.
- the junctions of the Ubi-1 promoter-endochitinase cDNA fusion of several clones were sequenced with primer UBI 1A (see Table 2); the endochitinase cDNA-NOS terminator fusion was sequenced with primer NOS A (see Table 2).
- Representative sense and antisense orientations of the cDNA in pUBKBglII- are shown in FIGS. 8 and 9, respectively.
- the sense clone of the endochitinase contains a BamHI restriction site at the Ubi-1-coding sequence junction and a BglII restriction site at the cDNA-NOS junction. BamHI and BglII sites are absent in the endochitinase antisense clone.
- an exochitinase clone was selected in which the 5′ end of the exochitinase coding sequence was proximal to the T3 primer site in pBKS+.
- About 3 ⁇ g of plasmid DNA was treated with 20 Units of BamHI in a final volume of 30 ⁇ L 150 mM sodium chloride, 10 mM TRIS-CL, pH 7.9, 1 mM magnesium chloride,1 mM dithiothreitol, and 100 ⁇ g/mL bovine serum albumin at 37° C. for 90 minutes.
- a DNA fragment of about 2.0 kb was purified from an agarose gel as described in Example 8.
- the ligation mixture contained about 60 ng of the cDNA fragment and 220 ng of pUBKBglII- DNA (see Example 16).
- Transformants of E. coli DH5-alpha were generated and grown as in Example 15. Plasmid DNA that migrated slower than the pUBKBglII- vector was subjected to nucleotide sequence analysis (Example 9) using UBI 1A and NOS A primers to determine the orientation of the coding sequence within pUBKBglII-. Additional exochitinase clones were identified using the colony blot procedure (see Example 15).
- FIGS. 10 and 11 Representative sequences for the sense and antisense orientations of the exochitinase cDNA in pUBKBglII- are shown in FIGS. 10 and 11, respectively.
- the sense clone of the exochitinase contains a BamHI restriction site at the Ubi-1-coding sequence junction and a BglII restriction site at the cDNA-NOS junction. These BamHI and BglII sites are absent in the exochitinase antisense clone.
- Embryos were placed scutellum-side up onto 15 mm ⁇ 100 mm plates containing 30 mL of MMS medium [4.3 g/L Murashige & Skoog salt mixture (Gibco BRL, Rockville, Md.), 0.5 mg/L thiamine hydrochloride, 0.15 g/L L-asparagine, 40 g/L maltose] solidified with 3.5 g/L Phytagel (Sigma Chemical Co., St. Louis, Mo.) and supplemented with 2 mg/L 2,4-D. The embryos were incubated at 25° C.
- MMS medium [4.3 g/L Murashige & Skoog salt mixture (Gibco BRL, Rockville, Md.), 0.5 mg/L thiamine hydrochloride, 0.15 g/L L-asparagine, 40 g/L maltose] solidified with 3.5 g/L Phytagel (Sigma Chemical Co., St. Louis
- embryogenic calli were transferred to “MMS2” medium (MMS media supplemented with 2 mg/L 2,4 D and solidified with 2.5 g/L Phytagel).
- MMS2 MMS media supplemented with 2 mg/L 2,4 D and solidified with 2.5 g/L Phytagel.
- the calli were placed at a density of 20 (FvGluS) or 40 (FvEndoS and FvExoS) per plate and incubated at 25° C. in darkness for two weeks.
- the calli were then selected for two 2-week periods by incubation in darkness at 25° C.
- Green shoots that formed during this time were transferred to 25 ⁇ 150 mm test tubes containing 18 mls of rooting medium [2.15 g/L Murashige & Skoog salt mixture, 0.25 mg/L thiamine hydrochloride, 0.075 g/L L-asparagine, 20 g/L maltose, 2.5 g/L Phytagel].
- the tubes were incubated in the light at 26° C.
- Shoots that formed roots were transferred to soil (Sunshine mix#1) and incubated in the light at 22° C. under saran wrap for one week. Humidity was lowered by poking an increasing number of holes in the saran wrap during a second week of incubation.
- the plants were then transplanted to the greenhouse maintained at 23° C. with supplemental lights set for 16/8-hour day/night cycle. Immature embryos about 21 dpa were excised from the seeds of these plants and precociously germinated in magenta boxes containing 100 mls of MMS medium solidified with 2.5 g/L phytagel.
- Genomic DNA or total DNA was extracted from wheat leaves to analyze candidate lines for stable incorporation of the modified, full-length glucanase, endochitinase and exochitinase cDNA constructs. Untransformed plants of the cultivar Bobwhite were sampled as negative controls.
- Genomic DNA was prepared by a modified method of d'Ovidio et al. (1992). Three to five grams fresh weight of healthy leaf tissue was excised from the plant, placed on ice immediately, then in liquid nitrogen, or placed directly in liquid nitrogen. Leaf samples were often stored at ⁇ 80° C. prior to extraction of genomic DNA. Leaf tissue was ground with a chilled mortar and pestle in the presence of liquid nitrogen.
- Powdered tissue was transferred to 35-40 mL of chilled homogenization buffer containing 0.5 M sucrose, 80 mM potassium chloride, 10 mM TRIS-chloride, 10 mM EDTA, 4 mM spermine, 1 mM spermidine, pH 9.5, 180 mg/L phenylmethylsulfonyl fluoride (added immediately before use), and 0.1% (v:v) beta-mercaptoethanol (added immediately before use).
- the leaf material was further homogenized with three to four 5-second pulses in a chilled blender. The homogenate was filtered through four layers of cheesecloth, followed by one layer of Miracloth filtration material (Calbiochem, La Jolla, Calif.).
- the clarified extract was centrifuged at 1000 ⁇ g for 20 minutes at 4° C. The supernatant was carefully decanted and the pellet containing nuclei washed twice in 20 to 40 mL homogenization buffer supplemented with 0.5% Triton X-100 (Sigma Chemical Co., St. Louis, Mo.). The washed nuclei pellet was resuspended in 1 mL of a buffer solution containing 50 mM TRIS-chloride, pH 8, 100 mM EDTA, 100 mM sodium chloride, and 600 ⁇ g of proteinase K. An additional 4 mL of buffer, pre-warmed to 65° C. without proteinase K, was added. The homogenate was incubated at 65° C.
- Total DNA was isolated from 1 to 1.5 cm 2 sections of leaf tissue by a modified method of Dellaporta et al. (1983). Sections were cut from healthy leaves with clean scissors, placed in 1.5 mL conical microcentrifuge tubes, and transferred directly to liquid nitrogen. Leaf sections were often stored at ⁇ 80° C. prior to DNA extraction. Leaf tissue was homogenized in liquid nitrogen with a microfuge pestle (Kontes Glass Co., Vineland, N.J.) in the microcentrifuge tube.
- a microfuge pestle Kontes Glass Co., Vineland, N.J.
- PCR amplification 200 to 400 ng of genomic DNA or 2 ⁇ L of total DNA was brought to 10 ⁇ L with water in a 0.2 mL thin-walled tube (MJ Research, Inc., Watertown, Mass.). To each sample was added 40 ⁇ L of a PCR mixture containing 25 picomoles of UBI A2 primer, 25 picomoles of transgene-specific primer (see Table 3), 50 mM potassium chloride, 10 mM TRIS-chloride, pH 9.0, 0.1% Triton X-100, 3 mM magnesium chloride, 1 mM each of dATP, dCTP, dGTP and TTP, and 2.5 Units of Taq DNA polymerase (Promega, Madison, Wis.).
- Polymerase chain reaction was carried out on a PTC-100 or PTC-200 Programmable Thermal Controller (MJ Research, Watertown, Mass.) with the following protocol: 96° C. for 30 sec; 35 cycles of 95° C. for 1 minute, 62° C. for 1 minute, 72° C. for 1 minute; 72° C. for 15 minutes; 4° C. soak.
- PCR products (10-13 ⁇ L of each PCR reaction mixture) were partitioned on 1% to 1.6% agarose gels and visualized by ethidium bromide staining and ultraviolet light as described in the Example.
- Positive PCR controls included 10 to 20 ng of pUBKBglII- plasmid DNA containing the glucanase, endochitinase or exochitinase cDNAs.
- At least three transgenic wheat lines showing amplification of the predicted 425 bp Ubi-exochitinase PCR product, or the predicted 375 bp Ubi-glucanase PCR product, or the predicted 340 bp Ubi-endochitinase PCR product have been obtained in our experiments (FIG. 12).
- Transgenic lines were propagated as described in Example 19. Progeny were analyzed for bialaphos resistance and/or for the presence of the F. venenatum transgenes using PCR. Populations in which all of the individual progeny were bialaphos resistant and/or were positive in the PCR transgene assay in two successive generations were considered to be derived from a line homozygous for the transgene.
- mRNA transgene messenger RNA
- RNA was isolated using a modified method of Altenbach (1998). Essentially, frozen endosperm was ground to a fine powder in a chilled mortar and pestle, in the presence of liquid nitrogen. The frozen homogenate was transferred to a 50-mL conical centrifuge tube containing 5 mL extraction buffer (10 mM sodium chloride, 10 mM TRIS-Cl, pH 9.0, 1 mM EDTA), 2.5 mL phenol (saturated with TRIS buffer at pH 4.3, Fisher #BP1751), and 2.5 mL chloroform. The tube was inverted 40 times, then centrifuged at 6500 rpm, 4° C. for 10 minutes in a Sorvall HS-4 rotor.
- extraction buffer 10 mM sodium chloride, 10 mM TRIS-Cl, pH 9.0, 1 mM EDTA
- 2.5 phenol saturated with TRIS buffer at pH 4.3, Fisher #BP1751
- chloroform 2.5 mL chloroform
- the aqueous phase was transferred to a fresh tube, extracted once with an equal volume of phenol/chloroform (1:1 v/v), and once with an equal volume of chloroform. The mixture was centrifuged after each extraction as described. To the aqueous extract, 8M LiCl was added to give a final concentration of 2M LiCl and incubated at ⁇ 20° C. overnight. The precipitate containing RNA was collected by centrifugation at 12,500 rpm, 4oC for 30 minutes in a Sorvall SS-34 rotor. The RNA pellet was washed with ice cold 2M LiCl, air dried, and resuspended in autoclaved water.
- RNA was stored at ⁇ 80° C.
- RNA samples were brought to 10 ⁇ L with sample buffer containing 50% formamide, 6% formaldehyde (Fisher #BP531), 20 mM MOPS, pH 7.0, 5 mM sodium acetate, 10 mM EDTA, and 0.01% bromophenol blue. RNA samples were heated at 60° C. for 10 to 15 minutes, then immediately placed on ice prior to partitioning on agarose.
- sample buffer containing 50% formamide, 6% formaldehyde (Fisher #BP531), 20 mM MOPS, pH 7.0, 5 mM sodium acetate, 10 mM EDTA, and 0.01% bromophenol blue.
- the agarose (0.3 g) was dissolved in 30 mL of 20 mM MOPS, 5 mM sodium acetate, 10 mM EDTA, pH 7.0, to which was added 37% formaldehyde for a final concentration of 6% and 10 ⁇ g/ ⁇ L ethidium bromide for a final concentration of 166 ng/mL. Electrophoresis was carried out at 50-70 V for up to 2 hours. Partitioned RNA was transferred to Zeta-Probe GT nylon membrane (Bio-Rad, Hercules, Calif.) in either 10 ⁇ SSC (150 mM sodium chloride, 15 mM sodium citrate, pH 7.0) or 50 mM sodium hydroxide for 3 hours.
- 10 ⁇ SSC 150 mM sodium chloride, 15 mM sodium citrate, pH 7.0
- 50 mM sodium hydroxide for 3 hours.
- the nylon membrane containing RNA was rinsed in 2 ⁇ SSC (30 mM sodium chloride, 3 mM sodium citrate, pH 7.0) and either treated with ultraviolet light using a UV Stratalinker 2400 (Stratagene, La Jolla, Calif.) and baked at 80° C. under vacuum (neutral transfer), or air dried and used directly (alkaline transfer).
- 2 ⁇ SSC 30 mM sodium chloride, 3 mM sodium citrate, pH 7.0
- UV Stratalinker 2400 Stratagene, La Jolla, Calif.
- Northern blots were incubated in a Hybaid hybridization oven (National Labnet, Woodbridge, N.J.) for 6 to 7 hours at 42° C. in 5 to 10 mL of a solution containing 50% (v:v) formamide, 7% (w:v) sodium dodecyl sulfate (SDS), 0.25 M sodium phosphate buffer, pH 7.0, 0.25 M sodium chloride, and 1 mM EDTA, pH 8.0, as recommended by Bio-Rad.
- SDS sodium dodecyl sulfate
- Probes for northern blots consisted of partial or full-length cDNAs as follows: 0.4 kb BamHI GLU fragment from FvGluS; 1.2 kb BamHI +BglII Endo fragment from FvEndoS; or 1.8 kb BamHI+BglII Exo fragment from FvExoS. Probe fragments were purified following agarose gel partitioning as described in Example 12. About 25-50 ng of probe DNA was radiolabeled to a specific activity of 1-2 ⁇ 10 6 cpm/ng with 32 P- ⁇ -dCTP (3000 Ci/ummol), using the Multiprime DNA Labelling System (Amersham Pharmacia Biotech, Inc., Piscataway, N.J.) as recommended.
- Radiolabeled fragments were added directly to the northern blots and hybridized at 42° C. for up to 36-40 hours. Blots were washed in 200 to 300 mL of 2 ⁇ SSC, 0.1% (w:v) SDS at room temperature for 15 minutes; in 200 to 300 mL of 0.5 ⁇ SSC, 0.1% (w:v) SDS at room temperature for 15 minutes; and in 200 to 300 mL of 0.1 ⁇ SSC, 0.1% (w:v) SDS at 62° C. for 15 minutes. Autoradiography was performed at ⁇ 80° C.
- RT-PCR reverse transcription-polymerase chain reaction
- RT-PCR was carried out on 600 ng of total RNA using either the GeneAmp PCR Core kit (Perkin Elmer, Foster City, Calif.) or a OneStep RT-PCR Kit (Qiagen, Inc., Valencia, Calif.), as recommended by the manufacturers.
- Transgene-specific transcripts were amplified using primers RTUBI and either RTGLU (for the 5′ end of the endochitinase transcript), RTEND (for the 5′ end of the endochitinase transcript), or RTEXO (for the 5′ end of the exochitinase transcript). See Table 3 for a list of RT-PCR primers.
- the 3′ portions of the transgene transcripts were amplified with RTNOS and either RTGLU2 (glucanase), RTEND2 (endochitinase), or RTEXO2 (exochitinase).
- the annealing temperatures were 58°-60° C.
- Endosperm and glume total RNA from Line AB9-59b showed very low but detectable levels of a single 700 bp RT-PCR product (FIG. 14), representing the 5′ end of the transcript, and including the Ubi exon.
- the size of this product compared closely with the expected product size of 705 bp, which was derived from the predicted nucleotide sequence of the transgene mRNA.
- the 3′ portion of the glucanase transcript was also amplified at very low, but detectable levels (data not shown), yielding a single 330 bp PCR product (expected size 340 bp).
- RNA from endosperm of Line C9-25a a single ⁇ 800 bp RT-PCR product, representing the 5′ end of the exochitinase MRNA (expected size 795 bp) (FIG. 15A, right panel), and a single 300 bp RT-PCR product representing the 3′ end of the exochitinase mRNA (expected size 310 bp)(FIG. 15B) was detectable.
- Total RNA from untransformed cultivar Bobwhite (bw) did not produce any RT-PCR products.
- the endochitinase 5′ RT-PCR product was cleaved exhaustively with either Clal or SspI restriction endonuclease according to the manufacturer's specifications (New England BioLabs, Beverly, Mass.).
- the resulting cleavage products were partitioned on a 6% TBE acrylamide gel (Novex/Invitrogen, Carlsbad, Calif.), 100V for ⁇ 1 hr, in running buffer consisting of 89 mM TRIS-Cl, pH 8.3, 89 mM borate, 1.5 mM EDTA.
- the resulting endochitinase cleavage products shown in FIG.
- the FvEndo and FvExo RT-PCR products were cDNA-dependent, and were therefore generated from RNA rather than from contaminating genomic DNA in the RNA preparations.
- the RT-PCR products were amplified from both the 5′ and 3′ regions of the FvEndo transcripts in endosperm from three independent lines (FIG. 18A) only if cDNA synthesis (+) was first carried out. In the absence of cDNA synthesis( ⁇ ), no 5′ or 3′ RT-PCR products were obtained.
- FvEndo transcripts were not detected in endosperm of untransformed Bobwhite (Bw) either with or without cDNA synthesis (FIG. 18B).
- F. venenatum CC1-3 a morphological mutant of strain A3/5 (Wiebe et al. 1991) was used for the DNA blots described in this example. Genomic DNA (5 ⁇ g per treatment) was cleaved exhaustively with HindIII, BamHI, or EcoRI restriction endonuclease, partitioned on agarose gels as described in EXAMPLE 8, then transferred to Nytran SuPerCharge (Schleicher and Schuell, Keene, N.H.).
- the hybridization probes consisted of the following: 1) the 0.4 kb 5′ BamHI fragment of FvGlu cDNA (from plasmid described in EXAMPLE 15); 2) 1.2 kb BamHI/BglII FvEndo cDNA fragment from FvEndoS (FIG.
- the FvGlu probe that was used for this experiment consisted of cDNA sequences 5′ to the BamHI site, so a single hybridizing BamHI fragment was expected and was obtained.
- the sizes of the hybridizing FvEndo fragments were 9.3 kb (HindIII), 8.9 kb (BamHI), and 6.6 kb (EcoRI).
- the sizes of the hybridizing FvExo fragments were 4.3 kb (HindIII), 11 kb (BamHI), and 4.7 kb (EcoRI).
- Neither FvEndo nor FvExo coding regions carry restriction sites for these three endonucleases.
- Plasmids identified below were introduced into the host Escherichia coli and the transformed Escherichia coli were deposited under terms of the Budapest Treaty with Agricultural Research Service Culture Collection (NRRL) National Center for Agricultural Utilization Research, Agricultural Research Service, U.S. Department of Agriculture, 1815 North University Street, Peoria, Ill. 61604 USA on the date listed and given the following accession numbers: Deposit Accession No. FIG./Seq No Date of Deposit GLU2 NRRL B-30201 SEQ16 Aug. 26, 1999 Endol67 NRRL B-30202 SEQ18 Aug. 26, 1999 Exo9 NRRL B-30203 SEQ20 Aug. 26, 1999 FvGluS NRRL B-30204 Aug.
- NRRL Agricultural Research Service Culture Collection
- Cecropin A-derived peptides are potent inhibitors of fungal plant pathogens. Mol. Plant-Microbe Inter. 11: 218-227.
- CDS (108)..(1853) misc_difference (231) Unsure, n is A or C; Xaa at amino acid 42 is Ile or Leu 12 actggtactt tggacacttt tgtctcttcc ttaaagatta gcctccctcg ctcagcttct 60 atctaccatt gttagcaatt atctcactca cctcacctct aggcgta atg tgg tcc 116 Met Trp Ser 1 aag gct ctt ctg gcc gtt gcc gcc ttt gcc ttc aca cccc gcc aat gct 164 Lys Ala Leu Leu Ala Val Ala Ala Phe Ala Phe Thr Pro Ala Asn Ala 5 10 15 ata tgg cca
- Xaa is Ile or Leu 13 Met Trp Ser Lys Ala Leu Leu Ala Val Ala Ala Phe Ala Phe Thr Pro 1 5 10 15 Ala Asn Ala Ile Trp Pro Val Pro Lys Lys Ile Ser Thr Gly Asp Lys 20 25 30 Ala Leu Phe Ile Asp Gln Thr Ile Asp Xaa Thr Tyr Asn Gly Asp Phe 35 40 45 Ile Pro Tyr Thr Tyr Asn Tyr Gln Pro Asp Ala Gly Ser Lys Phe Ser 50 55 60 Ser Lys Gln Ile Ile Gln Ala Gly Val Ser Arg Ala Leu Gln Gly Val 65 70 75 80 Phe Gln Asp Asn Phe Val Pro Trp Met Leu Arg Glu Arg Asp Ser Asp 85 90 95 Phe Glu Pro Asp Leu Gln Lys Lys Gln Trp Val Lys Ser Leu Lys Ile 100 105 110 Ile Gln Thr
Landscapes
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Cell Biology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Medicinal Chemistry (AREA)
- Pregnancy & Childbirth (AREA)
- Reproductive Health (AREA)
- Developmental Biology & Embryology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention is directed to nucleic acid sequences derived from Fusarium fungal genes which encode the cell wall-degrading enzymes glucanase, endochitinase, and exochitinase; isolated polypeptides having glucanase, endochitinase or exochitinase activity; recombinant nucleic acid molecules, vectors, and host cells comprising the nucleic acid sequences as well as methods for producing and using the polypeptides, including expression in plant cells to confer or enhance a plant's resistance to Fusarium and other pathogens.
Description
- This application claims the benefit of U.S. Provisional Applications Nos. 60/224,946, filed Aug. 11, 2000 and 60/151,582, filed Aug. 30, 1999. The disclosure of each of said provisional application is incorporated herein by reference in its entirety.
- 1. Field of the Invention
- The present invention relates to nucleic acid sequences derived from fungal genes which encode polypeptides having cell wall-degrading activity and isolated polypeptides having cell wall-degrading activity. The invention also relates to recombinant nucleic acid molecules, vectors, and host cells comprising the nucleic acid sequences as well as methods for producing and using the polypeptides, including expression in plant cells to confer or enhance a plant's resistance to Fusarium and other pathogens.
- 2. Description of the Art
- Overview.
- Wheat is one of the most important food crops for both domestic and export markets. The United States produces about 2.4 billion bushels of wheat per year with a value of over 7 billion dollars. Fusarium head blight or scab is a fungal disease of wheat, barley, oats, rye, and wheatgrasses that affects both grain yield and quality. It occurs worldwide, particularly when temperatures and humidity favor the proliferation of the causal agent,Fusarium graminearum, at the time of heading. Head blight has caused losses in the billions of dollars to United States and Canadian growers and processors within this decade. Yield and grain quality losses of wheat due to Fusarium head blight approached one billion dollars in Minnesota, North Dakota, and South Dakota in 1993 and 200-400 million dollars across the region in subsequent years. Losses were in excess of 300 million dollars in Ohio, Michigan, Indiana, and Illinois in 1995 and 1996. Quality of grain is also compromised since infected grain is usually contaminated with a mycotoxin, vomitoxin or DON, produced by the fungus that is detrimental to humans and livestock. In addition, the disease has threatened barley production in the upper Midwest because brewers have imposed zero tolerance limits for vomitoxin in grain.
- The Head Blight Disease.
- Cereal crops, including wheat, maize, barley, oats, and rye, are susceptible to infection by many types of fungi and by many species of the pathogenic fungus Fusarium.Fusarium graminearum (Schwabe) and Fusarium culmorum are the primary causal agents of a disease known as Fusarium head blight, head blight, or scab, of wheat, barley, oats and rye (reviewed in Bai and Shaner 1994; Parry et al. 1995). The life cycle of the pathogen alternates between two hosts, wheat and maize. The teliomorph (sexual stage) of F. graminearum, called Gibberella zeae, causes stalk rot, ear rot and seedling blight of maize. Head blight is a problem of wheat and barley worldwide, and has occurred in epidemic proportions in the United States since 1991. Thus far, no effective resistance genes for Fusarium head blight have been identified in wheat, barley or their sexually-compatible relatives. Due to this deficiency, and as a consequence of no-till agriculture and unfavorable climate patterns, the wheat and barley industries in 12 states have sustained direct losses totaling 2.6 billion dollars. Increased accumulation of corn and wheat stubble has resulted from no-till agriculture and contributes to the propagation of spores and conidia in the field. Moreover, periods of rainfall and warm temperatures at the time of anthesis (pollen shed) favor germination and growth of the pathogen. F. graminearum causes death of the floral organs (florets) that harbor the developing grain, giving the head a bleached and “scabby” appearance, and resulting in moderate to severe reductions in grain yield. In addition, F. graminearum and other Fusarium species produce trichothecene mycotoxins, such as deoxynivalenol (DON), that exacerbate disease severity and pose a health threat to humans and livestock that ingest contaminated cereal products.
- The marked susceptibility of the wheat head to Fusarium was noted as early as 1891 by Arthur (Parry et al. 1995). Pugh and coworkers (1933) reported that wheat heads inoculated with cultured conidia ofF. roseum (culmorum) were most susceptible to infection during a 20-day window, from anthesis to the soft dough stage, depending upon the cultivar. Dehisced anthers and other degenerating tissue appeared to serve as foci for the proliferation of hyphae into the phloem and throughout the rest of the head. In histological studies, Pugh noted that hyphae usually invade intercellularly but can penetrate the thin walls of the inner parts of the floret. Although F. graminearum can become established on various parts of the flower, rapid spread of infection was correlated with the presence of extruded anthers both in laboratory and field studies (Andersen 1948, McKay and Loughnane 1945). Aqueous extracts of anthers were found to stimulate growth of hyphae in vitro (Strange and Smith 1971), whereas germination of macroconidia appeared to be unaffected (Strange and Smith 1978). Hyphal growth stimulation was almost entirely attributable to two quaternary ammonium compounds, glycinebetaine and its precursor choline (Strange et al. 1974). These compounds are present in other organs of the floret, but are most abundant in pollen (Pearce et al. 1976). Glycinebetaine, an osmoprotectant, accumulates over the normal course of pollen development during desiccation (reviewed in McCue and Hanson 1990). Along with choline, it is postulated to serve as a fortuitous source of carbon and nitrogen for F. graminearum and for other fungal pathogens. Whether fungal hyphae readily access these compounds at the pollen surface or must invade the pollen cytoplasm is not known at this time.
- Identification of genes that confer resistance to Fusarium head blight is essential if wheat and barley are to remain in production in Minnesota, North Dakota, and other states. If Fusarium infection can be curtailed by even 10%, the economic impact is expected to be millions of dollars saved by producers, processors and, ultimately, consumers. Multigenic loci for resistance to scab have been identified in germplasms of wheat and other cereals, but at present, the degree of tolerance obtained in adapted cultivars through traditional breeding is inadequate for control of the pathogen. Since no-till agriculture offers clear benefits for soil and water conservation and since Fusarium is so abundant in wheat-growing regions, alternative sources for host resistance are needed.
- There are no effective control measures for the disease. Resistance genes for Fusarium head blight have not been identified in wheat, barley or other sexually-compatible species, limiting the efforts of wheat breeders to develop resistant varieties. What are needed are ways to obtain crop varieties that are resistant to Fusarium head blight.
- Roles of Fungal Glucanases and Chitinases.
- In many fungi, including species of Fusarium, glucan and chitin are principle components of the cell wall. The outer wall layer of Fusarium is comprised of polymers of glucose (1,3/1,6-β-D-glucan) with β-1,3 and β-1,6 linkages. The basal-most inner layer consists primarily of chitin microfibrils, linear polymers of β-1,4-N-acetylglucosamine that account for about one-third of the mass of the hyphal wall (Barbosa and Kemmelmeier 1993). Fusarium cell walls appear to be more refractory to the action of hydrolytic enzymes, possibly because of high levels of protein and chitin (Sivan and Chet 1989a), and clustering of acetylated glucosamine residues (Fukamizo et al. 1992).
- Chitinases and glucanases are produced by naturally occurring bacteria, fungi and plants. In the fungi, these proteins have roles in the self-hydrolysis of cell wall chitin and glucan, respectively, and in the hydrolysis of cell wall components of other microorganisms (Srivastava et al. 1985; Sivan and Chet 1989b, Chérif and Benhamou 1990, Vázquez-Garciduefias et al. 1998). This latter feature has been applied to the discovery of anti-microbial biocontrol agents. Chitin is also found in the cuticles of insects and in nematode egg shells. For fungi that can utilize chitin as a carbon source, chitinase might have a role in fungal metabolism (Flach et al. 1992). Endochitinases cleave randomly between C1 and C4 linkages within the chitin polymer (Flach et al. 1992, Graham and Sticklen 1994), whereas exochitinases cleave sequentially between each linkage, releasing chitobiose from the terminus of the chitin polymer. N-acetyl-glucosaminidase also cleaves terminally, releasing N-acetylglucosamine (Flach et al. 1992). Likewise, endoglucanases randomly cleave β-linkages within the glucan polymer, generating short oligosaccharides, whereas the exoglucanases cleave single glucose residues from the nonreducing end of the polymer (Vázquez-Garciduefias et al. 1998). Hydrolysis of the glucan layer of fungal cell walls is attributed to the combined action of both endo- and exo-glucanases.
- Glucanases and Chitinases as Antifungal Proteins.
- The distinctive glucan and chitin composition of the fungal cell wall has led to extensive engineering of chitinases and glucanases as antifungal proteins. Chitinases and glucanases produced by plants have been widely characterized as pathogenesis-related (Pr) proteins involved in defense against pathogens and insect pests. Pr protein genes in plants are induced upon exposure to microbial pathogens and insect pests. Cell wall-degrading proteins have been grouped into classes on the basis of their biochemical and structural properties. Both endo- and exochitinases appear to be effective in the hydrolysis of fungal cell wall chitin. Class I chitinases have chitinolytic activity against bacterial cell walls and are known to bind chitin directly. By contrast, the class II chitinases do not act on bacterial cell walls and lack chitin binding activity; they are postulated to play a role in production of fungal elicitors that trigger the host defense response (Graham and Sticklen 1994, Fritig et al. 1998). In general, the basic chitinases and glucanases (class I) accumulate intracellularly in the vacuole, and the acidic isoforms (class II) are extracellular, with some exceptions (e.g., Wu et al. 1994; Graham and Sticklen 1994). Cornelissen and coworkers (Sela-Buurlage et al. 1993) and others (Graham and Sticklen 1994) observed that the specific activities of class I chitinases and glucanases are higher than those of the class II enzymes. Class I chitinases are encoded by small gene families in most plants and in the fungi. The structure of various chitinase genes and proteins has been reviewed (Graham and Sticklen 1994).
- Of the five major classes of -glucanases, only the β-1,3-glucanases have been shown to exhibit antifungal activity (Simmons 1994). The β-1,3-glucanases are members of small multigene families in plants (Payne et al. 1990, Xu et al. 1992, Beffa and Meins 1996, Simmons 1994), but are single-copy genes inFusarium sporotrichioides (FIG. 27). Class I glucanases accumulate in the vacuoles, whereas Class II and III glucanases are acidic and extracellular (see Beffa and Meins 1996). Class I glucanases have been widely studied in the context of pathogen defense and stress responses in tobacco (Linthorst et al. 1990; Linthorst 1991; Payne et al. 1990), barley (Jutidamrongphan et al. 1991; Xu et al. 1992; Malehorn et al. 1993), wheat (Jutidamrongphan et al. 1991; Cruz-Ortega et al. 1997), and other species (Krishnaveni et al. 1999a, reviewed in Simmons 1994). Chitinases and glucanases act preferentially on the tips of growing fungal hypha (for instance, Broekaert et al. 1988, Collinge et al. 1993). Both chitinases and β-1,3-glucanases are differentially expressed during plant development (Lotan et al. 1989) as well as in response to pathogen attack. Glucanases appear to have roles in cell division and flower development (Beffa and Meins 1996) and the stress response (Simmons 1994) in plants. The role of chitinases in plant development is not as well-characterized; however, they have been implicated in embryogenesis and cell division (for review, see Collinge et al. 1993)
- Some of the chitinases and β-1,3-glucanases produced by naturally occurring bacteria and fungi have anti-Fusarium properties (Mitchell and Alexander 1961; Michael and Nelson 1972, Chérif and Benhamou 1990). Glucanases and chitinases from plants can degrade isolated cell walls ofFusarium solani (Mauch et al. 1988). Chitinases from tobacco were inhibitory to the growth of F. oxysporum (Yun et al. 1996) and F. solani (Sela-Buurlage et al. 1993) in culture. Krishnaveni et al. (1999b) have described three chitinases from sorghum seeds that inhibit the growth of F. moniliforme. The synergistic action of chitinases and glucanases against fungal pathogens is widely reported (reviewed in Graham and Sticklen 1994, Van Loon 1997). For instance, Mauch et al. (1988) observed that a chitinase and a β-1,3-glucanase from pea wee active against a wide range of fungi. Melchers et al (1994) reported the combined action of a Class V endochitinase plus a Class I β-1,3-glucanase, both from tobacco, synergistically inhibited the growth of F. solani. Expression of a tobacco acidic chitinase with a tobacco β-1,3-glucanase conferred resistance to F. oxysporum in tomato, whereas each protein had much less effect when expressed singly (Jongedijk et al. 1995). Likewise, the fungal pathogen Cercospora nicotiana was curtailed on tobacco expressing both a rice basic chitinase and an alfalfa acidic β-1,3-glucanase (Zhu et al. 1994). The synergistic action of a barley Class II chitinase and a barley Class II b-1,3-glucanase conferred protection to tobacco against Rhizoctonia solani (Jach et al. 1995). This chitinase in combination with a barley ribosome inactivating protein also inhibited R. solani infection.
- Apparent Specificity of Glucanases and Chitinases.
- Although fungi and many, if not all, plants express glucanases and chitinases, not all of the enzymes are equally effective against all types of microorganisms (Graham and Sticklen 1994, and references therein). For example, Broekaert and co-workers (1988) observed that chitinases from thorn-apple, tobacco and wheat had antifungal activity againstTrichoderma harzianum and Phycomyces blakesleeanus but not against Botrytis cinerea. Mauch et al. (1988) reported the differential action of a chitinase and a glucanase from pea on Trichoderma viride and F. solani, respectively. In combination, the enzymes were active against a wide range of other fungi. A tobacco chitinase with activity against Fusarium and Trichoderma were inactive against Aspergillus flavus, Phytophthora parasitica and other pathogens (Yun et al. (1996). The differential activities of the chitinases are attributed to inherent properties of the enzymes (Sela-Buurlage et al. 1993, Brunner et al. 1998), to differences in cell wall architecture (Sivan and Chet 1989a, Van Loon 1997) among the fungi, or to other factors.
- Other Anti-Fusarium Proteins.
- Additional types of proteins have been found to have anti-Fusarium activity in vitro or in planta. Boyapati et al. (1994) reported a cysteine protease inhibitor from pearl millet that inhibited the growth ofFusarium moniliforme in culture. A cysteine-rich polypeptide from Impatiens balsamina seeds was active against F. culmorum (Tailor et al. 1997). Cecropin A, a polypeptide from the Cecropia moth, was a potent inhibitor of both F. moniliforme and F. oxysporum (deLucca et al. 1997, Cavallarin et al. 1998). Antifungal proteins from seeds of sorghum had activity against F. moniliforme (Seetharaman et al. 1997), and two wheat seed proteins of the PR4 family of pathogenesis-related proteins inhibited hyphal growth of F. culmorum and F. graminearum (Caruso et al. 1996). Hu and Reddy (1997) isolated a thaumatin-like protein from Arabidopsis thaliana with activity against F. oxysporum. Non-specific lipid transfer proteins from barley and maize leaves were inhibitory to F. solani (Molina et al., 1993). A combination of a wheat purothionin and a 2S albumin from radish or oilseed rape was effective against the growth of F. culmorum in vitro (Terras et al. 1993).
- Microbial Genes as Anti-Fungal Transgenes in Plants.
- A majority of antifungal genes that have been examined both in vitro and in planta are of plant origin. To our knowledge, there are two examples of genes from fungi that exhibit antifungal activity. Endochitinases from the parasitic fungusTrichoderma harzianum conferred activity against Alternaria alternata and B. cinerea in transgenic tobacco, and against A. solani and Rhizoctonia solani in transgenic potato (Lorito et al. 1998). Terakawa et al. (1997) observed protection of transgenic tobacco against Sclerotinia sclerotiorum and B. cinerea, using a chitinase gene from the fungus Rhizopus oligosporus. A chitinase from the bacteria Serratia marcescens showed antifungal activity when expressed in tobacco (Suslow et al. 1988).
- The present invention is directed to nucleic acid sequences derived from Fusarium fungal genes which encode polypeptides having cell wall-degrading activity as well as isolated polypeptides having cell wall-degrading activity. The invention is also directed to recombinant nucleic acid molecules, vectors, and host cells comprising the nucleic acid sequences and methods for producing and using the polypeptides, including expression in plant cells to confer or enhance a plant's resistance to Fusarium and other pathogens.
- More particularly, the invention provides isolated nucleic acid molecules that encode polypeptides having cell wall-degrading activity comprising glucanase, endochitinase or exochitinase activity. Genomic sequences encoding glucanase and exochitinase and cDNA sequences encoding glucanase, endochitinase and exochitinase are specifically exemplified herein. Included within the scope of this invention are nucleic acid sequences encoding a polypeptide having the glucanase, endochitinase or exochitinase polypeptide sequences exemplified below and nucleic acid molecules encoding polypeptides having glucanase, endochitinase or exochitinase activity.
- Nucleic acid sequences which hybridize specifically to an enzyme coding sequence or its complement under medium or high stringency conditions and which encode a polypeptide having glucanase, endochitinase or exochitinase activity are also encompassed by the present invention.
- Nucleic acid sequences having at least 70% sequence identity with the exemplified glucanase sequences as described in detail, below, and which encode a polypeptide having glucanase activity are also encompassed by the present invention. Nucleic acid sequences encoding a polypeptide having at least 80% sequence identity with the exemplified glucanase polypeptide sequences as described in detail, below, and which encode a polypeptide having glucanase activity are also encompassed by the present invention.
- Nucleic acid sequences having at least 75% sequence identity with the exemplified endochitinase or exochitinase sequences as described in detail, below, and which encode a polypeptide having endochitinase or exochitinase activity are also encompassed by the present invention. Nucleic acid sequences encoding a polypeptide having at least 85% sequence identity with the exemplified endochitinase or exochitinase polypeptide sequences as described in detail, below, and which encode a polypeptide having endochitinase or exochitinase activity are also encompassed by the present invention.
- The present invention is also directed to isolated polypeptides having glucanase, endochitinase or exochitinase activity. A polypeptide having an amino acid sequence which has at least 80% sequence identity with exemplified glucanase polypeptides as described in detail, below, is encompassed by the invention. A polypeptide having an amino acid sequence which has at least 85% sequence identity with exemplified endochitinase or exochitinase polypeptides as described in detail, below, is also encompassed by the invention. Polypeptides encoded by a nucleic acid sequence which hybridizes under medium or high stringency conditions with exemplified nucleic acid sequences as discussed in detail, below, are also encompassed by the invention. Variants of the polypeptides are encompassed by the invention as well as fragments having glucanase, endochitinase or exochitinase activity.
- The invention is also directed to methods of producing and using the polypeptides of the invention.
- A further aspect of the invention is the provision of recombinant nucleic acid molecules containing the sequences encoding polypeptides having the fungal cell wall-degrading activity, including glucanase, endochitinase or exochitinase activity. Such molecules include, for example, recombinant vectors, such as cloning, expression or transformation vectors, which contain a DNA sequence encoding a glucanase, endochitinase or exochitinase.
- Another aspect of the invention is the provision of cells which are transformed by the above vectors or DNA sequences.
- A particular use of the invention is the provision of cells transformed with one or more nucleic acid sequences of the invention. A more particular use of the invention is the provision of plants, plant seeds or plant cells transformed with one or more nucleic acid sequences encoding a polypeptide having glucanase, endochitinase or exochitinase coding activity to provide plants having resistance to plant pathogens, including fungi, particularly, Fusarium species or to provide plants having enhanced resistance to plant pathogens.
- A further aspect of the invention is the provision of oligonucleotide probes capable of detecting a glucanase, endochitinase or exochitinase gene or functional equivalents thereof in fungi of the genus Fusarium and the use of the probes to isolate nucleic acid sequences encoding a glucanase, endochitinase or exochitinase gene or functional equivalent thereof. The nucleic acid sequences which specifically hybridize to the probes and which encode a functional glucanase, endochitinase or exochitinase are encompassed by the present invention.
- Using the nucleic acid sequences of the invention facilitates the isolation of homologous genes from fungi to obtain genes which protect host cells, including fungi, bacteria, and plants against related fungal pathogens.
- The invention also includes the application of transgene constructs in combination with each other, that is, glucanase and endochitinase; glucanase and exochitinase; endochitinase and exochitinase; and glucanase, endochitinase and exochitinase. Also included are transgenic monocot or dicot lines, plant cells, and progeny obtained by sexual or asexual propagation, that carry any combination of the transgene constructs.
- In accordance with this discovery, it is an object of the invention to provide nucleic acid sequences encoding fungal cell wall-degrading enzymes selected from the group consisting of glucanase, exochitinase, and endochitinase; isolated polypeptides having glucanase, endochitinase or exochitinase activity; recombinant nucleic acid molecules including expression vectors encoding polypeptides having cell wall-degrading activity; and cells harboring the recombinant nucleic acid molecules or expression vectors.
- It is also an object of the invention to provide transformation vectors comprising a cell wall-degrading recombinant molecule, which vectors are effective for stably introducing the recombinant molecule into a plant.
- It is also an object of the invention to provide methods of producing and using polypeptides having glucanase, endochitinase or exochitinase activity.
- It is another object of the invention to provide transgenic plants having bacterial or fungal resistance, wherein the resistance is a result of expression of a recombinant nucleic acid molecule of the invention.
- A further object of the invention is to provide fungal genes which generate cell wall-degrading enzymes, including proteins having the capability of degrading the glucan and chitin cell wall components ofF. venenatum and other Fusarium species, including F. graminearum and F. culmorum, the principle causal agents of head blight (scab) in the U.S.
- Another object of the invention is expression of the cell wall-degrading enzymes in transgenic monocots, including wheat, barley or oats, to confer partial or complete resistance to Fusarium species and/or to other fungal pathogens of wheat and other cereal crops. Such transgenic lines will be useful genetic stocks for generating improved crops.
- A still further object of the invention is the provision of novel wheat germplasms that express genes designed to limit the spread of the pathogenic fungus Fusarium and indirectly to curtail the accumulation of DON in infected heads.
- Other objects and advantages of the invention will become readily apparent from the ensuing description.
- FIG. 1 is a comparison of glucanases encoded byF. venenatum cDNA (upper) and F. sporotrichioides genomic DNA (lower) (SEQ ID NOS:5 and 4, respectively).
- FIG. 2 shows the
F. venenatum endochitinase 5′ cDNA PCR product and the deduced polypeptide sequence (SEQ ID NOS:6 and 7, respectively). Bold type indicates new sequence obtained in 5′ PCR product. Non-bold type shows the portion of theF. venenatum endochitinase 5′ cDNA sequence that matches the F. venenatum endochitinase partial cDNA sequence. - FIG. 3 shows theF. sporotrichioides exochitinase 5′ genomic PCR product and a portion of the deduced polypeptide sequence (SEQ ID NOS:8 and 9, respectively). The underlined segment indicates an intron. The nucleotide segment before the ATG (first M) is the promoter and 5′ untranslated region. Bold type indicates new sequence obtained in 5′ PCR product. Non-bold type shows the portion of the F. sporotrichioides genomic sequence that matches the F. venenalum exochitinase cDNA sequence.
- FIG. 4 shows the comparison of exochitinases encoded byF. venenatum cDNA (upper) and F. sporotrichioides genomic DNA (lower) (SEQ ID NOS:14 and 15, respectively).
- FIG. 5 illustrates the modification to the Ubiquitin-1 promoter.
- FIG. 6 shows the map of plasmid FvGluS; monocot expression vector having the modified full-length glucanase cDNA in the sense orientation.
- FIG. 7 shows the map of plasmid FvGluAS; monocot expression vector having the modified full-length glucanase cDNA in the antisense orientation.
- FIG. 8 shows the map of plasmid FvEndoS; monocot expression vector having the modified full-length endochitinase cDNA in the sense orientation.
- FIG. 9 shows the map of plasmid FvEndoAS;.monocot expression vector having the modified full-length endochitinase cDNA in the antisense orientation.
- FIG. 10 shows the map of plasmid FvExoS; monocot expression vector having the modified full-length exochitinase cDNA in the sense orientation.
- FIG. 11 shows the map of plasmid FvExoAS; monocot expression vector having the modified full-length exochitinase cDNA in the antisense orientation.
- FIG. 12 shows the PCR analyses of transgenic lines for stably integrated transgene DNA. UT indicates untransformed plant; T1-T3 indicates transformed plants; C indicates plasmid (construct) control; mw indicates 100 bp ladder.
- FIG. 13 shows the Northern blot of FvEndo mRNA inTriticum aestivum line AB8-108.
- FIG. 14 shows detection of 5 ′portion of glucanase transcripts in endosperm and glume ofTriticum aestivum using RT-PCR.
- FIG. 15 shows detection of 5′ and 3′ portions of endo- and exo-chitinase transcripts in endosperm and glume ofTriticum aestivum using RT-PCR.
- FIG. 16 shows authentication of
endochitinase 5′ RT-PCR product using restriction endonucleases. - FIG. 17 shows authentication of
exochitinase 5′ RT-PCR product using restriction exonucleases. - FIG. 18 shows
FvEndo 5′ and 3′ RT-PCR products are cDNA-dependent. - FIG. 19 shows
FvExo 5′ and 3′ RT-PCR products are cDNA-dependent. - FIG. 20 shows Southern blots ofF. venenatum genomic DNA hybridized with FvGlu, FvEndo, FvExo cDNAs.
- SEQ ID NO:1 is aF. venenatum glucanase full-length unmodified cDNA sequence.
- SEQ ID NO:2 is the glucanase encoded by SEQ ID NO:1.
- SEQ ID NO:3 is the genomic DNA segment containing theF. sporotrichioides glucanase gene.
- SEQ ID NO:4 is the glucanase sequence encoded by SEQ ID. NO:3.
- SEQ ID NO:5 is a glucanase encoded by aF. venenatum cDNA sequence.
- SEQ ID NO:6 is a
F. venenatum endochitinase 5′ cDNA PCR product. - SEQ ID NO:7 is the polypeptide encoded by SEQ ID NO:6.
- SEQ ID NO:8 is aF. sporotrichioides exochitinase 5′ genomic PCR product.
- SEQ ID NO:9 is a portion of the polypeptide sequence encoded by SEQ ID NO:8.
- SEQ ID NO:10 is aF. venenatum endochitinase unmodified full-length cDNA sequence.
- SEQ ID NO:11 is the endochitinase encoded by SEQ ID NO:10.
- SEQ ID NO:12 is aF. sporotrichioides-F. venenatum chimeric sequence.
- SEQ ID NO:13 is the exochitinase encoded by SEQ ID NO:12.
- SEQ ID NO:14 comprises
amino acids 1 to 249 of an exochitinase encoded by a F. venenatum cDNA. - SEQ ID NO:15 comprises
amino acids 1 to 249 of a F. sporotrichioides genomic DNA. - SEQ ID NO:16 is aF. venenatum glucanase modified full-length cDNA sequence.
- SEQ ID NO:17 is the glucanase encoded by SEQ ID NO:16.
- SEQ ID NO:18 is aF. venenatum endochitinase modified full-length cDNA sequence.
- SEQ ID NO:19 is the endochitinase encoded by SEQ ID NO:18.
- SEQ ID NO:20 is aF. venenatum exochitinase modified full-length cDNA sequence.
- SEQ ID NO:21 is the exochitinase encoded by SEQ ID NO:20.
- SEQ ID NOS:22-24 are the native Ubiquitin-1 and modified Ubiquitin-1 sequences shown in FIG. 5 (upper, middle, and lower, respectively).
- SEQ ID NO:25 is a leader sequence.
- SEQ ID NO:26 is primer M13F.
- SEQ ID NO:27 is primer M13R.
- SEQ ID NOS:28-74 are the primers shown in Table 2.
- SEQ ID NOS:75-82 are the PCR primers shown in Table 3.
- Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY (2d ed. 1994); THE CAMBRIDGE DICTIONARY OF SCIENCE AND TECHNOLOGY (Walker ed., 1988); THE GLOSSARY OF GENETICS, 5TH ED., Rieger, R., et al.(eds.), Springer Verlag (1991); and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY (1991).
- To facilitate understanding of the invention, a number of terms are defined below.
- “Glucanase” refers to a protein or polypeptide with hydrolytic activity on glucan, a component of fungal cell walls. In particular, glucanase refers to a polypeptide having the enzymatic ability to degrade the β-1,3 linkages of glucan.
- The term “glucanase activity” is defined herein as hydrolytic activity which catalyzes the degradation of the β-1,3 linkages of glucan. Glucanase activity can be measured by adding a suitable substrate (e.g., glucan, laminarin) to a cellular extract, e.g., wheat endosperm tissue, leaf tissue, according to published methods. Assays are described in Keen and Yoshikawa (1983) and Fontaine et al. (1997).
- “Chitinase” refers to a protein or polypeptide with hydrolytic activity on chitin, a macromolecule composed primarily of N-acetyl-glucosamine and found in cell walls of fungal hyphae and spores.
- “Endochitinase” refers to a protein or polypeptide that enzymatically degrades chitin by cleaving randomly between C1 and C4 linkage with the chitin polymer.
- The term “endochitinase activity” is defined herein to mean the ability to degrade chitin by cleaving randomly between C1 and C4 linkage with the chitin polymer.
- “Exochitinase” refers to a protein or polypeptide that enzymatically degrades chitin by cleaving sequentially between each C1 and C4 linkages from the terminus of the chitin polymer.
- The term “exochitinase activity” is defined herein to mean ability to degrade chitin by cleaving sequentially between each C1 and C4 linkages from the terminus of the chitin polymer.
- Chitinase (endochitinase or exochitinase) activity can be measured by adding a suitable substrate to a cellular extract, e.g., wheat endosperm tissue, leaf tissue, according to published methods. Assays are described in Harman et al. (1993); McCreath and Gooday (1992); Tronsmo and Harman (1993), Bolar et al. (2000), and U.S. Pat. No. 5,378,821.
- The term “transgenic” when used in reference to a cell refers to a cell which contains a transgene, or whose genome has been altered by the introduction of a transgene. The term “transgenic” when used in reference to a tissue or to a plant refers to a tissue or plant, respectively, which comprises one or more cells that contain a transgene, or whose genome has been altered by the introduction of a transgene. Transgenic cells, tissues and plants may be produced by several methods including the introduction of a “transgene” comprising nucleic acid (usually DNA) into a target cell or integration of the transgene into a chromosome of a target cell by way of human intervention, such as by the methods described herein.
- The term “transgene” as used herein refers to any nucleic acid sequence which is introduced into the genome of a cell by experimental manipulations. A transgene may be a “native DNA sequence,” or a “heterologous DNA sequence” (i.e., “foreign DNA”). The term “native DNA sequence” refers to a nucleotide sequence which is naturally found in the cell into which it is introduced so long as it does not contain some modification (e.g., a point mutation, the presence of a selectable marker gene, etc.) relative to the naturally-occurring sequence.
- The term “heterologous DNA sequence” refers to a nucleotide sequence which is ligated to, or is manipulated to become ligated to, a nucleic acid sequence to which it is not ligated in nature, or to which it is ligated at a different location in nature. Heterologous DNA is not endogenous to the cell into which it is introduced, but has been obtained from another cell. Heterologous DNA also includes a native DNA sequence which contains some modification. Generally, although not necessarily, heterologous DNA encodes RNA and proteins that are not normally produced by the cell into which it is expressed. Examples of heterologous DNA include reporter genes, transcriptional and translational regulatory sequences, selectable marker proteins (e.g., proteins which confer drug resistance), etc.
- The term “transformation” as used herein refers to the introduction of a transgene into a cell. Transformation of a cell may be stable or transient.
- The term “stable transformation” or “stably transformed” refers to the introduction and integration of one or more transgenes into the genome of a cell. Stable transformation of a cell may be detected by Southern blot hybridization of genomic DNA of the cell with nucleic acid sequences which are capable of binding to one or more of the transgenes. Stable transformation of a plant may also be detected by using the polymerase chain reaction to amplify transgene sequences from genomic DNA from cells of the progeny of that plant. The term “stable transformant” refers to a cell which has stably integrated one or more transgenes into the genomic DNA. Thus, a stable transformant is distinguished from a transient transformant in that, whereas genomic DNA from the stable transformant contains one or more transgenes, genomic DNA from the transient transformant does not contain a transgene.
- The term “isolated” when used in relation to a nucleic acid molecule, as in “an isolated nucleic acid sequence” refers to a nucleic acid sequence that is identified and separated from at least one contaminant nucleic acid with which it is ordinarily associated in its natural source. Isolated nucleic acid is nucleic acid present in a form or setting that is different from that in which it is found in nature. The isolated nucleic acid sequence may be present in single-stranded or double-stranded form. When an isolated nucleic acid sequence is to be utilized to express a protein, the nucleic acid sequence will contain at a minimum at least a portion of the sense or coding strand (i.e., the nucleic acid sequence may be single-stranded). Alternatively, it may contain both the sense and anti-sense strands (i.e., the nucleic acid sequence may be double-stranded).
- The techniques used to isolate or clone a nucleic acid sequence encoding a polypeptide are known in the art and include isolation from genomic DNA, preparation from cDNA, or a combination thereof. The cloning of the nucleic acid sequences of the present invention from such genomic DNA can be effected, e.g., by using the well known polymerase chain reaction (PCR) or antibody screening of expression libraries to detect cloned DNA fragments with shared structural features. See, e.g., Innis et al., 1990,PCR: A Guide to Methods and Application, Academic Press, New York. Other nucleic acid amplification procedures such as ligase chain reaction (LCR), ligated activated transcription (LAT) and nucleic acid sequence-based amplification (NASBA) may be used. The nucleic acid sequence may be cloned from a strain of Fusarium, or another or related organism and thus, for example, may be an allelic or species variant of the polypeptide encoding region of the nucleic acid sequence.
- As used herein, the term “purified” refers to molecules, either nucleic or amino acid sequences, that are removed from their natural environment, isolated or separated. An “isolated nucleic acid sequence” is therefore a purified nucleic acid sequence. “Substantially purified” molecules are at least about 20% pure, preferably at least about 40% pure, more preferably at least about 60% pure, even more preferably at least about 80% pure, and most preferably at least about 90% or 95% pure. Purity may be determined by agarose electrophoresis. For example, an isolated nucleic acid sequence can be obtained by standard cloning procedures used in genetic engineering to relocate the nucleic acid sequence from its natural location to a different site where it will be reproduced. The cloning procedures may involve excision and isolation of a desired nucleic acid fragment comprising the nucleic acid sequence encoding the polypeptide, insertion of the fragment into a vector molecule, and incorporation of the recombinant vector into a host cell where multiple copies or clones of the nucleic acid sequence will be replicated. The nucleic acid sequence may be of genomic, cDNA, RNA, semisynthetic, synthetic origin, or any combinations thereof.
- The term “identity,” as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by a comparison of the sequences. In the art, “identity” also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as determined by the match between strings of such sequences. “Identity” can be readily calculated by known methods, including but not limited to those described in Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; and Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987. Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA (Altschul, S. F. et al., J. Molec. Biol. 215: 403-410 (1990). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990) and Altschul et al., Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25: 3389-3402 (1997).), ALIGN (http://dot.imgen.bcm.tmc.edu:9331/seq-search/alignment.html), and ClustalW (http://dot.imgen.bcm.edu:9331 /cgi-bin/multi-align/multi-align.pl) (Higgens, 1989).
- “Percentage of sequence identity” is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. It is preferred that the comparison window is at least 50% of the coding sequence, preferably 60%, more preferably 75% or 85%, and even more preferably 95% to 100%.
- The term “hybridization” as used herein includes “any process by which a strand of nucleic acid joins with a complementary strand through base pairing.” [Coombs J (1994)Dictionary of Biotechnology, Stockton Press, New York N.Y.]. Hybridization and the strength of hybridization (i. e., the strength of the association between the nucleic acids) is impacted by such factors as the degree of complementarity between the nucleic acids, stringency of the conditions involved, the Tm of the formed hybrid, and the G:C ratio within the nucleic acids.
- The phrase “hybridizes under stringent conditions” refers to the formation of a double-stranded duplex from two single-stranded nucleic acids. The region of double-strandedness can include the full-length of one or both of the single-stranded nucleic acids, or all of one single stranded nucleic acid and a subsequence of the other single stranded nucleic acid, or the region of double-strandedness can include a subsequence of each nucleic acid.
- Nucleic acid probes to identify and clone DNA encoding polypeptides having the desired enzyme activity from strains of different genera or species can be prepared according to methods well known in the art. Such probes can be used for hybridization with the genomic or cDNA of the genus or species of interest, following standard Southern blotting procedures, in order to identify and isolate the corresponding gene therein. Both DNA and RNA probes can be used. The probes are typically labeled for detecting the corresponding gene (for example, with32P, 3H, 35S, biotin, or avidin).
- For long probes of at least 100 nucleotides in length, high or medium stringency conditions are used. High stringency conditions when used in reference to nucleic acid hybridization comprise conditions equivalent to binding or hybridization at 68° C. in a solution consisting of 5×SSPE, 1% SDS, 5×Denhardt's reagent and 100 μg/ml denatured salmon sperm DNA followed by washing in a solution comprising 0.1×SSPE, and 0.1% SDS at 68° C. when a probe of about 100 to about 1000 nucleotides in length is employed, or the above-mentioned conditions with 50% formamide at 42° C. High stringency washes can include 0.1×SSC to 0.2×SSC, 1% SDS, 65° C., 15-20 min. An example of stringent wash conditions for a Southern blot of such nucleic acids is a 0.2×SSC wash at 65° C. for 15 minutes (see, Sambrook et al., Molecular Cloning—A Laboratory Manual (2nd ed.) Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY, 1989, for a description of SSC buffer). Other exemplary high stringency hybridization conditions include, for example, 7% SDS, 0.25 M sodium phosphate buffer, pH 7.0-7.2, 0.25 M sodium chloride at 65° C.-68° C. or the above-mentioned conditions with 50% formamide at 42° C. Exemplary medium stringency conditions are as described above for high stringency except that 35% formamide at 42° C. is used, and the washes are carried out at 55° C.
- For short probes which are about 15 nucleotides to about 70 nucleotides in length, stringency conditions are defined as prehybridization, hybridization, and washing post-hybridization at about 5° C. to about 10° C. below the calculated Tm using the calculation according to Bolton and McCarthy (1962, Proceedings of the National Academy of Sciences USA 48:1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA, 0.5% NP-40, 1×Denhardt's solution, 1 mM sodium pyrophosphate, 1 mM sodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per ml following standard Southern blotting procedures.
- For short probes which are about 15 nucleotides to about 70 nucleotides in length, the material with immobilized DNA is washed once in 6×SCC plus 0.1% SDS for 15 minutes and twice each for 15 minutes using 6×SSC at 5° C. to 10° C. below the calculated Tm.
- A genomic DNA or cDNA library prepared from other organisms may be screened for DNA which hybridizes with the probes described above and which encodes a polypeptide having the desired enzyme activity. Genomic or other DNA from such other organisms may be separated by agarose or polyacrylamide gel electrophoresis, or other separation techniques. DNA from the libraries or the separated DNA may be transferred to and immobilized on nitrocellulose or other suitable material. In order to identify a clone or DNA which is homologous with a selected sequence or a subsequence thereof, the material with immobilized DNA is used in a Southern blot. For purposes of the present invention, hybridization indicates that the nucleic acid sequence hybridizes to a labeled nucleic acid probe corresponding to the selected nucleic acid sequence, its complementary strand, or a subsequence thereof, under medium to high stringency conditions. Molecules to which the nucleic acid probe hybridizes under these conditions are detected using X-ray film.
- The term “nucleic acid construct” refers to a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or which has been modified to contain segments of nucleic acid which are combined and juxtaposed in a manner which would not otherwise exist in nature. The nucleic acid construct can include, for example, a coding sequence of the invention, and control sequences such as a promoter, and transcriptional and translation stop signals. The term nucleic acid construct is synonymous with the term expression cassette when the nucleic acid construct contains all the control sequences required for expression of a coding sequence of the present invention. Exemplary constructs include plasmids and vectors, including cloning vectors, recombinant expression vectors. A “vector” is a nucleic acid composition which can transduce, transform or infect a cell and generally be replicated in the cell, thereby causing the cell to express vector-encoded nucleic acids and, optionally, proteins other than those native to the cell, or in a manner not native to the cell. A vector includes a nucleic acid (ordinarily RNA or DNA) to be expressed by the cell. A vector optionally includes materials to aid in achieving entry of the nucleic acid into the cell, such as a retroviral particle, liposome, protein coating or the like. Vectors contain nucleic acid sequences which allow their propagation and selection in bacteria or other non-plant organisms. For a description of vectors and molecular biology techniques, see CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel, et al., (eds.), Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (through and including the 1998 Supplement) (Ausubel).
- The term “coding sequence” is defined herein as a nucleic acid sequence which directly specifies the amino acid sequence of its protein product, e.g., a sequence which is transcribed into mRNA and translated into a polypeptide. The boundaries of the coding sequence are generally determined by the ATG start codon (eukaryotes) and a translation terminator (stop codon). A coding sequence can include, but is not limited to, DNA, cDNA, and recombinant nucleic acid sequences.
- The term “sense orientation” refers to the orientation of a cDNA sequence or coding sequence with respect to the promoter in a construct, such that the 5′ end of the cDNA sequence or coding sequence is adjoined to the promoter. The term “antisense” (or reverse) refers to the orientation of a nucleic acid sequence such as the cDNA or coding sequence with respect to the promoter in a construct, such that the 3′ end of the sequence is adjoined to the promoter.
- The term “control sequences” is defined to include all components which are necessary or advantageous for the expression of a polypeptide. Such control sequences include, but are not limited to, a leader, a polypeptide sequence, a promoter, a signal peptide sequence or a targeting sequence, an enhancer, and a transcription terminator. At a minimum, the control sequences include a promoter, and a transcriptional terminator sequence. The portion of a gene or nucleic acid construct containing the 5′ leader sequence, generally 5 to 15 nucleotides in length immediately upstream of the ATG start codon, can also be considered a control sequence as it can affect the efficiency of translation. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the nucleic acid sequence encoding a polypeptide. The term “operably linked” is defined here as a configuration in which a control sequence is placed at a position relative to the coding sequence of the nucleic acid sequence such that the control sequence directs the production of a messenger RNA and/or a polypeptide.
- The term “promoter,” “promoter element,” or “promoter sequence” as used herein, refers to a DNA sequence which when ligated to a nucleotide sequence of interest is capable of controlling the transcription of the nucleotide sequence of interest into mRNA. A promoter is typically, though not necessarily, located 5′ (i.e., upstream) of a nucleotide sequence of interest whose transcription into mRNA it controls, and provides a site for specific binding by RNA polymerase and other transcription factors for initiation of transcription. A DNA sequence is “operatively associated” with the promoter in a cell when RNA polymerase which binds the promoter sequence transcribes the coding sequence into mRNA which is then in turn translated into the protein encoded by the coding sequence.
- The control sequence may also be a suitable transcription terminator sequence, a sequence recognized by a host cell to terminate transcription. The terminator sequence is operably linked to the 3′ terminus of the nucleic acid sequence encoding the polypeptide.
- The control sequence may also be a suitable leader sequence, a nontranslated region of an mRNA which is important for translation by the host cell. The leader sequence is operably linked to the 5′ terminus of the nucleic acid sequence encoding the polypeptide.
- The control sequence may also be a polyadenylation sequence, a sequence operably linked to the 3′ terminus of the nucleic acid sequence and which, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA.
- The control sequence may also be a signal peptide coding region that codes for an amino acid sequence linked to the amino terminus of a polypeptide and directs the encoded polypeptide into the cell's secretory pathway. The 5′ end of the coding sequence of the nucleic acid sequence may inherently contain a signal peptide coding region naturally linked in translation reading frame with the segment of the coding region which encodes the secreted polypeptide. Alternatively, the 5′ end of the coding sequence may contain a signal peptide coding region which is foreign to the coding sequence. The foreign signal peptide coding region may be required where the coding sequence does not naturally contain a signal peptide coding region. Alternatively, the foreign signal peptide coding region may simply replace the natural signal peptide coding region in order to enhance secretion of the polypeptide.
- The control sequence may also be a localization peptide that codes for an amino acid sequence linked to the carboxy terminus of a polypeptide and directs the encoded polypeptide to specific locations in a cell.
- Antisense technology comprises cloning a nucleic acid segment and operatively linking it to a promoter such that the antisense (or complementary) strand of RNA will be transcribed. The construct is then transformed into the host cell and the antisense strand of RNA is produced.
- The term “expression vector” refers to a vector comprising a nucleic acid construct and sequences for delivery into and autonomous replication in microbial host cells. The various nucleic acid and control sequences described above may be joined together to produce a recombinant expression vector which may include one or more convenient restriction sites to allow for insertion or substitution of the nucleic acid sequence encoding the polypeptide at such sites. Alternatively, the nucleic acid sequence of the present invention may be expressed by inserting the nucleic acid sequence or a nucleic acid construct comprising the sequence into an appropriate vector for expression. In creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.
- Modification of a nucleic acid sequence encoding a polypeptide of the present invention may be necessary for the synthesis of polypeptides substantially similar to the polypeptide. The term “substantially similar” to the polypeptide refers to non-naturally occurring forms of the polypeptide. These polypeptides may differ in some engineered way from the polypeptide isolated from its native source, e.g., variants that differ in specific activity, therrnostability, pH optimum, subcellular localization or the like. The variant sequence may be constructed on the basis of the nucleic acid sequence presented as the polypeptide encoding part of an exemplified sequence, a subsequence thereof, and/or by introduction of nucleotide substitutions which do not give rise to another amino acid sequence of the polypeptide encoded by the nucleic acid sequence, but which correspond to the codon usage of the host organism intended for production of the enzyme, or by introduction of nucleotide substitutions which may give rise to a different amino acid sequence. For a general description of nucleotide substitution, see, e.g., Ford et al., 1991,Protein Expression and Purification 2: 95-107.
- It will be apparent to those skilled in the art that such substitutions can be made outside the regions critical to the function of the molecule and still result in an active polypeptide. Amino acid residues essential to the activity of the polypeptide encoded by the isolated nucleic acid sequence of the invention, and therefore preferably not subject to substitution, may be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (see, e.g., Cunningham and Wells, 1989,Science 244: 1081-1085). In the latter technique, mutations are introduced at every positively charged residue in the molecule, and the resultant mutant molecules are tested for enzyme activity to identify amino acid residues that are critical to the activity of the molecule. Sites of substrate-enzyme interaction can also be determined by analysis of the three-dimensional structure as determined by such techniques as nuclear magnetic resonance analysis, crystallography or photoaffinity labelling (see, e.g., de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, Journal of Molecular Biology 224: 899-904; Wlodaver et al., 1992, FEBS Letters 309: 59-64).
- The term “host cell” is a cell which has been transformed or transfected, or is capable of transformation or transfection by an exogenous polynucleotide sequence. The host cell may be a unicellular microorganism, e.g., a prokaryote, or a non-unicellular microorganism, e.g., a eukaryote. Eukaryote cells can include any cell such as from an insect, fungus or plant. Exemplary host cells, introduction of a vector into a host cell, and cloning are described in U.S. Pat. No. 5,374,540 which is hereby incorporated by reference.
- Plant host cells include but are not limited to, somatic cells, gametes or embryos. “Embryo” refers to a sporophytic plant before the start of germination. Embryos can be formed by fertilization of gametes by sexual crossing or by selfing. A “sexual cross” is pollination of one plant by another. “Selfing” is the production of seed by self-pollenization, i.e., pollen and ovule are from the same plant. The term “backcrossing” refers to crossing a F1 hybrid plant to one of its parents. Typically, backcrossing is used to transfer genes which confer a simply inherited, highly heritable trait into an inbred line. The inbred line is termed the recurrent parent. The source of the desired trait is the donor parent. After the donor and the recurrent parents have been sexually crossed, F1 hybrid plants which possess the desired trait of the donor parent are selected and repeatedly crossed (i.e., backcrossed) to the recurrent parent or inbred line.
- Embryos can also be formed by “embryo somatogenesis” and “cloning.” Somatic embryogenesis is the direct or indirect production of embryos from cells, tissues and organs of plants. Indirect somatic embryogenesis is characterized by growth of a callus and the formation of embryos on the surface of the callus. Direct somatic embryogenesis is the formation of an asexual embryo from a single cell or group of cells on an explant tissue without an intervening callus phase. Because abnormal plants tend to be derived from a callus, direct somatic embryogenesis is preferred.
- The term “plant” as used herein refers to a plurality of plant cells which are largely differentiated into a structure that is present at any stage of a plant's development. Such structures include, but are not limited to, whole plants, plant parts or organs, e.g., callus, root, fruit, seed, shoot, stem, tuber, leaf, floret organs including glume, lemma, palea, tapetum, and pollen, and progeny of same. Plant progeny includes progeny of plants, plant parts and plant cells. The class of plants which can be used in the methods of the invention is generally as broad as the class of higher plants amenable to transformation techniques, including both monocotyledonous (a monocot) and dicotyledonous (a dicot) plants.
- The term “progeny” refers to the descendants of a particular plant or regenerant (self-cross) or pair of plants (crossed or backcrossed). The descendants by self-fertilization can be of the T1, the T2, or any subsequent generation, and descendants by crossing can be of the F1, the F2, or any subsequent generation. Typically, the parents are the pollen donor and the ovule donor which are crossed to make the progeny plant of this invention. Parents also refer to F1 parents of a hybrid plant of this invention (the F2 plants). Finally, parents refer to a recurrent parent which is backcrossed to hybrid plants of this invention to produce another hybrid plant of this invention.
- The term “plant tissue” includes differentiated and undifferentiated tissues of plants including, but not limited to, roots, shoots, leaves, pollen, seeds, tumor tissue and various types of cells in culture (e.g., single cells, protoplasts, embryos, callus, etc.). Plant tissue may be in planta, in organ culture, tissue culture, or cell culture.
- Examples of plant parts are stem, callus, leaves, root, fruits, seeds, and tubers. Also specific plant tissues, such as chloroplast, apoplast, mitochondria, vacuole, peroxisomes, and cytoplasm are considered to be a plant part. Furthermore, any plant cell, whatever the tissue origin, is considered to be a plant part.
- “Transgenic plants” are plants into which a nucleic acid sequence has been introduced through recombinant techniques, i.e., nucleic acid-containing vectors, cloning, somatic embryogenesis or any other technique used by those of skill to produce plants.
- “Line” pertains to a plant or its germplasm, a primary regenerant from transformation, (To) plant or its progeny, resulting from genetic transformation, in which all or a portion of transgene has been stably integrated.
- The term “monocot” refers to a plant species having a single cotyledon, including wheat, oat, barley, rice, rye, triticale, maize (corn), and other cereals, as well as sugarcane, sorghum, pineapple, yam, onion, banana, coconut, date, hops, and grasses such as meadow grass and forage grass.
- The common names of cereal crop plants used throughout this disclosure refer to varieties of plants of the following genera:
Common Name Genera Wheat (soft, hard and durum varieties) Triticum Sorghum Sorghum Rice Oryza Barley Hordeum Maize or corn Zea Rye Secale Triticale Triticale Oat Avena - Dicotyledonous refers to plant species characterized by a pair of embryonic seed leaves that appear at germination, including tobacco, potato, tomato, soybean, pea, bean, sugar beet, papaya, Cucurbita (squash, cucumber, melon, pumpkin, zucchini), stone fruit trees, cotton, sweet potato, cruciferous plants (family Brassicaceae), such as cauliflower, rape seed, and the closely related model organismArabidopsis thaliana.
- Other plants susceptible to fungal or bacterial diseases are also encompassed, including, for example, grapes, coca beans, and nuts.
- “EST” refers to Expressed sequence tag; nucleotide sequence of a complete or partial cDNA, representing a transcript or messenger RNA that is expressed in an organ, tissue or cell type; usually 100 or more nucleotides in length.
- I. Fungal Cell Wall-Degrading Enzymes And Nucleic Acid Molecules Encoding the Enzymes
- The present invention is directed to isolated nucleic acid sequences derived from Fusarium fungal genes which encode polypeptides having cell wall-degrading activity comprising glucanase, endochitinase or exochitinase activity as well as isolated polypeptides having cell wall-degrading activity.
- A1. Nucleic Acid Molecules Which Encode Glucanase Activity
- Glucanase refers to a polypeptide having the ability to degrade the β-1,3 linkages of glucan. In a first embodiment, the present invention is directed to isolated nucleic acid molecules which encode a polypeptide having glucanase activity, selected from the group consisting of:
- (a) a nucleic acid sequence having at least 70% nucleotide sequence identity with SEQ ID NO:1 from nucleotide 37 through nucleotide 942, SEQ ID NO:3 from nucleotide 1801 through nucleotide 2761 or SEQ ID NO:16 from nucleotide 18 to nucleotide 923;
- (b) a nucleic acid sequence encoding a polypeptide having an amino acid sequence which has at least 80% identity with
amino acids 1 to 301 of SEQ ID NO:2, 4, 5 or 17; - (c) a nucleic acid sequence which hybridizes under medium or high -stringency conditions with (i) SEQ ID NO:1 from nucleotide 37 through nucleotide 942, SEQ ID NO:3 from nucleotide 1801 through nucleotide 2761 or SEQ ID NO:16 from nucleotide 18 to nucleotide 923; (ii) a subsequence of (i) of at least 100 contiguous nucleotides, or (iii) a complementary strand of (i) or (ii); and
- (d) a subsequence of (a), (b) or (c) wherein the subsequence encodes a polypeptide fragment which has glucanase activity.
- The isolated nucleic acid molecules which encode a polypeptide having glucanase activity include genomic sequences which encode a glucanase and which direct and regulate the transcriptional and translational expression of the glucanase coding sequences, and cDNA sequences which encode a polypeptide having glucanase activity.
- Specific embodiments of nucleotide sequences which encode polypeptides having glucanase activity are given in SEQ ID NOS:1, 3, and 16. An exemplified glucanase gene product has a predicted amino acid sequence as given in SEQ ID NOS:2, 4, 5, and 17. A genomic DNA sequence containing the full-length glucanase gene is presented in SEQ ID NO:3. The genomic DNA sequence is 3622 bp in length, and nucleotide sequence analysis reveals 2 exons and 1 intron. The coding region comprises nucleotide 1801 to 2014 and 2070 to 2761 which encodes a
protein 301 amino acids in length (SEQ ID NO:4). A F. venenatum glucanase full-length unmodified cDNA sequence is given in SEQ ID NO:1. The cDNA sequence is 1023 bp in length. The resulting open reading frame (coding portion), initiating at base 37 and terminating at base 942 encodes aprotein 301 amino acids in length (SEQ ID NO:2). A F. venenatum modified full length cDNA sequence which encodes a glucanase is given in SEQ ID NO:16. This cDNA is 932 bp in length. The open reading frame initiating at base 18 and terminating at base 923 encodes a protein of 301 amino acids (SEQ ID NO:17). One modification made to the unmodified glucanase sequence is the substitution of a CA-rich 5′ leader sequence, GGATCCACCAACCAGCG, (bases #1-#17 ofGLUC 5′ primer, Table 2). The modified 5′ leader sequence occurs immediately upstream of the ATG start codon of the glucanase coding sequence. The modification renders the leader sequence rich in C and A nucleotides, and minimizes the number of T nucleotides. These characteristics are reported to enhance the translational efficiency of genes in plants. SEQ ID NO:5 shows a glucanase having 301 amino acids which is encoded by a F. venenatum cDNA. - In another embodiment, the nucleic acid molecule is the sequence contained in plasmid GLU2 (SEQ ID NO:16 which is aF. venenatum glucanase modified full-length cDNA sequence) that is contained in Escherichia coli NRRL B-30201.
- In another embodiment, the nucleic acid molecule is the sequence contained in plasmids FvGluS or FvGluAS that are contained inEscherichia coli NRRL B-30204 and NRRL B-30205, respectively.
- The invention also encompasses nucleic acid sequences which have a degree of sequence identity to the coding region of SEQ ID NO:1, 3 or 16 or coding region plus intron of SEQ ID NO:3 of at least 70%, preferably at least about 75%, preferably at least about 80%, more preferably at least about 85%, more preferably at least about 90%, and even more preferably about 95% and which encode a polypeptide effective to degrade β-1,3-linkages of glucan. For purposes of the present invention, the degree of identity between two nucleic acid sequences is determined by the Clustal method (Thompson et al. 1994), using ClustalW 1.7 or 1.8 (http://dot.imgen.bcm.tmc.edu:9331 /multi-align/multi-align.html) with the following pairwise alignment parameters: K-tuple=2; gap penalty=5; window size=4; diagonals−4. Multiple alignment parameters were: gap opening penalty=10; gap extension penalty=5.
- Further, nucleic acid sequences which hybridize under medium stringency or high stringency conditions, as defined above, with the coding region of the DNA sequence of SEQ ID NO:1, 3 or 16 or coding region plus intron of SEQ ID NO:3 and which encode a polypeptide effective to degrade β-1,3-linkages of glucan are included in the present invention.
- Also encompassed by the invention are subsequences of at least 100 contiguous nucleotides and preferably at least 200 contiguous nucleotides which hybridize under medium stringency or high stringency conditions, as defined above, with the coding region of the DNA sequence of SEQ ID NO:1, 3 or 16 or coding region plus intron of SEQ ID NO:3.
- The invention further encompasses a complementary strand of a nucleic acid sequence or subsequence of at least 100 contiguous nucleotides and preferably at least 200 contiguous nucleotides of a nucleic acid sequence which hybridizes under medium stringency or high stringency conditions, as defined above, with the coding region of the DNA sequence of SEQ ID NO:1, 3 or 16 or coding region plus intron of SEQ ID NO:3.
- The invention is further directed to a subsequence of the aforenamed nucleic acid sequences wherein the subsequence encodes a polypeptide fragment which has glucanase activity.
- A2. Polypeptides Having Glucanase Activity
- The present invention is also directed to isolated polypeptides having glucanase activity which are encoded by the nucleic acid molecules described above. Isolated polypeptides having glucanase activity comprise:
- (a) a polypeptide having an amino acid sequence which has at least 80% identity with
amino acids 1 to 301 of SEQ ID NO:2, 4, 5 or 17; - (b) a polypeptide encoded by a nucleic acid sequence which hybridizes under medium stringency or high stringency conditions with (i) SEQ ID NO:1 from nucleotide 37 through nucleotide 942, SEQ ID NO:3 from nucleotide 1801 through nucleotide 2761 or SEQ ID NO:16 from nucleotide 18 to nucleotide 923; (ii) a subsequence of (i) of at least 100 nucleotides, or (iii) a complementary strand of (i) or (ii); and
- (c) a fragment of (a), or (b) that has glucanase activity.
- FIG. 1 is a comparison of full-length glucanases encoded byF. venenatum cDNA (upper) and F. sporotrichioides genomic DNA (lower) (SEQ ID NOS:5 and 4, respectively).
- Isolated nucleic acid sequences encoding polypeptides comprising an amino acid sequence which has a degree of identity to the amino acid sequence of SEQ ID NO:2, 4, 5 or 17 with
amino acids 1 to 301 of at least about 80%, preferably at least 85%, more preferably at least about 90%, further more preferably at least about 95%, and most preferably at least about 97%, and which have glucanase activity (homologous polypeptides), are encompassed by the invention. For purposes of the present invention, the degree of identity between two amino acids is determined by the FASTA/FASTP method of Pearson (1990), using ALIGN (http://dot.imgen.bcm.tmc.edu:9331 /seq-search/alignment.html), with the BLOSUM50 or PAM250 scoring matrix and the following pairwise alignment parameters: gap penalty=−12 for the first residue in the gap, and −2 for additional residues; K-tuple=2. Multiple alignments are carried out with the BLOSUM matrix, using the ClustalW 1.7 algorithm (Thompson et al. 1994). Multiple alignment parameters are: gap opening penalty=10, gap extension penalty=0.05; hydrophilic gap penalties on (hydrophilic residues GPSNDQERK); with residue-specific gap penalties. - Preferably, the polypeptides of the present invention comprise an amino acid sequence of SEQ ID NO:2, 4, 5 or 17 or a fragment thereof that has glucanase activity. In another preferred embodiment, the polypeptide of the present invention comprises
amino acids 1 to 301 of SEQ ID NO:2, 4, 5 or 17 or a fragment thereof that has glucanase activity. - A fragment of SEQ ID NO:2, 4, 5 or 17 is a polypeptide having one or more amino acids deleted from the amino and/or carboxyl terminus of this amino acid sequence. Preferably, a fragment contains at least 256 amino acid residues, more preferably at least 271 amino acid residues, and most preferably at least 286 amino acid residues.
- The present invention relates to isolated polypeptides having glucanase activity which are encoded by nucleic acid sequences which hybridize under medium stringency conditions or high stringency conditions, as described in detail above, with (i) SEQ ID NO:1 from nucleotide 37 through nucleotide 942, SEQ ID NO:3 from nucleotide 1801 through nucleotide 2761 or SEQ ID NO:16 from nucleotide 18 to nucleotide 923; (ii) a subsequence of (i) of at least 100 nucleotides, or (iii) a complementary strand of (i) or (ii). The subsequence of SEQ ID NO:1, 3 or 16 may be at least 100 contiguous nucleotides or preferably at least 200 contiguous nucleotides. Moreover, the subsequence may encode a polypeptide fragment which has glucanase activity.
- The degeneracy of the genetic code is well known to the art; therefore, synonymous coding sequences with one or more codon substitutions can be readily determined by one of ordinary skill in the art. Synonymous coding sequences vary from the exemplified coding sequences but encode proteins of the same amino acid sequences as those specifically provided herein.
- Examples of conservative substitutions are within the groups of basic amino acids (such as arginine, lysine and histidine), acidic amino acids (such as glutamic acid and aspartic acid), polar amino acids (such as glutamine and asparagine), hydrophobic amino acids (such as leucine isoleucine and valine), aromatic amino acids (such as phenylalanine, tryptophan and tyrosine), and small amino acids (such as glycine, alanine, serine, threonine and methionine). Amino acid substitutions which do not generally alter the specific activity are known in the art as described, for example, by H. Neurath and R. L. Hill, 1979, In,The Proteins, Academic Press, New York. The most commonly occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly as well as these in reverse.
- The polypeptides of the present invention have at least 20%, preferably at least 40%, more preferably at least 60%, even more preferably at least 80%, even more preferably at least 90%, and most preferably at least 100% of the glucanase activity of the polypeptide of SEQ ID NO:2, 4, 5 or 17.
- A polypeptide of the present invention may be obtained from microorganisms of any genus. For purposes of the present invention, the term “obtained from” as used herein in connection with a given source shall mean that the polypeptide encoded by the nucleic acid sequence is produced by the source or by a cell in which the nucleic acid sequence from the source has been inserted.
- A polypeptide of the present invention may be a fungal polypeptide, for example, Aspergillus, Fusarium, Magnaporthe, or Phytophthora. In a preferred embodiment, the polypeptide is aFusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium helerosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, or Fusarium venenatum polypeptide.
- In a more preferred embodiment, theFusarium venenatum cell is Fusarium venenatum A3/5, which was originally deposited as Fusarium graminearum ATCC 20334 and recently reclassified as Fusarium venenatum by Yoder and Christianson, 1998, Fungal Genetics and Biology 23: 62-80 and O'Donnell et al., 1998, Fungal Genetics and Biology 23: 57-67; as well as taxonomic equivalents of Fusarium venenatum regardless of the species name by which they are currently known. In another preferred embodiment, the Fusarium venenatum cell is a morphological mutant of Fusarium venenatum A3/5 or Fusarium venenatum ATCC 20334, as disclosed in WO 97/26330.
- It will be understood that for the aforementioned species, the invention encompasses both the perfect and imperfect states, and other taxonomic equivalents, e.g., anamorphs, regardless of the species name by which they are known. Those skilled in the art will readily recognize the identity of appropriate equivalents. For example, taxonomic equivalents of Fusarium are defined by D. L. Hawksworth, P. M. Kirk, B. C. Sutton, and D. N. Pegler (editors), 1995, In Ainsworth & Bisby'sDictionary of the Fungi, Eighth Edition, CAB International, University Press, Cambridge, England, pp. 173- 174.
- Strains of these species are readily accessible to the public in a number of culture collections, such as the American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), and Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).
- Furthermore, such polypeptides may be identified and obtained from other sources including microorganisms isolated from nature (e.g., soil, composts, water, etc.) using the above-mentioned probes. Techniques for isolating microorganisms from natural habitats are well known in the art. The nucleic acid sequence may then be derived by similarly screening a genomic or cDNA library of another microorganism. Once a nucleic acid sequence encoding a polypeptide has been detected with the probe(s), the sequence may be isolated or cloned by utilizing techniques which are known to those of ordinary skill in the art (see, e.g., Sambrook et al., 1989, supra).
- As defined herein, an “isolated” polypeptide is a polypeptide which is essentially free of other non-glucanase polypeptides, e.g., at least about 20% pure, preferably at least about 40% pure, more preferably about 60% pure, even more preferably about 80% pure, most preferably about 90% pure, and even most preferably about 95% pure, as determined by SDS-PAGE.
- Polypeptides encoded by nucleic acid sequences of the present invention also include fused polypeptides or cleavable fusion polypeptides in which another polypeptide is fused at the N-terminus or the C-terminus of the polypeptide or fragment thereof. A fused polypeptide is produced by fusing a nucleic acid sequence (or a portion thereof) encoding another polypeptide to a nucleic acid sequence (or a portion thereof) of the present invention. Techniques for producing fusion polypeptides are known in the art, and include ligating the coding sequences encoding the polypeptides so that they are in frame and that expression of the fused polypeptide is under control of the same promoter(s) and terminator.
- B1. Nucleic Acid Molecules Which Encode Endochitinase Activity
- Endochitinase refers to a polypeptide that degrades chitin by cleaving randomly between C1 and C4 linkages within the chitin polymer. In a first embodiment, the present invention is directed to isolated nucleic acid molecules which encode a polypeptide having endochitinase activity, selected from the group consisting of:
- (a) a nucleic acid sequence having at least 75% nucleotide sequence identity with SEQ ID NO:10 from nucleotide 186 through nucleotide 1385 or SEQ ID NO:18 from nucleotide 18 to nucleotide 1217;
- (b) a nucleic acid sequence encoding a polypeptide having endochitinase activity, said polypeptide having an amino acid sequence which has at least 85% identity with
amino acids 1 to 399 of SEQ ID NO:11 or 19; - (c) a nucleic acid sequence which hybridizes under medium stringency or high stringency conditions with (i) SEQ ID NO:10 from nucleotide 186 through nucleotide 1385 or SEQ ID NO:18 from nucleotide 18 to nucleotide 1217; (ii) a subsequence of (i) of at least 100 contiguous nucleotides, or (iii) a complementary strand of (i) or (ii); and
- (d) a subsequence of (a), (b) or (c) wherein the subsequence encodes a polypeptide fragment which has endochitinase activity.
- The fungal endochitinase nucleic acid molecules include genomic sequences which encode an endochitinase and which direct and regulate the transcriptional and translational expression of the endochitinase coding sequences, and cDNA sequences which encode an endochitinase.
- Specific embodiments of nucleotide sequences which encode a polypeptide having endochitinase activity are given in SEQ ID NOS :10 and 18. An exemplified endochitinase gene product has a predicted amino acid sequence as given in SEQ ID NOS:11 AND 19. AF. venenatum endochitinase full-length unmodified cDNA sequence is given in SEQ ID NO:10. The cDNA sequence is 1494 bp in length. The resulting open reading frame (coding portion), initiating at base 186 and terminating at base 1385 encodes a protein 399 amino acids in length (SEQ ID NO:11). A modified full length cDNA sequence which encodes a polypeptide having endochitinase activity is given in SEQ ID NO:18. This cDNA is 1227 bp in length. The open reading frame initiating at base 18 and terminating at base 1217 encodes a protein of 399 amino acids (SEQ ID NO:19). One modification made to the unmodified endochitinase sequence is the substitution of a CA-rich 5′ leader sequence, GGATCCACCAACCAGCG, (bases #1-#17 of
ENDO 5′ primer, Table 2). The modified 5′ leader sequence occurs immediately upstream of the ATG start codon of the endochitinase coding sequence. The modification renders the leader sequence rich in C and A nucleotides, and minimizes the number of T nucleotides. These characteristics are reported to enhance the translational efficiency of genes in plants. The DNA sequence containing the 5′ portion of the endochitinase cDNA (FIG. 2), from which the remainder of the primer was designed, is presented in SEQ ID NO:6. The partial cDNA is 467 bp in length. - In another embodiment, the nucleic acid molecule is the sequence contained in plasmid Endo 167 (SEQ ID NO:18 which is aF. venenatum endochitinase modified full-length cDNA sequence) that is contained in Escherichia coli NRRL B-30202.
- In another embodiment, the nucleic acid molecule is the sequence contained in plasmids FvEndoS or FvEndoAS that are contained inEscherichia coli NRRL B-30206 and NRRL B-30207, respectively.
- The invention also encompasses nucleic acid sequences which have a degree of sequence identity to the coding region of SEQ ID NO:I0 or 18 of at least 75%, preferably at least about 80%, more preferably at least about 85%, more preferably at least about 90%, and even more preferably about 95% and which encode a polypeptide having endochitinase activity, that is, the ability to degrade chitin by cleavage of internal glycosidic linkages. For purposes of the present invention, the degree of identity between two nucleic acid sequences is determined by the Clustal method (Thompson et al. 1994), using ClustalW 1.7 or 1.8 (http://dot.imgen.bcm.tmc.edu:9331 /multi-align/multi-align.html) with the following pairwise alignment parameters: K-tuple=2; gap penalty=5; window size=4; diagonals−4. Multiple alignment parameters were: gap opening penalty=10; gap extension penalty=5.
- Further, nucleic acid sequences which hybridize under medium stringency or high stringency conditions, as defined above, with the coding region of the DNA sequence of SEQ ID NO:10 or 18 and which encode a polypeptide having endochitinase activity are encompassed by this invention.
- Also encompassed by the invention are subsequences of at least 100 contiguous nucleotides and preferably at least 200 contiguous nucleotides which hybridize under medium stringency or high stringency conditions, as defined above, with the coding region of the DNA sequence of SEQ ID NO:10 or 18.
- The invention further encompasses a complementary strand of a nucleic acid sequence or subsequence of at least 100 contiguous nucleotides and preferably at least 200 contiguous nucleotides of a nucleic acid sequence which hybridizes under medium stringency or high stringency conditions, as defined above, with the coding region of the DNA sequence of SEQ ID NO:10 or 18.
- The invention is further directed to a subsequence of the aforenamed nucleic acid sequences wherein the subsequence encodes a polypeptide fragment which has endochitinase activity.
- B2. Polypeptides Having Endochitinase Activity
- The present invention is also directed to isolated polypeptides having endochitinase activity which are encoded by the nucleic acid molecules described above. Isolated polypeptides having endochitinase activity comprise:
- (a) a polypeptide having an amino acid sequence which has at least 85% identity with
amino acids 1 to 399 of SEQ ID NO:11 or 19; - (b) a polypeptide encoded by a nucleic acid sequence which hybridizes under medium stringency or high stringency conditions with (i) SEQ ID NO:10 from nucleotide 186 through nucleotide 1385 or SEQ ID NO:18 from nucleotide 18 to nucleotide 1217; (ii) a subsequence of (i) of at least 100 contiguous nucleotides, or (iii) a complementary strand of (i) or (ii); and
- (c) a fragment of (a), or (b) that has endochitinase activity.
- Isolated nucleic acid sequences encoding polypeptides comprising an amino acid sequence which has a degree of identity to the amino acid sequence of SEQ ID NO:11 or 19 of at least about 85%, preferably at least about 90%, more preferably at last about 95%, and most preferably at least about 97%, and which have endochitinase activity, (homologous polypeptides), are encompassed by the invention. For purposes of the present invention, the degree of identity between two amino acids is determined by the FASTA/FASTP method of Pearson (1990), using ALIGN (http://dot.imgen.bcm.tmc.edu:9331 /seq-search/alignment.html), with the BLOSUM50 or PAM250 scoring matrix and the following pairwise alignment parameters: gap penalty=−12 for the first residue in the gap, and −2 for additional residues; K-tuple=2. Multiple alignments are carried out with the BLOSUM matrix, using the ClustalW 1.7 algorithm (Thompson et al. 1994). Multiple alignment parameters are: gap opening penalty=10, gap extension penalty=0.05; hydrophilic gap penalties on (hydrophilic residues GPSNDQERK); with residue-specific gap penalties.
- Preferably, the polypeptides of the present invention comprise an amino acid sequence of SEQ ID NO:11 or 19 or a fragment thereof that has endochitinase activity. In another preferred embodiment, the polypeptide of the present invention comprises
amino acids 1 to 399 of SEQ ID NO:11 or 19 or a fragment thereof that has endochitinase activity. - A fragment of SEQ ID NO:11 or 19 is a polypeptide having one or more amino acids deleted from the amino and/or carboxyl terminus of this amino acid sequence. Preferably, a fragment contains at least 339 amino acid residues, more preferably at least 359 amino acid residues, and most preferably at least 379 amino acid residues.
- The present invention relates to isolated polypeptides having endochitinase activity which are encoded by nucleic acid sequences which hybridize under medium stringency conditions or high stringency conditions, as described in detail above, with (i) SEQ ID NO:10 from nucleotide 186 through nucleotide 1385 or SEQ ID NO:18 from nucleotide 18 to nucleotide 1217; (ii) a subsequence of (i) of at least 100 contiguous nucleotides, or (iii) a complementary strand of (i) or (ii).
- The subsequence of SEQ ID NO:10 or 18 may be at least 100 contiguous nucleotides or preferably at least 200 contiguous nucleotides. Moreover, the subsequence may encode a polypeptide fragment which has endochitinase activity. Synonymous coding sequences and conservative amino acid substitutions, as described in detail, above, are incorporated herein by reference.
- The polypeptides of the present invention have at least 20%, preferably at least 40%, more preferably at least 60%, even more preferably at least 80%, even more preferably at least 90%, and most preferably at least 100% of the endochitinase activity of the polypeptide of SEQ ID NO:11 or 19.
- A polypeptide of the present invention may be obtained from microorganisms of any genus as described in detail above and which is incorporated herein by reference.
- As defined herein, an “isolated” polypeptide is a polypeptide which is essentially free of other non-endochitinase polypeptides, e.g., at least about 20% pure, preferably at least about 40% pure, more preferably about 60% pure, even more preferably about 80% pure, most preferably about 90% pure, and even most preferably about 95% pure, as determined by SDS-PAGE.
- Polypeptides encoded by nucleic acid sequences of the present invention also include fused polypeptides or cleavable fusion polypeptides as described in detail above and which is incorporated herein by reference.
- C1. Nucleic Acid Molecules Which Encode Exochitinase Activity
- Exochitinase refers to a polypeptide having the ability to degrade chitin by cleaving sequentially between each C1-C4 linkage initiating from the terminus of the chitin polymer. In a first embodiment, the present invention is directed to isolated nucleic acid molecules which encode a polypeptide having endochitinase activity, selected from the group consisting of:
- (a) a nucleic acid sequence having at least 75% nucleotide sequence identity with SEQ ID NO:12 from nucleotide 108 to 1853 or SEQ ID NO:20 from nucleotide 18 to 1763;
- (b) a nucleic acid sequence encoding a polypeptide having endochitinase activity, said polypeptide having an amino acid sequence which has at least 85% identity with
amino acids 1 to 581 of SEQ ID NO:13 or 21; - (c) a nucleic acid sequence which hybridizes under medium stringency or high stringency conditions with (i) SEQ ID NO:12 from nucleotide 108 to 1853 or SEQ ID NO:20 from nucleotide 18 to 1763; (ii) a subsequence of (i) of at least 100 contiguous nucleotides, or (iii) a complementary strand of (i) or (ii); and
- (d) a subsequence of (a), (b) or (c) wherein the subsequence encodes a polypeptide fragment which has endochitinase activity.
- The isolated nucleic acid molecules which encode a polypeptide having exochitinase activity include genomic sequences which encode an exochitinase and which direct and regulate the transcriptional and translational expression of the exochitinase coding sequences, and cDNA sequences which encode an exochitinase.
- Specific embodiments of nucleotide sequences which encode exochitinase are given in SEQ ID NOS:8, 12 and 20. An exemplified exochitinase gene product has a predicted amino acid sequence as given in SEQ ID NOS:13 and 21. A Fusarium exochitinase full-length unmodified cDNA sequence is given in SEQ ID NO:12. The cDNA sequence is 1949 bp in length. The resulting open reading frame (coding portion), initiating at base 108 and terminating at base 1853 encodes a protein 581 amino acids in length. The encoded protein is described in SEQ ID NO:13. AF. venenatum modified full length cDNA sequence which encodes exochitinase is given in SEQ ID NO:20. This cDNA is 1781 bp in length. The open reading frame, initiating at base 18 and terminating at base 1763 encodes a protein of 581 amino acids (SEQ ID NO:21). One modification made to the unmodified exochitinase sequence is the substitution of a CA-rich 5′ leader sequence, GGATCCACCAACCAGCG, (bases #1-#17 of
EXO 5′ primer, Table 2). The modified 5′ leader sequence occurs immediately upstream of the ATG start codon of the exochitinase coding sequence. The modification renders the leader sequence rich in C and A nucleotides, and minimizes the number of T nucleotides. These characteristics are reported to enhance the translational efficiency of genes in plants. - The genomic DNA sequence containing a 5′ portion of the exochitinase gene ofF. sporotrichioides is presented in SEQ ID NO:8 (FIG. 3). The deduced polypeptide sequence is given in SEQ ID NO:9. The genomic DNA sequence is 995 bp in length, and nucleotide sequence analysis reveals 2 exons and 1 intron.
- In another embodiment, the nucleic acid molecule is the sequence contained in plasmid Exo9 (SEQ ID NO:20 which is aF. venenatum exochitinase modified full-length cDNA sequence) that is contained in Escherichia coli NRRL B-30203.
- In another embodiment, the nucleic acid molecule is the sequence contained in plasmids FvExoS or FvExoAS that are contained inEscherichia coli NRRL B-30208 and NRRL B-30209, respectively.
- The invention also encompasses nucleic acid sequences which have a degree of sequence identity to the coding region of SEQ ID NO:12 or 20 of at least 75%, preferably at least about 80%, more preferably at least about 85%, more preferably about 90%, and even more preferably about 95% and which encode a polypeptide having exochitinase activity, that is, ability to degrade chitin by cleavage of terminal glycosidic linkages. For purposes of the present invention, the degree of identity between two nucleic acid sequences is determined by the Clustal method (Thompson et al. 1994), using ClustalW 1.7 or 1.8 (http://dot.impen.bcm.tmc.edu:9331/multi-align/multi-align.html) with the following pairwise alignment parameters: K-tuple=2; gap penalty=5; window size=4; diagonals−4. Multiple alignment parameters were: gap opening penalty=10; gap extension penalty=5.
- Further, nucleic acid sequences which hybridize under medium stringency or high stringency conditions, as defined above, with the coding region of the DNA sequence of SEQ ID NO:12 or 20 and which encode a polypeptide having exochitinase activity are encompassed by this invention.
- Also encompassed by the invention are subsequences of at least 100 contiguous nucleotides and preferably at least 200 contiguous nucleotides which hybridize under medium stringency or high stringency conditions, as defined above, with the coding region of the DNA sequence of SEQ ID NO:12 or 20.
- The invention further encompasses a complementary strand of a nucleic acid sequence or subsequence of at least 100 contiguous nucleotides and preferably at least 200 contiguous nucleotides of a nucleic acid sequence which hybridizes under medium stringency or high stringency conditions, as defined above, with the coding region of the DNA sequence of SEQ ID NO:12 or 20.
- The invention is further directed to a subsequence of the aforenamed nucleic acid sequences wherein the subsequence encodes a polypeptide fragment which has exochitinase activity.
- C2. Polypeptides Having Exochitinase Activity
- The present invention is also directed to isolated polypeptides having exochitinase activity which are encoded by the nucleic acid molecules described above. Isolated polypeptides having exochitinase activity comprise:
- (a) a polypeptide having an amino acid sequence which has at least 85% identity with
amino acids 1 to 581 of SEQ ID NO:13 or 21; - (b) a polypeptide encoded by a nucleic acid sequence which hybridizes under medium stringency or high stringency conditions with (i) SEQ ID NO:12 from nucleotide 108 to 1853 or SEQ ID NO:20 from nucleotide 18 to 1763; (ii) a subsequence of (i) of at least 100 contiguous nucleotides, or (iii) a complementary strand of (i) or (ii); and
- (c) a fragment of (a), or (b) that has exochitinase activity.
- FIG. 4 shows the comparison of exochitinases encoded byF. venenatum cDNA (upper) and F. sporotrichioides genomic DNA (lower) (SEQ ID NOS:14 and 15, respectively).
- Isolated nucleic acid sequences encoding polypeptides comprising an amino acid sequence which has a degree of identity to the amino acid sequence of SEQ ID NO:13 or 21 of at least about 85%, preferably at least about 90%, more preferably at last about 95%, and most preferably at least about 97%, and which have exochitinase activity, (homologous polypeptides), are encompassed by the invention. For purposes of the present invention, the degree of identity between two amino acids is determined by the FASTA/FASTP method of Pearson (1990), using ALIGN (http://dot.imgen.bcm.tmc.edu:9331 /seq-search/alignment.html), with the BLOSUM50 or PAM250 scoring matrix and the following pairwise alignment parameters: gap penalty=−12 for the first residue in the gap, and −2 for additional residues; K-tuple=2. Multiple alignments are carried out with the BLOSUM matrix, using the ClustalW 1.7 algorithm (Thompson et al. 1994). Multiple alignment parameters are: gap opening penalty=10, gap extension penalty=0.05; hydrophilic gap penalties on (hydrophilic residues GPSNDQERK); with residue-specific gap penalties.
- Preferably, the polypeptides of the present invention comprise an amino acid sequence of SEQ ID NO:13 or 21 or a fragment thereof that has exochitinase activity. In another preferred embodiment, the polypeptide of the present invention comprises
amino acids 1 to 581 of SEQ ID NO:13 or 21 or a fragment thereof that has exochitinase activity. - A fragment of SEQ ID NO:13 or 21 is a polypeptide having one or more amino acids deleted from the amino and/or carboxyl terminus of this amino acid sequence. Preferably, a fragment contains at least 494 amino acid residues, more preferably at least 523 amino acid residues, and most preferably at least 552 amino acid residues.
- The present invention relates to isolated polypeptides having exochitinase activity which are encoded by nucleic acid sequences which hybridize under medium stringency conditions or high stringency conditions, as described in detail above, with (i) SEQ ID NO:12 from nucleotide 108 to 1853 or SEQ ID NO:20 from nucleotide 18 to 1763; (ii) a subsequence of (i) of at least 100 contiguous nucleotides, or (iii) a complementary strand of (i) or (ii).
- The subsequence of SEQ ID NO:12 or 20 may be at least 100 contiguous nucleotides or preferably at least 200 contiguous nucleotides. Moreover, the subsequence may encode a polypeptide fragment which has exochitinase activity. Synonymous coding sequences and conservative amino acid substitutions, as described in detail, above, are incorporated herein by reference.
- A polypeptide of the present invention may be obtained from microorganisms of any genus as described in detail above and which is incorporated herein by reference.
- As defined herein, an “isolated” polypeptide is a polypeptide which is essentially free of other non-exochitinase polypeptides, e.g., at least about 20% pure, preferably at least about 40% pure, more preferably about 60% pure, even more preferably about 80% pure, most preferably about 90% pure, and even most preferably about 95% pure, as determined by SDS-PAGE.
- Polypeptides encoded by nucleic acid sequences of the present invention also include fused polypeptides or cleavable fusion polypeptides as described in detail above and which is incorporated herein by reference.
- D. Nucleic Acid Molecules Encoding Cell Wall-Degrading Enzymes
- Any isolated cell wall-degrading nucleic acid molecule encoding a polypeptide having glucanase, endochitinase or exochitinase activity can be used in the present invention. The particular polynucleotide and amino acid sequences used are not critical features of the invention, so long as the desired cell wall-degrading function is achieved.
- E. Comparison to Known Fungal Cell Wall Hydrolytic Enzymes
- Computerized searches of the GenBank/EMBL sequence databases (Altschul et al. 1997) indicated that theF. venenatum full length cDNA sequences encoded polypeptides that had identity to known fungal cell wall hydrolytic enzymes. The F. venenatum glucanase shares 37% amino acid sequence identity with
glucan 1,3-β-glucosidases from the yeast Schizosaccharomyces pombe (GenBank accession nos. Z99126 and AB000539), and 34% amino acid sequence identity withglucan 1,3-β-glucosidase precursors from the yeast Saccharomyces cerevisiae (SwissPro accession no. P15703; Klebl and Tanner 1989) and the yeast Candida albicans (SwissPro accession no. P43070). The F. venenatum endochitinase and exochitinase are similar to chitinases from other fungi. The endochitinase shares 56% amino acid sequence identity with an Aspergillus nidulans chitinase (GenBank accession no. D87063), 53% identity with chitinase antigens from Coccidioides immitis (Yang et al. 1996; Zimmerman et al. 1996), and 48% identity to a chitinase from Aphanocladium album (Blaiseau and Lafay 1992). The F. venenatum exochitinase had 66% and 58% amino acid sequence identity with two exochitinases from Trichoderma harzianum (Draborg et al. 1995), and 64% identity with a N-acetyl-β-D-glucosaminidase from T. harzianum (Peterbauer et al. 1996). - II. Using Probes to Identify And Isolate Homologs of The Sequences
- The isolation of other Fusarium cell wall-degrading genes or homolog genes, including non-functional sequence homologs, may be accomplished by a number of techniques. For instance, nucleic acid probes based on the disclosed sequences can be used to isolate the desired gene from a cDNA or genomic DNA library, as described in detail, above. Nucleic acid probes can be either DNA or RNA. The desired gene can be isolated by hybridization of the probes to target sequences, including genomic library clones, cDNA library clones, or uncloned isolated DNA or cDNA fragments. Hybridization can be carried out under stringent, highly stringent, or low-stringency conditions either on membranes or in solution. Nucleic acid probes can be used as restriction fragment length polymorphic (RFLP) markers for the genetic mapping of loci, especially in fungi, or for the identification of homologous genes or loci in fungi.
- III. Using Primers to Amplify Nucleotide Sequences
- Primers based on the disclosed sequences can be used to isolate the desired genes from a cDNA or genomic library. The nucleic acids of interest can be amplified from nucleic acid samples using amplification techniques. For instance, polymerase chain reaction (PCR) technology can be used to amplify the sequences of the Fusarium cell wall-degrading enzymes and related genes directly from genomic DNA, from cDNA, from genomic libraries or cDNA libraries. PCR and other in vitro amplification methods may also be useful, for example, to clone nucleic acid sequences that code for proteins to be expressed, to make nucleic acids to use as probes for detecting the presence of the desired DNA or mRNA in samples, for nucleic acid sequencing, or for other purposes. For a general overview of PCR see, PCR PROTOCOLS: A GUIDE TO METHODS AND APPLICATIONS, Innis, et al. (eds.), Academic Press, San Diego (1990).
- IV. Preparation of Recombinant Nucleic Acid Molecules (Constructs)
- In one embodiment of this invention, recombinant nucleic acid molecules which contain isolated cell wall-degrading sequences and are suitable for transformation of host cells are prepared. A nucleic acid sequence coding for the desired polypeptide, for example, a cDNA or a genomic sequence encoding a full length protein or, a nucleic acid sequence encoding a homologous polypeptide is conveniently used to construct a recombinant expression cassette which can be introduced into the desired host cell. An expression cassette will typically comprise the Fusarium cell wall-degrading nucleic acid sequence operably linked to a one or more control sequences which direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences, and may include other transcriptional and translational initiation regulatory sequences which will direct the transcription of the sequence from the sense or antisense gene in the intended tissues of the transformed host cell. Expression will be understood to include any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.
- An isolated nucleic acid sequence encoding a polypeptide of the present invention may be manipulated in a variety of ways to provide for expression of the polypeptide. Manipulation of the nucleic acid sequence prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying nucleic acid sequences utilizing recombinant DNA methods are well known in the art.
- The control sequences include all components which are necessary or advantageous for the expression of a polypeptide of the present invention. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator sequence, as described in detail above.
- Promoter sequence as used herein, refers to a DNA sequence which when ligated to a nucleotide sequence of interest is capable of controlling the transcription of the nucleotide sequence of interest into mRNA. This is discussed in detail, above.
- Promoters may be constitutive. The term “constitutive” when made in reference to a promoter means that the promoter is capable of directing transcription of an operably linked nucleic acid sequence under most environmental and developmental conditions in the absence of a stimulus (e.g., heat shock, chemicals, light, etc.), and/or in several or many tissues, cell types, or organs.
- Promoters may be tissue specific or cell specific. The term “tissue specific” as it applies to a promoter refers to a promoter that is capable of directing selective expression of a nucleotide sequence of interest to a specific type of tissue (e.g., petals) in the relative absence of expression of the same nucleotide sequence of interest in a different type of tissue (e.g., roots). Tissue specificity of a promoter may be evaluated by, for example, operably linking a reporter gene to the promoter sequence to generate a reporter construct, introducing the reporter construct into the genome of a plant such that the reporter construct is integrated into every tissue of the resulting transgenic plant, and detecting the expression of the reporter gene (e.g., detecting mRNA, protein, or the activity of a protein encoded by the reporter gene) in different tissues of the transgenic plant.
- For example, a constitutive plant promoter fragment may be employed which will direct expression of the Fusarium cell wall-degrading enzyme in some to many tissues of a plant. Such promoters are active under most environmental conditions and states of development or cell differentiation. Examples of constitutive promoters include the cauliflower mosaic virus (CaMV) 35S transcription initiation region, the 1′- or 2′- promoter derived from T-DNA ofAgrobacterium tumefaciens, and other transcription initiation regions from various plant genes known to those of skill.
- Promoters for constitutive expression of anti-Fusarium genes in cereals and the tissues in which they have been shown to be active are the following: maize ubiquitin-1, young leaf, root, pollen, seed (cereals) (Christensen and Quail, 1996); maize Adh-1, embryo shoots, roots, anther, pollen, seed (rice) (Freeling and Bennett, 1985); rice ACT-1, shoot leaf, root, floral parts, including pollen (rice) (Zhang et al., 1991); CaMV 35S, leaf, root (rice) (Battraw et al.); ScBv, stem, palea, lemma, other floret organs, anther (oat) (Tzafrir et al.),1998.
- Organ-specific promoters may be, for example, a promoter from storage sink tissues such as seeds, potato tubers, and fruits (Edwards & Coruzzi, 1990,Ann. Rev. Genet. 24:275-303), or from metabolic sink tissues such as meristems (Ito et al., 1994, Plant Mol. Biol. 24: 863-878), a seed specific promoter such as the glutelin, prolamin, globulin, or albumin promoter from rice (Wu et al., 1998, Plant and Cell Physiology 39:885-889), the storage protein napA promoter from Brassica napus, or any other seed specific promoter known in the art. Furthermore, the promoter may be a leaf specific promoter such as the rbcS promoter from rice or tomato (Kyozuka et al., 1993, Plant Physiology 102:991 -1000, or the aldP gene promoter from rice (Kagaya et al., 1995, Molecular and General Genetics 248: 668-674), or a wound inducible promoter such as the potato pin2 promoter (Xu et al., 1993, Plant Molecular Biology 22:573-588).
- Alternatively, the plant promoter may be under environmental or developmental control. Such promoters are referred to here as “inducible” promoters. Examples of environmental conditions that may effect transcription by inducible promoters include pathogen attack, anaerobic conditions, or the presence of light.
- A promoter enhancer element may also be used to achieve higher expression of the gene in the plant. For instance, the promoter enhancer element may be an intron which is placed between the promoter and the nucleotide sequence encoding a polypeptide of the present invention. For instance, Xu et al., 1993, supra, disclose the use of the first intron of the rice actin gene to enhance expression.
- In a preferred embodiment, regulatory sequences or promoters that are particularly suitable for expression of genes in monocots are used. For example, as illustrated in the Examples, below, we selected the promoter of the maize Polyubiquitin-1 gene (Ubi-1, Christensen and Quail 1996) for regulating the expression of the glucanase and chitinase genes in wheat. The Ubi-1 promoter is active in a number of cereal species. It is termed a “constitutive” promoter because it functions in many different organs and tissues, including callus, young leaves, endosperm (A. Blechl, unpub.), mature pollen, and the floret organs of wheat (P. Okubara and A. Blechl, unpub.), in dividing embryogenic calli, leaves, roots and mature pollen of rice (Cornejo et al. 1993), and plumules and radicals of maize seedlings (Liu et al. 1995). The use of the Ubi-1 promoter is envisioned to extend the application of the constructs to pathogens that attack other organs of wheat and to pathogens that attack other cereal crops, such as rice, maize, oat and barley.
- The promoters can also be used to initiate transcription of mRNA molecules to inhibit expression of the gene. Means for inhibiting gene expression in host cells using recombinant DNA techniques are well known. For instance, antisense Technology can be conveniently used. See, e.g., Sheehy et al.,Proc. Nat. Acad. Sci. USA, 85:8805-8809 (1988), and Hiatt et al., U.S. Pat. No. 4,801,340. Introduction of nucleic acid configured in the sense orientation has also been shown to be an effective means by which to block the transcription of target genes. For an example of the use of sense suppression to modulate expression of endogenous genes see, Napoli et al., The Plant Cell 2:279-289 (1990), and U.S. Pat. Nos. 5,034,323, 5,231,020, and 5,283,184.
- The control sequence may also be a suitable leader sequence, a nontranslated region of an mRNA which is important for translation by the host cell. The leader sequence is operably linked to the 5′ terminus of the nucleic acid sequence encoding the polypeptide. Any leader sequence that is functional in the host cell of choice may be used in the present invention.
- The control sequence may also be a polyadenylation sequence, a sequence operably linked to the 3′ terminus of the nucleic acid sequence and which, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence which is functional in the host cell of choice may be used in the present invention.
- The control sequence may also be a signal peptide coding region that codes for an amino acid sequence linked to the amino terminus of a polypeptide and directs the encoded polypeptide into the cell's secretory pathway. The 5′ end of the coding sequence of the nucleic acid sequence may inherently contain a signal peptide coding region naturally linked in translation reading frame with the segment of the coding region which encodes the secreted polypeptide. Alternatively, the 5′ end of the coding sequence may contain a signal peptide coding region which is foreign to the coding sequence. The foreign signal peptide coding region may be required where the coding sequence does not naturally contain a signal peptide coding region. Alternatively, the foreign signal peptide coding region may simply replace the natural signal peptide coding region in order to enhance secretion of the polypeptide. However, any signal peptide coding region which directs the expressed polypeptide into the secretory pathway of a host cell of choice may be used in the present invention.
- The vector comprising the sequences from the cell wall-degrading genes may comprise a marker gene which confers a selectable phenotype on plant cells. Alternatively, and more typically for cereal transformation, a marker gene may be co-transformed on a separate vector molecule. For example, the marker may encode biocide resistance, particularly antibiotic resistance, such as resistance to kanamycin, carbenicillin, ampicillin or herbicide resistance, such as resistance to chlorosulfuron, or phosphinothricin (the active ingredient in bialaphos and Basta).
- As described in the Examples below, for the expression the glucanase and chitinase genes, we constructed a vector derived from pAHC20 (Christensen and Quail 1996), in which the bar selectable marker gene is regulated by the maize Ubi-1 promoter (0.9 kb, including the first exon and first intron located in the 5′ untranslated region) and
NOS 3′ terminator. The bar gene, conferring resistance to the herbicide bialaphos (de Block et al. 1987), is successfully and routinely used in our laboratory as a selectable marker for transgenic wheat plants. pUBK, engineered by Dr. Kent McCue (USDA-ARS, Albany, Calif.) to avoid the use of ampicillin resistance, was used. To construct pUBK, Dr. McCue replaced the origin of replication and gene for ampicillin resistance (bla) of pAHC20 (Christensen and Quail 1996) with the corresponding portion of pBGS9 (Spratt et al. 1986), which encodes kanamycin resistance (nptII). pUBK is publicly available from Dr. McCue. - Another factor in making recombinant constructs of transgenes is the efficiency of translation of the transgene mRNA. Computerized analyses of gene sequences and in vitro experiments indicate that the efficiency of translation initiation is mediated by three nucleotides (−3 to −1) immediately preceding the ATG start (initiation) codon. The −3 to −1 start codon context consensus for eukaryotic genes was reported to be ACC (Kozak 1987); a survey of plant genes yielded a consensus of ACA (Fütterer and Hohn 1996); the start codon context for 85 maize genes was preferentially GGC or AAG (Luehrsen and Walbot 1994). To examine the hypothetical efficiency of translational initiation of leaf- or endosperm-specific mRNAs in pollen, we undertook a limited survey of start codon contexts for some monocot (T. aestivum, H. vulgare, O. sativa, A. sativa, Z. mays) genes from GenBank and EMBL databases. The consensus start codon context for wheat leaf mRNAs was GCC, and for monocot pollen mRNAs was G/A,A/C, not T.
- Additionally, the 15-20 nucleotides upstream of ATG, including −3 to −1, are AC-rich in many monocot genes. Enhancements of translational efficiency of heterologous genes, e.g., fungal genes in monocots have been engineered.
- V. Expression Vectors
- The present invention also relates to recombinant expression vectors comprising a nucleic acid sequence of the present invention, a promoter, and transcriptional and translational stop signals. The various nucleic acid and control sequences described above may be joined together to produce a recombinant expression vector which may include one or more convenient restriction sites to allow for insertion or substitution of the nucleic acid sequence encoding the polypeptide at such sites. Alternatively, the nucleic acid sequence of the present invention may be expressed by inserting the nucleic acid sequence or a nucleic acid construct comprising the sequence into an appropriate vector for expression. In creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.
- The recombinant expression vector may be any vector (e.g., a plasmid or virus) which can be conveniently subjected to recombinant DNA procedures and can bring about the expression of the nucleic acid sequence. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vectors may be linear or closed circular plasmids.
- The vector may be an autonomously replicating vector, i.e., a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids which together contain the total DNA to be introduced into the genome of the host cell, or a transposon may be used.
- The vector may also contain sequences that stabilize the integration and expression of the nucleic acid construct in a host cell. These include DNA sequences carrying matrix attachment regions also known as scaffold attachment regions, for example, for yeast or plant, e.g., Arabidopsis or wheat, sources.
- The vector may also contain one or more selectable markers which permit easy selection a transformed cells.
- The vector may also contain an element(s) that permits integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome.
- For integration into the host cell genome, the vector may rely on the nucleic acid sequence encoding the polypeptide or any other element of the vector for integration of the vector into the genome by homologous or nonhomologous recombination.
- For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question.
- More than one copy of a nucleic acid sequence of the present invention may be inserted into the host cell to increase production of the gene product. An increase in the copy number of the nucleic acid sequence can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the nucleic acid sequence where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the nucleic acid sequence, can be selected for by cultivating the cells in the presence of the appropriate selectable agent.
- The procedures used to ligate the elements described above to construct the recombinant expression vectors of the present invention are well known to one skilled in the art (see, e.g., Sambrook et al., 1989, supra).
- VI. Preparation of Transgenic Host Cells and Production of Polypeptide
- The present invention also relates to recombinant host cells, comprising a nucleic acid sequence of the invention, which are advantageously used in the recombinant production of the polypeptides. Preparation of transformed host cells and cloning methods are described by U.S. Pat. No. 5,374,540, which is incorporated herein by reference.
- The host cell may be a unicellular microorganism, e.g., a prokaryote, or a non-unicellular microorganism, e.g., a eukaryote. Eukaryote cells can include any cell such as from an insect, fimgus or plant. The term “plant” includes whole plants, plant parts or organs, plant tissue, as described in detail, above.
- The present invention also relates to methods for producing a polypeptide comprising cultivating a host cell under conditions suitable for production of the polypeptide and recovering the polypeptide. The cells are cultivated in nutrient medium suitable for production of the polypeptide using methods know in the art. The polypeptides may be detected and recovered using methods known in the art.
- The present invention also relates to methods for producing a polypeptide of the present invention comprising (a) cultivating a transgenic plant or a plant cell comprising a nucleic acid sequence encoding a polypeptide having enzyme activity of the present invention under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide.
- The polypeptides may be detected using methods known in the art that are specific for the polypeptides. These detection methods may include use of specific antibodies, formation of an enzyme product, or disappearance of an enzyme substrate. For example, an enzyme assay may be used to determine the activity of the polypeptide as described herein.
- The resulting polypeptide may be recovered by methods known in the art. For example, the polypeptide may be recovered from the nutrient medium by conventional procedures including, but not limited to, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation.
- The polypeptides of the present invention may be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g.,Protein Purification, J.-C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989).
- VIII. Preparation of Transgenic Plants
- The transgenic plant or plant cell expressing an RNA transcript or polypeptide of the present invention may be constructed in accordance with methods known in the art. In brief, the plant or plant cell is constructed by incorporating one or more expression constructs encoding a polypeptide of the present invention into the plant host genome and propagating the resulting modified plant or plant cell into a transgenic plant or plant cell.
- In a preferred embodiment of this invention, the transgenic plants of this invention are cereal crop plants, including but not limited to, wheat, rye, triticale, barley, maize, sorghum and rice. In a more preferred embodiment, the transgenic plants are wheat, maize, barley, oats, and rye. In an alternate preferred embodiment, the transgenic plants of this invention are dicotyledonous plants.
- The DNA constructs described above may be introduced into the genome of the desired plant host by a variety of conventional techniques. Techniques for transforming a wide variety of higher plant species are well known and described in the technical and scientific literature. See, for example, Weising, et al.,Ann. Rev. Genet. 22:421-477 (1988).
- The DNA construct may be introduced directly into the genomic DNA of the plant cell using techniques such as biolistic methods, electroporation, PEG poration, and microinjection of plant cell protoplasts or embryogenic callus. Alternatively, the DNA constructs may be combined with suitable T-DNA flanking regions and introduced using anAgrobacterium tumefaciens or A. rhizogenes vector.
- The terms “bombarding, “bombardment,” and “biolistic bombardment” refer to the process of accelerating particles towards a target biological sample (e.g., cell, tissue, etc.) to effect wounding of the cell membrane of a cell in the target biological sample and/or entry of the particles into the target biological sample. Methods for biolistic bombardment are known in the art (e.g., U.S. Pat. No. 5,584,807, the contents of which are herein incorporated by reference), and are commercially available (e.g., the helium gas-driven microprojectile accelerator (PDS-1000/He) (BioRad).
- Particle bombardment techniques are described in Klein, et al.,Nature 327:70-73 (1987). A particularly preferred method of transforming wheat and other cereals is the bombardment of calli derived from immature embryos as described by Weeks, et al., Plant Physiol. 102:1077-1084 (1993).
- Microinjection techniques are known in the art and well described in the scientific and patent literature. The introduction of DNA constructs using polyethylene glycol precipitation is described in Paszkowski, et al.,EMBO J. 3:2717-2722 (1984). Electroporation techniques are described in Fromm, et al., Proc. Nat'l Acad. Sci. USA 82:5824 (1985).
-
- The present invention is particularly useful in wheat and other cereals. A number of methods of transforming cereals have been described in the literature. For instance, reliable methods for stable transformation of wheat, including the highly-regenerable cultivars such as the hard white spring wheat Bobwhite, are described (Vasil, et al., 1992, 1993; Weeks, et al., 1993; Becker et al., 1994; Nehra et al. 1994, Blechl and Anderson, 1996). U.S. Pat. Nos. 5,650,558 and 5,914,450 to Blechl et al. describe transformation of wheat and non-wheat cereal plants, which patents are incorporated herein by reference. Chen et al. (1998) introduced a rice chitinase gene under control of the 35S promoter into wheat. It was expressed in the first generation, but was subsequently silenced in progeny plants. Jensen et al. (1998) introduced a modified heat-stable 1,3-1,4-glucanase gene under control of its own promoter into barley and obtained plants that expressed the gene in germinating aleurone and scutellum. Bliffeld et al. (1998) introduced a barley seed class II chitinase gene under control of the maize Ubiquitin-1 promoter into wheat. Two lines of transgenic plants containing this construct showed increased resistance to infection by the powdery mildew-causing fungusErysiphe graminis. Transgenic maize regenerants have been described by Fromm, et al., Bio/Technology 8:833-839 (1990) and Gordon-Kamm, et al., Plant Cell 2:603-618 (1990)). Similarly, oats (Sommers, et al., Bio/Technology 10:1589-1594 (1992)), sorghum (Casas, et al., Proc. Nat'l Acad. Sci. USA 90:11212-11216 (1993)), rice (Li, et al., Plant Cell Rep. 12:250-255 (1993)), barley (Yuechun & Lemaux, Plant Physiol. 104:37-48 (1994)), and rye (Castillo, et al., Bio/Technology 12:1366-1371 (1994)) have been transformed via bombardment. Transformation of rice is described by Toriyama, et al., Bio/Technology 6:1072-1074 (1988), Zhang, et al., Theor. Appl. Gen. 76:835-840 (1988), and Shimamoto, et al., Nature 338:274-276 (1989).
- The present invention further relates to plants, seeds, plant tissues, plant organs, and plant cells transiently expressing the claimed sequences. Transient expression refers to the generation of mRNA and/or protein from the claimed DNA sequences in plants without stable integration of the plasmid into host genomes. Such expression might be observed after 1 to 14 days following introduction of DNA by particle gun bombardment, Agrobacterium, and other means used for plant transformation.
- Transformed plant cells that are derived by any of the above transformation techniques can be cultured to regenerate a whole plant which possesses the transformed genotype and thus the desired phenotype. Such regeneration techniques rely on manipulation of certain phytohormones in a tissue culture growth medium, typically relying on a biocide and/or herbicide marker which has been introduced together with the cell wall-degrading polynucleotide sequences. Plant regeneration from cultured protoplasts is described in Evans, et al., PROTOPLASTS ISOLATION AND CULTURE, HANDBOOK OF PLANT CELL CULTURE, Macmillian Publishing Company, New York, pp. 124-176 1983; and Binding, REGENERATION OF PLANTS, PLANT PROTOPLASTS, CRC Press, Boca Raton, pp. 21-73 1985. Regeneration can also be obtained from plant callus, explants, organs, or parts thereof. Such regeneration techniques are described generally in Klee, et al.Ann. Rev. of Plant Phys. 38:467-486 (1987).
- Transformed plants are evaluated for the presence of the desired properties and/or the extent to which the desired properties are expressed. A first evaluation may include the level of expression of the newly introduced genes, the level of fungal resistance of the transformed plants, and stable heritability of the desired properties.
- One of skill will recognize that after the expression cassette is stably incorporated in transgenic plants and confirmed to be operable, it can be introduced into other plants by sexual crossing. A technique used to transfer a desired phenotype to a breeding population of plants is through backcrossing. However, any of a number of standard breeding techniques can be used, depending upon the species to be crossed.
- Any plant species or variety that is subject to fungal attack may be transformed with one or more genetic constructs according to the invention in order to improve resistance to Fusarium or other pathogens, for example, to improve resistance to Fusarium head blight and other fungal diseases, including by (a) generation of cell wall-degrading enzymes, including proteins having the capability of degrading the glucan and chitin cell wall components ofF. venenatum and other Fusarium species, including F. graminearum and F. culmorum, the principle causal agents of head blight (scab) in the U.S., (b) expression of the cell wall-degrading enzymes in transgenic monocots, including wheat, barley or oats, to confer partial or complete resistance to Fusarium species and to other fungal pathogens of wheat and other cereal crops, (c) limiting the spread of the pathogenic fungus Fusarium and curtailing the accumulation of DON in infected heads, and (d) expression of the cell wall-degrading enzymes in dicots to confer resistance to plant pathogens.
- The following, non-limitative, list is illustrative of fungal diseases: Fusarium head blight, Fusarium wilt caused byFusarium oxysporum or Fusarium solani, stock rot caused by Fusarium moniliforme, fungal diseases caused by Phytophthora.
- As a matter of illustration the species of the following, non-limitative, list are moncots of particular interest: wheat, rice, barley, maize, rye, oat, sorghum, triticale.
- Analysis of transgenic lines may be done by northern blots, in situ hybridization, RT-PCR, S1 nuclease protection assays, RNase protection assays, Western blots, enzyme assays or other methods for detection of expressed mRNA or protein.
- IX. USES
- As discussed above, a particular use of the invention is the provision of plants or plant cells transformed with a DNA sequence encoding a glucanase, endochitinase or exochitinase coding sequence to provide plants having resistance to plant pathogens, particularly, Fusarium species.
- Also, another use of the invention is as probes and primers capable of detecting a glucanase, endochitinase or exochitinase gene or functional equivalents thereof in fungi of the genus Fusarium and the use of the probes to isolate DNA sequences encoding a glucanase, endochitinase or exochitinase gene or functional equivalent thereof.
- Using the nucleic acid sequences of the invention facilitates the isolation of homologous genes from hosts to obtain genes which protect host cells, including fungi and plants against related fungal pathogens.
- Another use of the sequences is in the antisense orientation as a gene knock-out.
- Another use is to generate Fusarium cell wall-degrading proteins for research purposes.
- Another use is to generate cell-wall degrading chitinases and glucanases for use in degrading seafood waste, such as shells that contain chitin, or for use for chemical modification of chitin or glucan.
- Overview: In our examples, below, we describe in detail work to combat Fusarium head blight (scab) and other Fusarium-mediated diseases of wheat and other crops. We isolated cDNAs and genes encoding enzymes that digest the glucan and chitin components of fungal cell walls, and identified three distinct cDNA clones (encoding a glucanase, an endochitinase, and an exochitinase) fromFusarium venenatum, and gene homologs for the endochitinase and exochitinase from F. sporotrichioides. F. venenatum is a close relative of the causal agent of head blight, F. graminearum. Both F. venenatum and F. sporotrichioides produce mycotoxins similar to that made by the head blight pathogen. Cell-wall degrading enzymes from a Fusarium species are required by the fungus for normal growth and development.
- The nucleotide sequences of two glucanase cDNA clones, an endochitinase cDNA clone, and 3 exochitinase cDNA clones from a collection ofF. venenatum ESTs were examined. Both glucanase cDNA clones were determined to be full-length, whereas all the chitinase clones were partial cDNA clones that were missing the 5′ ends, including the ATG translational start codons. To restore the missing sequences, we performed a polymerase chain reaction (PCR) method known as 5′ anchored PCR on a genomic library of F. sporotrichioides. The resulting 5′ genomic fragments for both the endo- and exochitinase could not be added directly to the partial cDNA clones because each fragment carried an intron. However, the nucleotide sequences of these 5′ fragments provided the necessary data for the design of oligonucleotide PCR primers with which full length clones were eventually obtained.
- To obtain full-length cDNA clones of the endo- and exochitinases, we performed a second polymerase chain reaction procedure using a cDNA library ofF. venenatum. The primers used to amplify the cDNAs eliminated most of the untranslated sequences flanking the coding region. The PCR primers specific to the 5′ ends of the genes included additional nucleotides that replaced the 15-16 bases immediately upstream of the ATG start codon with A or C nucleotides. The AC-rich start codon context (and 5′ flanking nucleotides) was expected to render the translation of the gene transcripts more facile in a variety of wheat and other monocot organs. The PCR primers added a 5′ BamHI restriction site and a 3′ BglII restriction site to the termini of the amplified portions of the cDNAs. These sites were useful for cloning the cDNAs into our monocot expression vector.
- The endo- and exochitinase PCR products, and the glucanase cDNA, were first cloned in bacterial vectors, either pCR2.1 or pBKS+. Production of appropriate amounts of the cDNA plasmids for subsequent cloning were readily obtained in the bacterial host. Also, nucleotide sequences were verified for all three cDNAs after cloning in bacterial vectors, to check that PCR amplification introduced modifications to the 5′ region and eliminated unwanted flanking sequences without altering the coding regions. The cDNAs were then introduced into the vector, pUBKBglII-, for expression in monocotyledonous plants. The monocot expression vector carried a promoter from the maize Polyubiquitin-1 (Ubi-1) gene, the bar gene for resistance to the herbicide bialaphos, a 3′ terminator segment from the nopaline synthase (nos) gene ofAgrobacterium tumefaciens, and the nptll marker gene for resistance to the antibiotic kanamycin. A BglII restriction enzyme site located in the Ubi-1 promoter was destroyed by site-directed mutagenesis. This allowed the removal of the bar gene from pUBKBglII- with restriction enzymes BglII and BamHI, and substitution of the F. venenatum cDNAs. Both sense and antisense orientations of the three cDNAs (6 total) were obtained, as determined from nucleotide sequences of the promoter-transgene and transgene-terminator junctions.
- Embryos ofTriticum aestivum cultivar Bobwhite were bombarded simultaneously with plasmids carrying the sense orientations of the cDNAs plus a selector marker plasmid carrying the bar gene. Calli and regenerated plants were cultured using methods developed in the laboratory. Candidate transgenic plants were selected on the basis of growth in the presence of the herbicide bialaphos (conferred by the bar gene). DNA from leaf sections of candidate tranformants were further analyzed by PCR with a primer designed from the maize Ubi-1 promoter sequence and a second primer specific for each of the transgene (glucanase, endochitinase, and exochitinase) sequences. Those plants carrying transgene DNA are being propagated for production of seed that are homozygous for the transgenes. Homozygous lines are tested for expression of the transgenes and tested for resistance to F. graminearum and other fungal pathogens.
- The following examples are intended only to further illustrate the invention and are not intended to limit the scope of the invention which is defined by the claims. The examples use many techniques well known and accessible to those skilled in the arts of molecular biology, in the manipulation of recombinant DNA in plant tissue and in the culture and regeneration of transgenic plants. Enzymes are obtained from commercial sources and are used according to the vendors' recommendations or other variations known to the art. Reagents, buffers and culture conditions are also known to the art. References providing standard molecular biological procedures include Sambrook et al. (1989)Molecular Cloning, second edition, Cold Spring Harbor Laboratory, Plainview, N.Y.; R. Wu (ed.) (1993) Methods in Enzymology 218; Wu et al. (eds.) Methods in
Enzymology 100, 101; Glover (ed.) (1985); DNA Cloning, Vols. I and II, IRL Press, Oxford, UK; and Hames and Higgins (eds.) (1985) Nucleic Acid Hybridization, IRL Press, Oxford, UK. References related to the manipulation and transformation of plant tissue include Kung and Arntzen (eds.) (1989) Plant Biotechnology, Butterworths, Stoneham, MA; R. A. Dixon (ed.) (1985) Plant Cell Culture: A Practical Approach, IRL Press, Oxford, UK; Schuler and Zielinski (1989) Methods in Plant Molecular Biology, Academic Press, San Diego, Calif.; Weissbach and Weissbach (eds.) (1988) Academic Press, San Diego, Calif.; I. Potrykus (1991) Ann. Rev. Plant Physiol. Plant Mol. Biol. 42:205; Weising et al. (1988) Annu. Rev. Genet. 22:421; van Wordragen et al. (1992) Plant Mol. Biol. Rep. 19:12; Davey et al. (1989) Plant Mol. Biol. 13:273; Walden and Schell (1990) Eur. J. Biochem. 192:563; Joersbo and Brunstedt (1991) Physiol. Plant. 81:256 and references cited in those references. Abbreviations and nomenclature, where employed, are deemed standard in the field and are commonly used in professional journal such as those cited herein. All references cited in the present application are expressly incorporated by reference herein. -
- Mycelial samples were harvested at 2, 4, 6, and 8 days post-inoculum and quick-frozen in liquid nitrogen. The samples were stored at −80° C. until they were disrupted for RNA extraction.
- Total cellular RNA was extracted from the mycelial samples described in Example 1 according to the method of Timberlake and Barnard (1981,Cell 26: 29-37), and the RNA samples were analyzed by Northern hybridization after blotting from 1% formaldehyde-agarose gels (Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier Science Publishing Co., Inc., New York). Polyadenylated mRNA fractions were isolated from total RNA with an mRNA Separator Kit™ (Clontech Laboratories, Inc., Palo Alto, Calif.) according to the manufacturer's instructions. Double-stranded cDNA was synthesized using approximately 5 μg of poly(A)+ mRNA according to the method of Gubler and Hoffman (1983, Gene 25: 263-269) except a NotI-(dT) 18 primer (Pharmacia Biotech, Inc., Piscataway, N.J.) was used to initiate first strand synthesis. The cDNA was treated with mung bean nuclease (Boehringer Mannheim Corporation, Indianapolis, Ind.) and the ends were made blunt with T4 DNA polymerase (New England Biolabs, Beverly, Mass.).
- The cDNA was digested with NotI, size selected by agarose gel electrophoresis (ca. 0.7-4.5 kb), and ligated with pZErO-2.1 (Invitrogen Corporation, Carlsbad, Calif.) which had been cleaved with NotI plus EcoRV and dephosphorylated with calf-intestine alkaline phosphatase (Boehringer Mannheim Corporation, Indianapolis, Ind.). The ligation mixture was used to transform competent
E. coli TOP 10 cells (Invitrogen Corporation, Carlsbad, Calif.). Transformants were selected on 2YT agar plates (Miller, 1992, A Short Course in Bacterial Genetics. A Laboratory Manual and Handbook for Escherichia coli and Related Bacteria, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.) which contained kanamycin at a final concentration of 50 μg/ml. - Two independent directional cDNA libraries were constructed using the plasmid cloning vector pZErO-2.1. Library A was made using mRNA from mycelia harvested at four days, and Library B was constructed with mRNA from the six day time point. Neither cDNA library was amplified in order to examine a representative “snapshot” of the gene expression profile in the cells. Instead the libraries were plated, titered, and independent clones from each were analyzed by DNA sequencing.
- Library A (4 day cells) consisted about 7.5×104 independent clones and Library B (6 day cells) consisted of roughly 1.2×105 clones. Miniprep DNA was isolated from forty colonies in each library and checked for the presence and size of cDNA inserts. In this analysis 39 of 40 colonies (97.5%) from Library A contained inserts with sizes ranging from 600 bp to 2200 bp (avg.=1050 bp). Similarly, 39 of 40 colonies (97.5%) picked from Library B had inserts with sizes ranging from 800 bp to 3600 bp (avg.=1380 bp).
- From each cDNA library described in Example 2, 1192 transformant colonies were picked directly from the transformation plates into 96-well microtiter dishes which contained 200 μl of 2YT broth (Miller, 1992, supra) with 50 μg/ml kanamycin. The plates were incubated overnight at 37° C. without shaking. After
incubation 100 μl of sterile 50% glycerol was added to each well. The transformants were replicated into secondary, deep-dish 96-well microculture plates (Advanced Genetic Technologies Corporation, Gaithersburg, Md.) containing 1 ml of Magnificent Broth™ (MacConnell Research, San Diego, Calif.) supplemented with 50 μg of kanamycin per ml in each well. The primary microtiter plates were stored frozen at −80° C. The secondary deep-dish plates were incubated at 37° C. overnight with vigorous agitation (300 rpm) on rotary shaker. To prevent spilling and cross-contamination, and to allow sufficient aeration, each secondary culture plate was covered with a polypropylene pad (Advanced Genetic Technologies Corporation, Gaithersburg, Md.) and a plastic microtiter dish cover. - DNA was isolated from each well using the 96-well Miniprep Kit protocol of Advanced Genetic Technologies Corporation (Gaithersburg, Md.) as modified by Utterback et al. (1995,Genome Sci. Technol. 1: 1-8). Single-pass DNA sequencing was done with a Perkin-Elmer Applied Biosystems Model 377 Sequencer XL (Perkin-Elmer Applied Biosystems, Inc., Foster City, Calif.) using dye-terminator chemistry (Giesecke et al., 1992, Journal of Virology Methods 38: 47-60) and the reverse lac sequencing primer (New England Biolabs, Inc., Beverly, Md.).
- Nucleotide sequence data were scrutinized for quality, and samples giving improper spacing or ambiguity levels exceeding 2% were discarded or re-run. Vector sequences were removed with assistance of FACTURA™ software (Perkin-Elmer Applied Biosystems, Inc., Foster City, Calif.). In addition, sequences were truncated at the end of each sample when the number of ambiguous base calls increased. All sequences were compared to each other to determine multiplicity using AutoAssembler™ software (Perkin-Elmer Applied Biosystems, Inc., Foster City, Calif.). Lastly, all sequences were translated in three frames and searched against a non-redundant data base (NRDB) using GeneAssist™ software (Perkin-Elmer Applied Biosystems, Inc., Foster City, Calif.) with a modified Smith-Waterman algorithm using the BLOSUM 62 matrix with a threshold score of 70. The NRDB was assembled from Genpept, Swiss-Prot, and PIR databases.
- EST clones encoding putative chitinolytic enzymes were identified by partial sequencing of random cDNA clones using an Applied Biosystems Model 377 XL Automated DNA Sequencer according to the manufacturer's instructions and comparison of the deduced amino acid sequence of each EST to the amino acid sequences in a NRDS made from publicly available protein and nucleic acid databases (e.g., GENBANK, EMBL, TREMBL, GENPEPT, SWISSPROT, PIR). EST translations showing deduced amino acid sequence identities toTrichoderma harzianum exochitinase (TREMBL P78739), the
endochitinase 1 precursor of Coccidioides immitis (SWISSPROT P54196), Coccidioides immitis chitinase (PIR JC4565), exo-beta-1,3-glucanase from Saccharomyces cerevisiae (SWISSPROT P15703), poly-beta-glucosaminidase from Clostridium thermocellum (TREMBL Q59326), Emericella nidulans chitinase (GENPEPT D87063), and the chitooligosaccharidolytic beta-N-acetylglucosaminidase from Bombyx mori (SWISSPROT P49010) were identified as described in Example 4. - TwoF. venenatum glucanase cDNA clones, an endochitinase cDNA clone, and 3 exochitinase cDNA clones, all in the vector pZErO-2 (Invitrogen, Carlsbad, Calif.), were selected from Example 5. These clones were assigned identities based on matches to known chitinases and glucanases in the GenBank database as described in Example 5. From preliminary nucleotide sequences (ESTs) of the 5′ ends of the cDNAs, one full-length glucanase clone, one partial endochitinase cDNA clone, and the longest of the 3 exochitinase partial clones were selected for all further work.
- Approximately 5 to 10 ng of plasmid DNAs carrying the above three cDNAs were introduced separately intoE. coli host strain NM522 by electroporation. Electroporation was performed on 50 μL aliquots of cells (3.5×107/μg) using the Bio-Rad Gene Pulser (Bio-Rad, Hercules, Calif.), in 0.2 cm gap cuvettes at 2500 Volts. Following electroporation, the cells were suspended in 1
mL 2% (w:v) Bacto tryptone, 0.5% (w:v) yeast extract, 0.05% (w:v) sodium chloride, 20 mM glucose, 10 mM magnesium chloride, and 2.5 mM potassium chloride. The cells were maintained at 37° C. for 60 minutes with agitation at 200-300 rpm. Aliquots of 50 to 200 μL of cells were spread onto agar plates of Luria broth (LB) containing 50 μg/mL kanamycin sulfate. After culture at 37° C. for 16-20 hr, bacterial colonies showing good growth were transferred to a fresh LB-kanamycin plate. From this stock plate, additional cultures of individual colonies were grown for plasmid DNA isolation. - For plasmid DNA isolation, 30 mL cultures ofE. coli containing the above cDNA clones were incubated at 37° C., 16-18 hr, with agitation. Culture medium was a modified LB containing 1.5% (w:v) Bacto tryptone, 0.75% (w:v) yeast extract, 0.75% (w:v) sodium chloride, 15 mM TRIS-Cl buffer, pH 7.0, 1.5 mM magnesium chloride, and 50 μg/mL kanamycin sulfate. Cells were collected by centrifugation at 8000×g for 10 minutes at 4° C. Plasmid DNA was isolated according to the protocol described in the Qiagen Plasmid Midi Kit (Qiagen, Inc., Valencia, Calif.). Plasmid DNA was isolated from 1.5 mL cultures using the
QIAprep 8 kit and QIAvac Manifold-6S (Qiagen, Inc., Valencia, Calif.). - Preliminary nucleotide sequence (EST) data were analyzed for the presence of restriction enzyme recognition sites using the Lasergene software programs EditSeq and MapDraw (DNASTAR, Inc, Madison, Wis.). Restriction enzymes that cleaved in one or more of the cDNA coding sequences or in the polylinker region of pZErO-2 were used. Plasmid DNAs (˜0.5-1 μg), prepared as described in Example 7, were incubated with Apal, BamHI, EcoRI or Kpn I in a total volume of 15 μL as prescribed by the enzyme manufacturers. The cleavage products were partitioned on 1% agarose in 40 mM TRIS acetate, pH 8.2, 1 mM EDTA, stained with an ethidium bromide solution (˜1 μg/mL), and visualized by irradiation with an ultraviolet light source (UVT 400-M transilluminator, IBI Kodak, Rochester, N.Y.). The results are shown in the Table 1, below.
TABLE 1 Restriction Fragments Theoretical Plasmid/cDNA Enzyme Obtained (kb) Fragments (kb) Glucanase Apa I 3.7, 0.6 3.6, 0.7 BamHI 4.4 ˜4.3 EcoRI 4.4 ˜4.3 Kpn I no digestion no digestion Endochitinase Apa I 3.7, 0.9 3.7, 1.0 BamHI 4.6 4.5-4.7 EcoRI 4.5 4.5-4.7 Kpn I 4.5, 0.3 4.4, 0.2 Exochitinase Apa I 4.7 4.5-4.7 BamHI 4.7 4.5-4.7 EcoRI 4.7 4.5-4.7 Kpn I 3.8, 0.9 3.3, 1.4 - One glucanase cDNA clone, the endochitinase cDNA clone, and the longest exochitinase cDNA clone were selected for more rigorous, double strand nucleotide sequence determinations. Each cDNA was sequenced by a modified method of Sanger et al. (1977), using 250-500 ng of plasmid DNA in 2 μL, 8 μL of BigDye Terminator Mix and 1 μL of primer (4 uM) in a total volume of 20 μL, according to the ABI Prism BigDye™ Terminator Cycle Sequencing Ready Reaction Kit (Perkin Elmer Applied Biosystems, Foster City, Calif.). The PCR protocol (25 cycles of 96° C., 30 sec, 50° C. for 15 sec, 60° C. for 4 min) was performed in a PTC-100 Programmable Thermal Controller (MJ Research, Watertown, Mass.). Fluorescently-labeled PCR products were enriched from unincorporated fluorescent dideoxynucleotides by passage through a column containing 50 mg (dry weight) of Sephadex G-50 Fine, DNA grade, (Amersham Pharmacia, Biotech AB, Uppsala, Sweden) imbibed in water. Sequence data of these clones and all other DNA templates were obtained with the
ABI Prism 310 Genetic Analyzer. Best results were obtained with an injection time of 45 seconds at 3 kV injection voltage, and a run time of 90-120 minutes at 15 kV and 42° C., using the Seq POP6 (1 mL) E module. Sequence data was assembled and proofread using theABI Prism 310 Genetic Analyzer Data Collection software, version 1.0.2 (Perkin Elmer Applied Biosystems, Foster City, Calif.). - Nucleotide sequences of the 5′ and 3′ ends of the cDNA inserts in pZErO-2 were obtained with primers M13F (5′ GTAAAACGACGGCCAG) and M13R (5′ AGCGAATAACAATTTCACACAGGA). Additional sequences were obtained by primer walking. Primers synthesized for this purpose, shown in Table 2, were 902A and 902B for the glucanase cDNA; 958A-D for the endochitinase clone; and 1082 A-D for the exochitinase clone. The glucanase cDNA was 1023 bp. The full length unmodified glucanase cDNA is shown in SEQ ID NO:1. Using agarose gel electrophoresis, the unmodified partial cDNA endochitinase was 1290 bp, and the unmodified partial exochitinase cDNA was 1390 bp.
- Sequences were compared to entries in the GenBank database using BLASTN and BLASTX algorithms (Altschul et al., 1997). Portions of theF. venenatum glucanase cDNA showed 60-70% amino acid sequence identity to a 1,3-beta-glucosidase gene from Schizosaccharomyces pombe (Accession Number Z99126). The glucanase cDNA was full-length, having an ATG start codon at approximately the same position as that observed in the S. pombe gene. Segments of the F. venenatum endochitinase partial cDNA had 70-80% amino acid sequence identity to a chitinase antigen from Coccidioides immitis [Accession Number U33265, Yang et al., Infect. Immun., 64:1992-1997 (1996)]. Segments of the exochitinase partial cDNA sequence shared about 80% amino acid sequence identity with an exochitinase gene from Trichoderma harzianum [Accession Number S80069, Draborg et al., Biochem. Mol. Biol. Int. 36:781-791 (1995)]. Both the endo- and exochitinase cDNAs appeared to be partial clones based on the absence of
candidate 5′ ATG start codons and on comparisons with the sequences identified in the database searches.TABLE 2 Primers Used for Polymerase Chain Reaction Experiments Name Purpose Target DNA Sequence (5′ to 3′) 128 Sequencing of 5′exo- λgt11 TCCTGGAGCCCGTCAGTATCGG chitinase PCR product cloning vector 129 PCR amplification of 5′ λgt11 TGCCTGAATATCGACGGTTTCC portion of exochitinase, cloning vector 410 Sequencing of 5′endo- pZER0-2 ATTTAGGTGACACTATAG chitinase PCR product cloning vector 720 PCR amplification of 5′ pZER0-2 CAGGACAGGAAACAGCTATGACC portion of endochitinase, cloning vector 902 A Sequencing of glucanase Glucanase cds CTATCAAGCAGCACGG cDNA, forward 902 B Sequencing of glucanase Glucanase cds CTGGAAGGTCTTGAGGC cDNA, reverse 958 A Sequencing of chitinase Endochitinase cds GATATCGATTGGGAGTACC cDNA, forward 958 B Sequencing of chitinase Endochitinase cds CATGGTACGTGACTGAGG cDNA, reverse 958 C Sequencing of chitinase Endochitinase cds CAACCCTCAGTCACGTAC cDNA, forward 958 D Sequencing of chitinase Endochitinase cds CCTCGTTGGCATCCTG cDNA, reverse 1003 PCR amplification of 5′ Exochitinase cds AGATCCTTATAGGCAAGCTCAACGACACCG portion of exochitinase 1009 Sequencing of 5′exo- 5′portion of exo- CATAAATACCGGCAAGATCC chitinase PCR product chitinase gene 1010 PCR amplification of 5′ Endochitinase cds ATCGATATCTATACCGTCAAAACCG portion of endochitinase 1011 Sequencing of 5′endo- 5′portion of endo- ATCGATATCTATACCGTCAAAACCG chitinase PCR product chitinase cDNA 1014 Sequencing of glucanase pFSC22-2 ATCTGCTGGGCGAGCTTC genomic fragment 1015 Sequencing of glucanase pFSC22-2 CAGAAACCACTGGCATCC genomic fragment 1016 Sequencing of glucanase pFSC22-2 AAGTTCGGCCTTACTTGC genomic fragment 1017 Sequencing of glucanase pFSC22-2 CTTTGGGCTCGTATTCAC genomic fragment 1018 Sequencing of glucanase pFSC22-2 CTTCAGCACTCTCAGCAC genomic fragment 1021 Sequencing of glucanase pFSC22-2 GGACTGAGGGAGTGTTGGTTC genomic fragment 1023 Sequencing of glucanase pFSC22-2 TCTACCCTACAACACTCATC genomic fragment 1038 Sequencing of glucanase pFSC22-2 GTCGATCATGGTGGAAAG genomic fragment 1040 Sequencing of glucanase pFSC22-2 TGTCCCTGTTACTAACGG genomic fragment 1042 Sequencing of glucanase pFSC22-2 TAGTCGCCTTTCTAAGCC genornic fragment 1047 Sequencing of 5′exo- 5′portion of exo- AAGAATGCCTCGATGAGGGTACTCG chitinase PCR product chitinase gene 1048 Sequencing of 5′exo- 5′portion of exo- ATCTCACTCACCTCACCTC chitinase PCR product chitinase gene 1082 A Sequencing of chitinase Exochitinase cds CCGGTATTTATGAGTATGG cDNA, forward 1082 B Sequencing of chitinase Exochitinase cds GATAGTCTCAGTCCATACGG cDNA, reverse 1082 C Sequencing of chitinase Exochitinase cds GTACCTTGACTGTGGGC cDNA, forward 1082 D Sequencing of chitinase Exochitinase cds GGAAATGCGAGGGAAG cDNA, reverse ENDO 3′ Start codon context Endochitinase gene AGATCTCGCCTCAATTGTCCGGGAACC modification ENDO 5′ Start codon context Endochitinase gene GGATCCAACACCACGCGATGGGTGGTGGA modification CCCGAAGG EXO 3′ Start codon context Exochitinase gene AGATCTGCGTCAAATTTATCATCCCTCG EXO 5′ Start codon context Exochitinase gene GGATCCACCAACCAGCGATGTGGTCCAAG modification GCTCTTCTGGCCGTTG FVEN B1 PCR amplification of Endochitinase cds GCTTCATGCAACCGTACAAGTTGG Ubiquitin-endochitinase gene fusion FVEX B1 PCR amplification of Exochitinase cds CGACTTCACCCACTGCTTCTTTTGC Ubiquitin-exochitinase gene fusion FVGLU B1 PCR amplification of Glucanase cds CCCAGACGCCAACAAGGATCTTC Ubiquitin-glucanase gene fusion GLUC 3′ Start codon context Glucanase gene AGATCTGGCTTAGCAAGTAAGGCTGAAC modification GLUC 5′ Start codon context Glucanase gene GGATCCACCAACCAGCGATGAAGTTCTTCA modification GCACTCTTAGC Glucgene-1 Sequencing of glucanase pFSC22-2 GTACTGGGTTGGTGAGAC genomic fragment Glucgene-2 Sequencing of glucanase pFSC22-2 CCATTCCAAGACCAGGC genomic fragment NOS A Sequencing across the NOS 3′UT CCCATCTCATAAATAACGTC Transgene-NOS fusion border UBI 1A Sequencing across the Ubiquitin promoter CCTGCCTTCATACGCTAT Ubiquitin-transgene fusion border UBI A2 PCR amplification of Ubiquitin promoter CCTGCCTTCATACGCTATTTATTTGC Ubiquitin-transgene fusion UBI 1B Sequencing mutated Ubiquitin promoter GACACCAACCAGCGAACCA BglII site in promoter UBI 1E Removal of Bgl II site Ubiquitin promoter CACACACAACCAGATTTCCCCCAAATCCACC from promoter by site-directed mutagenesis UBI 1F Removal of Bgl II site Ubiquitin promoter GGTGGATTTGGGGGAAATCTGGTTGTGTGTG from promoter by site-directed mutagenesis - Glucanase. The nucleotide sequence of aF. sporotrichioides genomic fragment encoding a glucanase was obtained as described in Example 9 from plasmid clone pFSC22-2. Primers used for sequencing were: 1014, 1015, 1016, 1017, 1018, 1021, 1023, 1038, 1040, 1042, Glucgene-1 and Glucgene-2 (Table 2). The nucleotide sequence was proofread and assembled as in Example 9.
- Endochitinase. The 5′ end of the endochitinase cDNA (FIG. 2) was obtained by PCR amplification of 0.5 μg of theF. venenatum cDNA library using primers 1010 (from the endochitinase EST) and 720 (from the pZERO-2 sequence for M13 reverse primer). PCR conditions for the 1010/720 reaction were 94° C., 30 sec (1 min. first cycle); 52° C., 25 sec; 72° C., 60 sec, for 25 cycles. The PCR product was purified after partitioning on an agarose gel. Nucleotide sequence data of the endochitinase PCR product (5′ end) was obtained using primers 410, corresponding to the SP6 promoter of the cloning vector pZERO-2 (Invitrogen), and 1011, corresponding to the endochitinase cDNA. PCR conditions for the 1011/410 cycle sequencing reactions were 94° C., 30 sec (1 min. first cycle); 52° C., 25 sec; 72° C., 50 sec, for 25 cycles. Note: the reaction for 1011/410 took advantage of internal primers to reduce non-specific amplification of the initial PCR reaction.
- Exochitinase. A genomic library ofFusarium sporotrichioides was constructed in phage Lambda λgt11 as described in Huynh et al. 1984. The 5′ portion of the exochitinase gene (FIG. 3) was amplified from the F. sporotrichioides library (0.5 μg of DNA in a 20 μL reaction) with primers 129, corresponding to sequence located upstream of the EcoRI site of the λgt11 cloning vector (Huynh et al. 1984), and 1003, corresponding to the exochitinase cDNA. PCR conditions for the 129/1003 reaction were 94° C., 30 sec (1 min. first cycle); 52° C., 25 sec; 72° C., 135 sec, for 25 cycles. The resulting PCR product was purified from an agarose gel. Nucleotide sequence data of the PCR product (5′ portion of the exochitinase gene) was obtained with primers 128, corresponding to sequence located upstream of the EcoRI site of λgt11, and 1009, corresponding to the exochitinase cDNA. PCR conditions for the 128/1009 cycle sequencing reactions were 94° C., 30 sec (1 min. first cycle); 52° C., 25 sec; 72° C., 50 sec, for 25 cycles. Note: the second PCR reaction (128/1009) took advantage of internal primers to reduce background from the initial PCR reaction.
- Overlaps in the nucleotide sequences of the partial cDNAs and the corresponding 5′ PCR products were detected both by visual examination and by analysis of open reading frames using MapDraw (DNASTAR, Inc, Madison, Wis.). For theF. venenatum full-length endochitinase, a composite was assembled directly from the partial cDNA sequence and from the sequence of the 5′ cDNA PCR product. The full-length exochitinase cDNA represents a composite of the F. sporotrichioides 5′ genomic sequence adjoined to the partial F. venenatum cDNA sequence. The transcriptional start of the exochitinase message is hypothesized to be the first A nucleotide following the putative TATAA consensus element.
- A 930 bp, full-length glucanase cDNA with an AC-rich 5′ untranslated region was obtained by PCR amplification. The PCR amplification reaction consisted of: 50 ng of glucanase plasmid DNA cleaved with EcoRI, 0.4
uM GLUC 3′ andGLUC 5′ primers (Table 2), 0.2 uM of each deoxyribonucleotide (dATP, dCTP, dGTP, TTP), and 1 Unit of Elongase (Gibco BRL, Rockville, Md.) in a total volume of 50 μL buffer (60 mM TRIS sulfate, pH 9.1, 18 mM ammonium sulfate, 1 mM magnesium sulfate). PCR was carried out for 5 cycles at 92° C. for 30 sec, 57° C. for 30 sec, 68° C. for 60 sec followed by 25 cycles at 92° C. for 30 sec, 62° C. for 30 sec, 68° C. for 60 sec. - Full-length cDNAs of the endochitinase and exochitinase were obtained by PCR amplification of genomic DNA
fragments using ENDO 3′ plusENDO 5′ primers orEXO 3′ plusEXO 5′ primers, respectively (Table 2). The final step in generating the full-length endochitinase cDNA was accomplished in a 100 μL reaction using 32 pmol each ofprimers ENDO 5′ andENDO 3′. Amplification was performed with approximately 0.5 μg of F. venenatum cDNA library “A” and Pfu polymerase (Stratagene, La Jolla, Calif.) in Pfu buffer according to the manufacturer's protocol. Conditions for amplification were: 94° C., 30 sec (1 minute for the first cycle); 56° C., 25 sec; 72° C., 150 sec, for 25 cycles. The PE 2400 thermocycler (Perkin Elmer) was used. The fragments were cloned into bacterial and monocot expression vectors as described in Examples 6 and 17, respectively. - PCR amplification of the exochitinase full-length cDNA was accomplished in a 100 μL reaction using 32 pmol each of
primers EXO 5′ andEXO 3′. Amplification of F. venenatum cDNA library “A” (approx. 0.5 μg) was performed with Pfu (Stratagene, La Jolla, Calif.) in Pfu buffer according to the manufacturers' protocol, using a PE 2400 thermocycler (Perkin Elmer). PCR conditions were 94° C., 30 sec (1 min first cycle); 56° C. 25 sec; 72° C., 150 sec, for 25 cycles. The resulting PCR product was gel-purified and cloned into bacterial and monocot expression vectors, as described in Examples 6 and 18. - cDNA fragments were separated from unincorporated primers and from other PCR products by partitioning on agarose gels (Example 8). PCR products of the desired size (930 bp for glucanase, 1240 bp for endochitinase, 1780 bp for exochitinase) were excised from gels with a clean razor blades, then recovered from the gel segments by adsorption onto silica beads (GENECLEAN Spin Kit, BIO 101, Inc., Vista, Calif.) as recommended. cDNA fragments were eluted from the silica matrix with water and stored at −20° C. for subsequent cloning (Examples 16-18).
- Removal of the BglII restriction enzyme recognition site in the Ubi-1 promoter region of pUBK was carried out using the QuikChange™ Site-Directed Mutagenesis Kit and protocols (Stratagene, La Jolla, Calif.). pUBK plasmid DNA (50 ng) was suspended in a reaction mixture of: 125 ng primer UBI 1E (Table 2), 125 ng primer UBI 1F (Table 2), 10 mM of each deoxynucleotide, and 2.5 Units of Pfu polymerase in a total volume of 50 μL 20 mM TRIS chloride, pH 8.0, 10 mM potassium chloride, 6 mM ammonium sulfate, 2 mM magnesium sulfate, 0.1% Triton X-100, and 10 μg/mL bovine serum albumin. PCR was performed with a DNA Thermal Cycler (Perkin Elmer Cetus, currently Foster City, Calif.) and an amplification protocol of 16 cycles of 96° C. for 30 sec, 55° C. for 60 sec, 68° C. for 13 min. The resulting amplified products were treated with 10 Units of Dpn I restriction enzyme for 1 hour at 37° C. CompetentE. coli XL 1-Blue host cells were incubated on ice with 1 μL of the Dpn-treated PCR mix for 30 minutes, immediately transferred to a water bath at 42° C. for 45 sec, then placed on ice for 2 minutes. Cells were suspended in 0.5 mL of liquid medium containing 1% (w:v) NZ amine, 0.5% (w:v) yeast extract, 0.5% (w:v) sodium chloride, 20 mM glucose, 12.5 mM magnesium chloride, and 12.5 mM magnesium sulfate. Cells were incubated at 37° C. for 1 hour with agitation at 225 rpm. Cells were concentrated by centrifugation at 4° C., 1700×g for 3 minutes, then spread onto an LB agar plate containing 50 /g/mL kanamycin sulfate.
- Bacterial colonies showing growth on LB-kanamycin were cultured in 1.5 mL of liquid LB-kanamycin for plasmid DNA isolation as described in Example 7. Plasmid DNA was treated with Bgl II restriction enzyme and the cleavage products visualized in an agarose gel as described in Example 8. A single fragment was obtained, indicating that one of the two Bgl II sites in pUBK had been ablated in the mutagenized plasmid. Nucleotide sequence data of a portion of the Ubi-1 promoter region spanning the mutated site was obtained as described in Example 9 with primer UBI 1B (Table 2). In addition to the conversion of the AGATCT (BglII site) to AGATTT, a 29 bp duplication of a portion of the Ubi-1 promoter was also noted (see FIG. 5).
- The mutagenized plasmid was further treated with BamHI+BglII, PstI and PvuII restriction enzymes as recommended by the manufacturer, and the cleavage products visualized on an agarose gel as described in Example 8. The mutagenized plasmid lacked about 0.7 kb of DNA that was present in pUBK prior to site-directed mutagenesis. To correct this deletion, a 2.0 kb portion of the mutagenized plasmid was excised with HindIII and BamHI restriction enzymes, then ligated to pUBK from which the native HindIII-BamHI fragment had been removed. The ligation reaction contained approximately 50 ng of 2.0 kb fragment, 50 ng of pUBK vector, 0.5 Units of T4 DNA ligase in 10 μL of 50 mM TRIS chloride, pH 7.8, 10 mM magnesium chloride, 10 mM dithiothreitol, 2 mM ATP, and 50 μg/mL bovine serum albumin. The reaction was incubated at 15° C. for 17 hours. ElectrocompetentE. coli NM522 cells were subjected to electroporation in the presence of 1 μL of the ligation mixture and further treated as described in Example 6. Six kanamycin-resistant bacterial colonies were individually cultured in 1.5 mL LB-kanamycin for plasmid isolation as described in Example 7. As expected, these plasmids gave 4.5 kb and 2.0 kb fragments upon digestion with HindIII+BamHI, and a single fragment of about 6.6 kb following BglII treatment. One plasmid, called the monocot expression vector, was selected for subsequent cloning experiments.
- To test the activity of the Ubi-1 promoter following mutagenesis, the 2.0 kb HindIII-BamHI fragment (Example 13) carrying the mutant region was substituted for the original region in a plasmid carrying a Ubi-1-beta-glucuronidase gene (uidA) fusion, such that the GUS reporter gene was under regulation of the modified Ubi-1 promoter. This plasmid was introduced into embryos ofTriticum aestivum (wheat), using procedures described in Example 19. The organs of the floret of uidA transgenic lines were tested for β-glucuronidase (GUS) activity.
- Whole heads of transgenic wheat carrying at least 1 copy of the Ubi::uidA transgene were surface-sterilized by treatment in 70% ethanol for 5 minutes and 20% (v/v) bleach for 15 minutes. Whole heads were rinsed 4 to 5 times in distilled water that had either been autoclaved or passed through 0.2 micron Millipore filter membranes. Florets were dissected in a sterile hood such that glumes, lemmas, paleas, developing seeds and anthers were separated. The floret organs were immediately immersed in a substrate solution containing 10 mM sodium phosphate buffer,
pH 7, 0.5 mM potassium ferrocyanide, 0.5 mM potassium ferricyanide, 0.5% (w:v) Triton X-100, and 330 μg/mL 5-bromo-4-chloro-3-indoyl-beta-D-glucuronide (Jersey Lab and Glove Supply, Livingston, N.J.). After 16-20 hours of incubation in darkness, the substrate solution was withdrawn. The plant materials were rinsed twice with 0.1 M sodium phosphate buffer,pH 7. Chlorophyll and other pigments were removed from the green tissues by successive immersions in 70%, 80% and 90% ethanol solutions, 20-24 hours per solution. Treatments with 90% ethanol were repeated until the tissues appeared translucent and nearly white. Activity of the GUS gene was evident as a dark blue pigment within the developing seed and pollen of wheat, indicating that the modified Ubi-1 promoter was active in these organs. GUS activity was also detected in the chlorophyll-containing cells and tissues of the glume, lemma and palea. No GUS activity was seen in the brush or in the anther. - PCR products representing full-length cDNA fragments of the glucanase, endochitinase, and exochitinase, with 15 to 16 bases of AC-rich 5′ untranslated sequence, were mobilized into either pCR2.1 (Invitrogen, Carlsbad, Calif.; for glucanase) or Bluescript vector pBKS+(Stratagene, La Jolla, Calif.; for endochitinase and exochitinase). Ligations were carried out as described in Example 13 with approximately 50-100 ng of bacterial vector DNA that was previously treated with SmaI restriction enzyme, and 60 ng of the PCR product. Transformations ofE. coli NM522 host cells (for glucanase) or DH5-alpha host cells (for endochitinase and exochitinase) were carried out as described in Example 6. Transformants were selected on LB agar plates containing 100 μg/mL carbenicillin.
- To facilitate the identification of transformants carrying the endochitinase and exochitinase cDNAs, colony blot hybridizations were performed (Sambrook et al., 1989). Carbenicillin-resistant colonies were transferred in an ordered array to both fresh agar plates and to 82 mm disks of Hybond N+nylon membrane (Amersham) laid atop LB-carbenicillin agar. Transferred colonies were grown at 37° C. for 16-18 hours. Nylon disks with colonies were placed on a sheet of 3MM (Whatman) paper that was saturated with 0.5 N sodium hydroxide, 1.5 M sodium chloride for 5 minutes, blotted briefly on clean paper, then transferred to paper saturated with 0.5 M TRIS-CL, pH 7.0, 3 M sodium chloride for 5 minutes. The colony disks were submerged in 2×SSC (0.3 M sodium chloride, 0.03 M sodium citrate, pH 7.0) for 10 minutes with agitation at 60-70 rpm to loosen bacterial debris, rinsed in fresh 2×SSC and dried in air. The colony disks were incubated in glass tubes in a Hybaid hybridization oven (National Labnet, Woodbridge, N.J.) at 65° C. for 17 to 18 hours in 20 mL of a hybridization solution containing 5×SSC, 0.1% (w:v) sodium lauryl sarcosine, 0.02% (w:v) sodium dodecyl sulfate, and blocking reagent (Boehringer Mannheim). Colony disks were transferred to 10 mL of fresh hybridization solution containing 20-25 ng of heat-denatured probe and incubated at 65° C. for 16 to 20 hours in the hybridization oven. Digoxygenin-labeled hybridization probes were made with ˜100 ng of the endochitinase and exochitinase PCR products and 4 μL of DIG HighPrime mix in a final volume of 20 μL (Boehringer Mannheim), incubated at 37° C. for 16 to 20 hours. Unincorporated label was removed by precipitation of the probe DNA in 0.5 M lithium chloride and 2.5 volumes of 95% or 100% ethanol at −80° C. for 20 minutes. Probe DNA was resuspended in water for use in hybridization solutions. Hybridized filters were washed in the hybridization tubes as follows: two washes of 50
mL 2×SSC, 0.1% SDS at room temperature for 5 minutes and two washes of 50 mL 0.5×SSC, 0.1% SDS, 65° C. for 25 minutes. Filters were transferred to 1 L beakers and incubated at 70 to 75 rpm, room temperature in: 100 mL 0.1 M maleic acid, 0.15 M sodium chloride, pH 7.5, 0.3% (v:v)Tween 20; 100 mL 0.1 M maleic acid, 0.15 M sodium chloride, pH 7.5, and 0.1 volumes of blocking solution, 30 minutes; 40 mL 0.1 M maleic acid, 0.15M sodium chloride, pH 7.5, 0.1 volumes of blocking solution, and 4 μL anti-DIG-AP conjugate (Boehringer Mannheim); two washes of 100 mL 0.1 M maleic acid, 0.15M sodium chloride, pH 7.5, 0.3% (v:v)Tween 20, 15 minutes each; 40 mL 0.1 M TRIS-Cl, pH 9.2, 0.1 M sodium chloride, 5 minutes. DIG hybridization to colonies on the filters was detected with CSPD reagent (Boehringer Mannheim) and subsequent exposure onXAR 5 scientific imaging film (Kodak, Rochester, N.Y.). - Candidate glucanase cDNA clones were treated with BamHI+BglII, Eco RI or EcoRV; candidate endochitinase cDNA clones were treated with BamHI, Bgl II, and BamHI+BglII; candidate exochitinase cDNA clones were treated with BamHI, Bgl II, BamHI+BglII, Clal, and XhoI. The restriction enzyme digestion patterns indicated that all three cDNAs had been cloned in both orientations within the bacterial plasmid vectors, and that a BamHI and a BglII site had been added to each cDNA.
- To verify the fidelity of the polymerase chain reaction and cloning, nucleotide sequences of both strands of each cDNA were obtained by automated fluorescent sequencing as described in Example 9 (see SEQ ID NOS. 16, 18, and 20). Double-stranded sequences of the cDNAs were analyzed for the presence of restriction sites as described in
Part 3, and for matches to entries in the GenBank database with BLASTX. - PCR-modified cDNAs having nucleotide sequences identical to the original cDNAs were introduced into a monocot expression vector, pUBKBglII-, for use in wheat transformation experiments. The monocot expression plasmid vector was digested extensively with BamHI and BglII restriction enzymes. The pUBKBglII- plasmid vector DNA was purified by partitioning on agarose as described in Example 8.
- A glucanase clone in which the 5′ end of the glucanase coding sequence was inserted proximal to the T7 promoter and M13 forward primer sites of pCR2.1 was selected to generate the modified glucanase cDNA. The modified glucanase cDNA was excised from pCR2.1 by partial digestion with BamHI restriction enzyme as follows: In a 1.5 mL conical bottom tube, 25 μg of glucanase plasmid DNA was suspended in 125 μL of 150 mM sodium chloride, 10 mM TRIS-Cl, pH 7.9, 1 mM magnesium chloride, 1 mM dithiothreitol, and 100 μg/mL bovine serum albumin. Thirty microliter aliquots of the mixture were dispensed to two other tubes. To the mix remaining in the first tube, 10 Units of BamHI enzyme was added and thoroughly mixed. Then, 30 μL of the plasmid-restriction enzyme mixture was added to a tube containing 30 μL of plasmid mix without Bam HI restriction enzyme, and mixed thoroughly. The resulting solution was diluted about two-fold for BamHI enzyme. A sequential dilution of the enzyme mix to about 1:4 was carried out with the second tube of plasmid mix (without BamHI enzyme). Incubation of all 3 tubes was performed at 37° C. for 85 minutes. Four different DNA fragments representing partial BamHI digestion products were visualized on a 1% agarose gel. About 6 to 8 μg of plasmid DNA from the digestion mixture containing 1:4 diluted BamHI was partitioned on 1% agarose. A fragment of about 1 kb carrying the modified glucanase cDNA was excised from the agarose gel and purified on silica beads as described in Example 12.
- About 36 ng of the glucanase cDNA fragment and 220 ng of pUBKBglII- vector DNA, digested with BamHI and BglII, were ligated in 10 μL at 15° C. for 22 hours. Two microliters of the ligation mixture was used to transformE. coli host DH5- alpha as described in Example 6. Bacterial colonies were selected for growth on LB agar containing 50 μg/mL kanamycin. Cells were prepared from 1.5 mL liquid cultures as described in Example 7. Plasmid DNA and pUBKBglII- DNA were partitioned on 1% agarose at 69V for about 1 hour. Plasmids that showed slower migration in the agarose gel were further treated with BamHI+BglII or BamHI restriction enzymes. Insertion of the modified glucanase cDNA in the sense orientation with respect to the Ubi-1 promoter gave BamHI fragments of 0.55 kb in size, and were distinguishable from the antisense orientation of the cDNA, which resulted in a 0.39 kb BamHI fragment.
- Several clones representing each orientation were selected for nucleotide sequence verification. The junction of the Ubi-1 promoter-glucanase gene fusion (Ubi::Glu) was sequenced with primer UBI 1A (see Table 2); the glucanase gene-NOS terminator fusion (Glu: :NOS) was sequenced with primer NOS A (see Table 2). Sequences of representative sense and antisense clones of the modified glucanase cDNA are given. In the sense orientation, a BamHI site at the Ubi-1 junction is present at the 5′ end of the cDNA (FIG. 6). A BglII site, and 47 bp of DNA from the polylinker region of pCR2.1 containing an EcoRI site, are present at the 3′ end. In the antisense orientation, the BamHI site at the Ubi-1-cDNA junction is followed by 47 bp of the pCR2.1 polylinker region (with an EcoRI site), then the BglII site that occurs at the 3′ end of the modified glucanase cDNA (FIG. 7). The antisense version contains a chimeric non-functional BamHI-BglII site at the cDNA-NOS terminator junction.
- The modified full-length endochitinase cDNA was obtained from a clone in which the 5′ end of the coding sequence was proximal to the T7 promoter site of pBKS+. About 4 μg of plasmid DNA was cleaved extensively with 30 Units of BamHI and 30 Units of BglII restriction enzymes in 50 μL of 150 mM sodium chloride, 10 mM TRIS-Cl, pH 7.9, 1 mM magnesium chloride, 1 mM dithiothreitol, and 100 μg/mL bovine serum albumin. The cDNA fragment was recovered from the agarose gel by partitioning on silica beads as described in Example 12. Ligation of 90 ng of cDNA fragment and 440 ng of pUBKBglII-, digested with BamHI and BglII, was carried out in 15 ,μL as described in Example 13.E. coli DH5-alpha cells were transformed with 3 μL of the ligation mixture (see Example 6) and grown at 37° C. in the presence of 50 μg/mL kanamycin sulfate. Plasmid DNA was extracted from candidate kanamycin-resistant colonies. Plasmids that migrated more slowly than pUBKBglII- on agarose gels were cleaved with BamHI and BglII restriction enzymes. A ˜1.2 kb fragment was seen in some (sense orientation) clones. The absence of any cleavage by BamHI+BglII was expected for antisense clones.
- The junctions of the Ubi-1 promoter-endochitinase cDNA fusion of several clones were sequenced with primer UBI 1A (see Table 2); the endochitinase cDNA-NOS terminator fusion was sequenced with primer NOS A (see Table 2). Representative sense and antisense orientations of the cDNA in pUBKBglII- are shown in FIGS. 8 and 9, respectively. The sense clone of the endochitinase contains a BamHI restriction site at the Ubi-1-coding sequence junction and a BglII restriction site at the cDNA-NOS junction. BamHI and BglII sites are absent in the endochitinase antisense clone.
- To obtain the modified exochitinase cDNA, an exochitinase clone was selected in which the 5′ end of the exochitinase coding sequence was proximal to the T3 primer site in pBKS+. About 3 μg of plasmid DNA was treated with 20 Units of BamHI in a final volume of 30
μL 150 mM sodium chloride, 10 mM TRIS-CL, pH 7.9, 1 mM magnesium chloride,1 mM dithiothreitol, and 100 μg/mL bovine serum albumin at 37° C. for 90 minutes. A DNA fragment of about 2.0 kb was purified from an agarose gel as described in Example 8. The ligation mixture contained about 60 ng of the cDNA fragment and 220 ng of pUBKBglII- DNA (see Example 16). Transformants of E. coli DH5-alpha were generated and grown as in Example 15. Plasmid DNA that migrated slower than the pUBKBglII- vector was subjected to nucleotide sequence analysis (Example 9) using UBI 1A and NOS A primers to determine the orientation of the coding sequence within pUBKBglII-. Additional exochitinase clones were identified using the colony blot procedure (see Example 15). Representative sequences for the sense and antisense orientations of the exochitinase cDNA in pUBKBglII- are shown in FIGS. 10 and 11, respectively. The sense clone of the exochitinase contains a BamHI restriction site at the Ubi-1-coding sequence junction and a BglII restriction site at the cDNA-NOS junction. These BamHI and BglII sites are absent in the exochitinase antisense clone. - The method of Weeks et al. (1993) was used to introduce the modified full-length cDNA clones (in the monocot expression vector, pUBKBglII-) into wheat, but with following additions and modifications. Embryos were excised from seeds ofT. aestivum cultivar Bobwhite at about 10-12 days post anthesis (dpa). Embryos were placed scutellum-side up onto 15 mm×100 mm plates containing 30 mL of MMS medium [4.3 g/L Murashige & Skoog salt mixture (Gibco BRL, Rockville, Md.), 0.5 mg/L thiamine hydrochloride, 0.15 g/L L-asparagine, 40 g/L maltose] solidified with 3.5 g/L Phytagel (Sigma Chemical Co., St. Louis, Mo.) and supplemented with 2 mg/
L 2,4-D. The embryos were incubated at 25° C. in darkness for 5 days to allow development of callus and then transferred to 15 mm×60 mm plates containing 20 mL of the above medium supplemented with 0.35 M mannitol. After 4 hours on mannitol-containing medium, embryos were subjected to bombardment with 0.7 mg per plate of 1-2 micron (average diameter) particles of gold (Bio-Rad, Hercules, Calif.) coated with 17.4 μg of pUBKBglII- and 19.5 μg of FvEndoS or with 15.5 μg pUBKBglII- and 16.2 μg of FvGluS or with 12.5 μg pUBK and 22.6 μg FvExoS. The next day, embryogenic calli were transferred to “MMS2” medium (MMS media supplemented with 2 mg/L 2,4 D and solidified with 2.5 g/L Phytagel). The calli were placed at a density of 20 (FvGluS) or 40 (FvEndoS and FvExoS) per plate and incubated at 25° C. in darkness for two weeks. The calli were then selected for two 2-week periods by incubation in darkness at 25° C. on plates containing MMS2 media supplemented with the following additions: for FvEndo, 0 or 1 mg/L bialaphos (Meiji Seika Kaisha Ltd., Tokyo, Japan), then 2 mg/L bialaphos; forFvExoS 1 or 1.5 mg/L, then 3 mg/L bialaphos; for FvGluS, 1 mg/L, then 2 mg/L bialaphos. The surviving calli were then transferred to MMS media solidified with 2.5 g/L Phytagel and containing 0.2 mg/L 2,4-D and 3 mg/L bialaphos and incubated in the light at 26° C. for six weeks. Green shoots that formed during this time were transferred to 25×150 mm test tubes containing 18 mls of rooting medium [2.15 g/L Murashige & Skoog salt mixture, 0.25 mg/L thiamine hydrochloride, 0.075 g/L L-asparagine, 20 g/L maltose, 2.5 g/L Phytagel]. The tubes were incubated in the light at 26° C. Shoots that formed roots were transferred to soil (Sunshine mix#1) and incubated in the light at 22° C. under saran wrap for one week. Humidity was lowered by poking an increasing number of holes in the saran wrap during a second week of incubation. The plants were then transplanted to the greenhouse maintained at 23° C. with supplemental lights set for 16/8-hour day/night cycle. Immature embryos about 21 dpa were excised from the seeds of these plants and precociously germinated in magenta boxes containing 100 mls of MMS medium solidified with 2.5 g/L phytagel. - Genomic DNA or total DNA was extracted from wheat leaves to analyze candidate lines for stable incorporation of the modified, full-length glucanase, endochitinase and exochitinase cDNA constructs. Untransformed plants of the cultivar Bobwhite were sampled as negative controls.
- Genomic DNA was prepared by a modified method of d'Ovidio et al. (1992). Three to five grams fresh weight of healthy leaf tissue was excised from the plant, placed on ice immediately, then in liquid nitrogen, or placed directly in liquid nitrogen. Leaf samples were often stored at −80° C. prior to extraction of genomic DNA. Leaf tissue was ground with a chilled mortar and pestle in the presence of liquid nitrogen. Powdered tissue was transferred to 35-40 mL of chilled homogenization buffer containing 0.5 M sucrose, 80 mM potassium chloride, 10 mM TRIS-chloride, 10 mM EDTA, 4 mM spermine, 1 mM spermidine, pH 9.5, 180 mg/L phenylmethylsulfonyl fluoride (added immediately before use), and 0.1% (v:v) beta-mercaptoethanol (added immediately before use). The leaf material was further homogenized with three to four 5-second pulses in a chilled blender. The homogenate was filtered through four layers of cheesecloth, followed by one layer of Miracloth filtration material (Calbiochem, La Jolla, Calif.). The clarified extract was centrifuged at 1000×g for 20 minutes at 4° C. The supernatant was carefully decanted and the pellet containing nuclei washed twice in 20 to 40 mL homogenization buffer supplemented with 0.5% Triton X-100 (Sigma Chemical Co., St. Louis, Mo.). The washed nuclei pellet was resuspended in 1 mL of a buffer solution containing 50 mM TRIS-chloride,
pH - Total DNA was isolated from 1 to 1.5 cm2 sections of leaf tissue by a modified method of Dellaporta et al. (1983). Sections were cut from healthy leaves with clean scissors, placed in 1.5 mL conical microcentrifuge tubes, and transferred directly to liquid nitrogen. Leaf sections were often stored at −80° C. prior to DNA extraction. Leaf tissue was homogenized in liquid nitrogen with a microfuge pestle (Kontes Glass Co., Vineland, N.J.) in the microcentrifuge tube. To a tube of the frozen powder was added 0.5 mL of an extraction buffer containing 50 mM TRIS-chloride,
pH pH DNA 110 Speed Vac, and dissolved in 50 μL of water. Best PCR amplification was obtained with freshly-prepared extracts. - For PCR amplification, 200 to 400 ng of genomic DNA or 2 μL of total DNA was brought to 10 μL with water in a 0.2 mL thin-walled tube (MJ Research, Inc., Watertown, Mass.). To each sample was added 40 μL of a PCR mixture containing 25 picomoles of UBI A2 primer, 25 picomoles of transgene-specific primer (see Table 3), 50 mM potassium chloride, 10 mM TRIS-chloride, pH 9.0, 0.1% Triton X-100, 3 mM magnesium chloride, 1 mM each of dATP, dCTP, dGTP and TTP, and 2.5 Units of Taq DNA polymerase (Promega, Madison, Wis.). Polymerase chain reaction was carried out on a PTC-100 or PTC-200 Programmable Thermal Controller (MJ Research, Watertown, Mass.) with the following protocol: 96° C. for 30 sec; 35 cycles of 95° C. for 1 minute, 62° C. for 1 minute, 72° C. for 1 minute; 72° C. for 15 minutes; 4° C. soak. PCR products (10-13 μL of each PCR reaction mixture) were partitioned on 1% to 1.6% agarose gels and visualized by ethidium bromide staining and ultraviolet light as described in the Example. Positive PCR controls included 10 to 20 ng of pUBKBglII- plasmid DNA containing the glucanase, endochitinase or exochitinase cDNAs.
- At least three transgenic wheat lines showing amplification of the predicted 425 bp Ubi-exochitinase PCR product, or the predicted 375 bp Ubi-glucanase PCR product, or the predicted 340 bp Ubi-endochitinase PCR product have been obtained in our experiments (FIG. 12).
- Transgenic lines were propagated as described in Example 19. Progeny were analyzed for bialaphos resistance and/or for the presence of theF. venenatum transgenes using PCR. Populations in which all of the individual progeny were bialaphos resistant and/or were positive in the PCR transgene assay in two successive generations were considered to be derived from a line homozygous for the transgene.
- Homozygous lines and/or their progeny, identified as described in Example 20, were analyzed for the expression of transgene messenger RNA (mRNA) by northern blot analysis. Seed was harvested from heads at 15-20 days post-anthesis and surface sterilized by immersion in 70% ethanol for 5 min., followed by 20% (v:v) hypochlorite solution for 15 min. Seeds were rinsed 4 to 5 times with sterile water. The embryo was removed from each seed with dissecting forceps, and endosperm material was squeezed from the seed coat with the flat side of a metal spatula. The endosperm was frozen in liquid nitrogen as it was harvested, and pooled endosperm material from 15-30 seeds was stored at −80° C. prior to RNA isolation. Total RNA was isolated using a modified method of Altenbach (1998). Essentially, frozen endosperm was ground to a fine powder in a chilled mortar and pestle, in the presence of liquid nitrogen. The frozen homogenate was transferred to a 50-mL conical centrifuge tube containing 5 mL extraction buffer (10 mM sodium chloride, 10 mM TRIS-Cl, pH 9.0, 1 mM EDTA), 2.5 mL phenol (saturated with TRIS buffer at pH 4.3, Fisher #BP1751), and 2.5 mL chloroform. The tube was inverted 40 times, then centrifuged at 6500 rpm, 4° C. for 10 minutes in a Sorvall HS-4 rotor. The aqueous phase was transferred to a fresh tube, extracted once with an equal volume of phenol/chloroform (1:1 v/v), and once with an equal volume of chloroform. The mixture was centrifuged after each extraction as described. To the aqueous extract, 8M LiCl was added to give a final concentration of 2M LiCl and incubated at −20° C. overnight. The precipitate containing RNA was collected by centrifugation at 12,500 rpm, 4oC for 30 minutes in a Sorvall SS-34 rotor. The RNA pellet was washed with ice cold 2M LiCl, air dried, and resuspended in autoclaved water. The 8M LiCl solution and water used to resuspend the RNA was previously treated with 0.05% (v:v) diethyl pyrocarbonate (Sigma Chemical Co., St. Louis, Mo.). About 100 μg of total RNA was further purified by passage through an RNeasy column (Qiagen Inc., Valencia, Calif.), as recommended by the manufacturer. Purified RNA was stored at −80° C.
- For northern blots, approximately 4-5 μg of purified RNA was brought to 10 μL with sample buffer containing 50% formamide, 6% formaldehyde (Fisher #BP531), 20 mM MOPS, pH 7.0, 5 mM sodium acetate, 10 mM EDTA, and 0.01% bromophenol blue. RNA samples were heated at 60° C. for 10 to 15 minutes, then immediately placed on ice prior to partitioning on agarose. The agarose (0.3 g) was dissolved in 30 mL of 20 mM MOPS, 5 mM sodium acetate, 10 mM EDTA, pH 7.0, to which was added 37% formaldehyde for a final concentration of 6% and 10 μg/μL ethidium bromide for a final concentration of 166 ng/mL. Electrophoresis was carried out at 50-70 V for up to 2 hours. Partitioned RNA was transferred to Zeta-Probe GT nylon membrane (Bio-Rad, Hercules, Calif.) in either 10×SSC (150 mM sodium chloride, 15 mM sodium citrate, pH 7.0) or 50 mM sodium hydroxide for 3 hours. The nylon membrane containing RNA (northern blot) was rinsed in 2×SSC (30 mM sodium chloride, 3 mM sodium citrate, pH 7.0) and either treated with ultraviolet light using a UV Stratalinker 2400 (Stratagene, La Jolla, Calif.) and baked at 80° C. under vacuum (neutral transfer), or air dried and used directly (alkaline transfer).
- Northern blots were incubated in a Hybaid hybridization oven (National Labnet, Woodbridge, N.J.) for 6 to 7 hours at 42° C. in 5 to 10 mL of a solution containing 50% (v:v) formamide, 7% (w:v) sodium dodecyl sulfate (SDS), 0.25 M sodium phosphate buffer, pH 7.0, 0.25 M sodium chloride, and 1 mM EDTA, pH 8.0, as recommended by Bio-Rad. Probes for northern blots consisted of partial or full-length cDNAs as follows: 0.4 kb BamHI GLU fragment from FvGluS; 1.2 kb BamHI +BglII Endo fragment from FvEndoS; or 1.8 kb BamHI+BglII Exo fragment from FvExoS. Probe fragments were purified following agarose gel partitioning as described in Example 12. About 25-50 ng of probe DNA was radiolabeled to a specific activity of 1-2×106 cpm/ng with 32P-α-dCTP (3000 Ci/ummol), using the Multiprime DNA Labelling System (Amersham Pharmacia Biotech, Inc., Piscataway, N.J.) as recommended. Radiolabeled fragments were added directly to the northern blots and hybridized at 42° C. for up to 36-40 hours. Blots were washed in 200 to 300 mL of 2×SSC, 0.1% (w:v) SDS at room temperature for 15 minutes; in 200 to 300 mL of 0.5×SSC, 0.1% (w:v) SDS at room temperature for 15 minutes; and in 200 to 300 mL of 0.1×SSC, 0.1% (w:v) SDS at 62° C. for 15 minutes. Autoradiography was performed at −80° C. on
preflashed XAR 5 film (Kodak, Rochester, N.Y.), using an X-ray film cassette and Spectroline L-Plus intensifying screen (Spectronics Corp., Westbury, N.Y.), or at room temperature on a phosphor screen using a Storm 820 phosphoimager (Molecular Dynamics, Sunnyvale, Calif.). Northern blot analysis detected the presence of mRNA transcripts from the endochitinase transgene in line AB8-108 (FIG. 13). - Heterozygous and homozygous lines and/or their progeny, identified as described in Example 20, were also analyzed for the accumulation of transgene mRNAs using reverse transcription-polymerase chain reaction (RT-PCR). RT-PCR is a more sensitive means by which to detect mRNA accumulation in various tissues and organs (Altenbach 1998; Wang et. al. 1998). Total RNA was obtained from glumes of wheat at 2 to 3 weeks post anthesis using the same procedure described for endosperm RNA isolation (above). RT-PCR was carried out on 600 ng of total RNA using either the GeneAmp PCR Core kit (Perkin Elmer, Foster City, Calif.) or a OneStep RT-PCR Kit (Qiagen, Inc., Valencia, Calif.), as recommended by the manufacturers. Transgene-specific transcripts were amplified using primers RTUBI and either RTGLU (for the 5′ end of the endochitinase transcript), RTEND (for the 5′ end of the endochitinase transcript), or RTEXO (for the 5′ end of the exochitinase transcript). See Table 3 for a list of RT-PCR primers. The 3′ portions of the transgene transcripts were amplified with RTNOS and either RTGLU2 (glucanase), RTEND2 (endochitinase), or RTEXO2 (exochitinase). The annealing temperatures were 58°-60° C.
- Endosperm and glume total RNA from Line AB9-59b showed very low but detectable levels of a single 700 bp RT-PCR product (FIG. 14), representing the 5′ end of the transcript, and including the Ubi exon. The size of this product compared closely with the expected product size of 705 bp, which was derived from the predicted nucleotide sequence of the transgene mRNA. The 3′ portion of the glucanase transcript was also amplified at very low, but detectable levels (data not shown), yielding a single 330 bp PCR product (expected
size 340 bp). - In endosperm and glume total RNA isolated from two successive generations of Line AB8-108, a single ˜700 bp RT-PCR product, representing the 5′ end of the endochitinase mRNA, (expected
size 685 bp) (FIG. 15A, left and right panels), and a single 320 bp RT-PCR product, representing the 3′ end of the endochitinase mRNA, (expectedsize 310 bp) (FIG. 15B) were strongly amplified. In total RNA from endosperm of Line C9-25a, a single ˜800 bp RT-PCR product, representing the 5′ end of the exochitinase MRNA (expectedsize 795 bp) (FIG. 15A, right panel), and a single 300 bp RT-PCR product representing the 3′ end of the exochitinase mRNA (expectedsize 310 bp)(FIG. 15B) was detectable. Total RNA from untransformed cultivar Bobwhite (bw) did not produce any RT-PCR products. - For the purpose of authentication, the
endochitinase 5′ RT-PCR product was cleaved exhaustively with either Clal or SspI restriction endonuclease according to the manufacturer's specifications (New England BioLabs, Beverly, Mass.). The resulting cleavage products were partitioned on a 6% TBE acrylamide gel (Novex/Invitrogen, Carlsbad, Calif.), 100V for ˜1 hr, in running buffer consisting of 89 mM TRIS-Cl, pH 8.3, 89 mM borate, 1.5 mM EDTA. The resulting endochitinase cleavage products, shown in FIG. 16, closely matched those expected from the predicted nucleotide sequence of the endochitinase mRNA, and confirmed the identity of the RT-PCR product. Likewise, theexochitinase 5′ RT-PCR product was cleaved with either BglII, EcoRV, or NcoI restriction enzymes (New England BioLabs, Beverly, Mass.). The resulting cleavage products, partitioned on either a 6% acrylamide gel (FIG. 17A) or a 2% agarose gel (FIG. 17B), as previously described in Example 8, were very close in size to those expected from the predicted nucleotide sequence of the exochitinase mRNA. Due to the presence of a minor amplification product in the starting material (lane labeled “none”, FIG. 17A), additional cleavage fragments were observed in the first experiment. The major fragment at ˜800 bp in the BglII lane of the second experiment (FIG. 17B) is attributed to incomplete cleavage by BglII. - The FvEndo and FvExo RT-PCR products were cDNA-dependent, and were therefore generated from RNA rather than from contaminating genomic DNA in the RNA preparations. The RT-PCR products were amplified from both the 5′ and 3′ regions of the FvEndo transcripts in endosperm from three independent lines (FIG. 18A) only if cDNA synthesis (+) was first carried out. In the absence of cDNA synthesis(−), no 5′ or 3′ RT-PCR products were obtained. As expected, FvEndo transcripts were not detected in endosperm of untransformed Bobwhite (Bw) either with or without cDNA synthesis (FIG. 18B). Likewise, FvExo RT-PCR products were amplified from both 5′ and 3′ regions of the transcripts only after cDNA synthesis (FIG. 19). No products were obtained in untransformed Bobwhite samples.
TABLE 3 PCR primers used for RT-PCR experiments Primer Sequence (5′ to 3′) Amplification target RTEND CTGGAAGTTTGTCGCACCG 5′end of FvEndo transcript RTEND2 GAGATAGAGGATGCTGTGCC 3′end of FvEndo transcript RTEXO CAGTGATGTGAAGGTGAAGGC 5′end of FvExo transcript RTEXO2 CGTATGGACTGAGACTATCGAC 3′end of FvExo transcript RTGLU GAGCATCCTTGATGGGAACAC 5′end of FvGlu transcript RTGLU2 GTCAACTCCAAGGCTGTCGTC 3′end of FvGlu transcript RTNOS GCCAAATGTTTGAACGATCTGC 3′end of transcripts RTUBI CAACCTCGTGTTGTTCGGAG 5′end of transcripts -
kb 5′ BamHI fragment of FvGlu cDNA (from plasmid described in EXAMPLE 15); 2) 1.2 kb BamHI/BglII FvEndo cDNA fragment from FvEndoS (FIG. 15); (3) the 1.8 kb BamHI/BglII FvExo cDNA fragment (from plasmid described in EXAMPLE 15). The probes were made radioactive with 32P-alpha-dCTP [specific activity 3000 Ci (111tBq)/mmol] using the Multiprime DNA labelling system (Amersham Pharmacia Biotech, Inc., Piscataway, N.J.). - Our Southern blot results indicate that each of the three genes are present in a single copy in theF. venenatum genome (FIG. 20). The sizes of the hybridizing bands for FvGlu were estimated to be 12.5 kb (HindIII), 1.6 kb (BamHI), and 11 kb (EcoRI), using phage Lambda DNA cleaved with HindIII as a migration standard (see “kb” in FIG. 20). The FvGlu coding region contains a single BamHI site, so two hybridizing BamHI fragments would have been obtained with a full-length cDNA probe. However, the FvGlu probe that was used for this experiment consisted of
cDNA sequences 5′ to the BamHI site, so a single hybridizing BamHI fragment was expected and was obtained. The sizes of the hybridizing FvEndo fragments were 9.3 kb (HindIII), 8.9 kb (BamHI), and 6.6 kb (EcoRI). The sizes of the hybridizing FvExo fragments were 4.3 kb (HindIII), 11 kb (BamHI), and 4.7 kb (EcoRI). Neither FvEndo nor FvExo coding regions carry restriction sites for these three endonucleases. - Statement of Deposit
- Plasmids identified below were introduced into the hostEscherichia coli and the transformed Escherichia coli were deposited under terms of the Budapest Treaty with Agricultural Research Service Culture Collection (NRRL) National Center for Agricultural Utilization Research, Agricultural Research Service, U.S. Department of Agriculture, 1815 North University Street, Peoria, Ill. 61604 USA on the date listed and given the following accession numbers:
Deposit Accession No. FIG./Seq No Date of Deposit GLU2 NRRL B-30201 SEQ16 Aug. 26, 1999 Endol67 NRRL B-30202 SEQ18 Aug. 26, 1999 Exo9 NRRL B-30203 SEQ20 Aug. 26, 1999 FvGluS NRRL B-30204 Aug. 26, 1999 FvGluAS NRRL B-30205 Aug. 26, 1999 FvEndoS NRRL B-30206 Aug. 26, 1999 FvEndoAS NRRL B-30207 Aug. 26, 1999 FvExoS NRRL B-30208 Aug. 26, 1999 FvExoAS NRRL B-30209 Aug. 26, 1999 - It is understood that the foregoing detailed description is given merely by way of illustration and that modification and variations may be made within, without departing from the spirit and scope of the invention. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.
- Andersen, A. (1948). The development of Gibberella zeae headblight of wheat. Phytopathology, 38: 595-611
- Altenbach, S. B. (1998). Quantification of individual low-molecular-weight glutenin subunit transcripts in developing wheat grains by competitive RT-PCR. Theor Appl Genet 97: 413-421
- Altschul S. F., Madden T. L., Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25: 3389-3402
- Bai and Shaner (1994). Scab of wheat: prospects for control. Plant Disease 78: 760-766
- Barbosa, I. P. and C. Kemmelmeier (1993). Chemical composition of the hyphal wall fromFusarium graminearum. Exp. Mycol. 17: 274-283
- Battraw, M. J. and T. C. Hall (1990). Histochemical analysis of CaMV 35S promoter-β-glucuronidase gene expression in transgenic rice plants. Plant Mol. Biol. 15: 527-538
- Becker, D., R. Brettschneider, and H. Lorz (1994). Fertile transgenic wheat from microprojectile bombardment of scutellar tissue. Plant J. 5: 299-307
- Beffa, R. and F. Meins, Jr. (1996). Pathogenesis-related functions of plant β-1,3-glucanases investigated by antisense transformation—a review. Gene 179: 97-103
- Blaiseau, P-L. and J-F. Lafay (1992). Primary structure of a chitinase-encoding gene (chi1) from the filamentous fungusAphanocladium album: similarity to bacterial chitinases. Gene 120: 243-248.
- Blechl, A. E. and O. D. Anderson (1996). Expression of a novel high-molecular-weight glutenin subunit gene in transgenic wheat. Nat. Biotech. 14: 875-879
- Bliffeld, M., J. Mundy, I. Potrykus and J. Futterer (1998). Genetic engineering of wheat for increased resistance to powdery mildew disease. Theor. Appl. Genet. 98: 1079-1086
- Bolar J. P., J. L. Norelli, K-W Wong, C. K. Hayes, G. E. Harman, and H. S. Aldwinckle (2000). Expression of endochitinase fromTrichoderma harzianum in transgenic apple increases resistance to apple scab and reduces vigor. Phytopathology 90: 72-77
- Boyapati, R., A. L. Moyne, T. E. Cleveland and S. Tuzun (1994). Cloning of putative chitinase genes from Bacillus to control toxin producing fungi. Phytopathology 84: 1081
- Broekaert, W. F., J. Van Parijs, A. K. Allen and W. J. Peumans (1988). Comparison of some molecular, enzymatic and antifungal properties of chitinases from thorn-apple, tobacco and wheat. Physiol. Mol. Plant Path. 33: 319-331
- Brunner, F., A. Stintzi, B. Fritig, and M. Legrand (1998). Substrate specificities of tobacco chitinases. Plant J. 14: 225-234
- Caruso, C., C. Caporale, G. Chilosi, F. Vacca, L. Bertini, P. Magro, E. Poerio and V. Buonocore (1996). Structural and antifungal properties of a pathogenesis-related protein from wheat kernel. J. Prot. Chem. 15: 35-44.
- Cavallarin, L., D. Andreu and B. San Segundo (1998). Cecropin A-derived peptides are potent inhibitors of fungal plant pathogens. Mol. Plant-Microbe Inter. 11: 218-227.
- Chen, W. P., X. Gu, G. H. Liang, S. Muthukrishnan, P. D. Chen, D. J. Liu and B. S. Gill (1998). Introduction and constitutive expression of a rice chitinase gene in bread wheat using biolistic bombardment and the bar gene as a selectable marker. Theor. Appl. Genet. 97: 1296-1306
- Chérif, M. and N. Benhamou (1990). Cytochemical aspects of chitin breakdown during the parasitic action of a Trichoderma sp. onFusarium oxysporum f. sp. radicis-lycospersici. Phytopathology 80: 1406-1414
- Christensen, A. H. and P. H. Quail (1996). Ubiquitin promoter-based vectors for high-level expression of selectable and/or screenable marker genes in monocotyledonous plants. Trans. Res. 5: 213-218
- Collinge, D. B., K. M. Kragh, J. D. Mikkelsen, K. K. Nielsen, U. Rasmussen, K. Vad. (1993). Plant chitinases. The Plant Journal 3:31-40
- Cornejo, M-J., D. Luth, K. M. Blankenship, O. D. Anderson and A. E. Blechl (1993). Activity of a maize ubiquitin promoter in transgenic rice. Plant Mol. Biol. 23: 567-581
- Cruz-Ortega, R., J. C. Cushman and J. D. Ownby (1997). cDNA clones encoding 1,3-β-glucanase and a fimbrin-like cytoskeletal protein are induced by A1 toxicity in wheat roots. Plant Physiol. 114: 1453-1460
- de Block, M., J. Botterman, M. Vandewiele, J. Dockx, C. Thoen, V. Gosselé, N. Rao Movva, C. Thompson, M. Van Montagu and J. Leemans (1987). Engineering herbicide resistance in plants by expression of a detoxifying enzyme. EMBO J. 6: 2513-2518
- Dellaporta, S. L., J. Wood and J. B. Hicks (1983). A plant DNA minipreparation: version II. Plant Mol. Biol. Rep. 1: 19-21
- deLucca, A. J., J. M. Bland, T. J. Jacks, C. Grimm, T. E. Cleveland, and T. J. Walsh (1997). Fungicidal activity of cecropin A. Antimicrob. Agents Chemother. 41: 481-483
- D'Ovidio, R., O. A. Tanzarella and E. Porceddu (1992). Isolation of an alpha-type gliadin gene fromTriticum durum Desf. and genetic polymorphism at the Gli-2 loci. J. Genet. Breed. 46: 41-48.
- Draborg, H., S. Kauppinen, H. Dalbøge and S. Christgau (1995). Molecular cloning and expression inS. cerevisiae of two exochitinases from Trichoderma harzianum. Biochem. Mol. Biol. Int. 36: 81-791
- Flach, J., P.-E. Pilet, and P. Jollès (1992). What's new in chitinase research? Experientia 48: 701-716
- Fontaine, T., R. P. Hartland, M. Diaquin, C. Simenel, and J. P. Latge (1997). Differential patterns of activity displayed by two exo-β-1,3-glucanases associated with theAspergillus fumigatus cell wall. J Bact 179: 3154-3163
- Freeling, M. and D. C. Bennett (1985). Maize Adhl. Annu Rev Genet 19:297-323
- Fritig, B., T. Heitz and M. Legrand (1998). Antimicrobial proteins in induced plant defense. Curr. Opinion Immun. 10: 16-22
- Fukamizo, T., T. Ohkawa, K. Sonoda, H. Toyoda, T. Nishiguchi, S. Ouchi and S. Goto (1992). Chitinous components of the cell wall ofFusarium oxysporum. Biosci. Biotech. Biochem. 56: 1632-1636
- Fütterer, J. and T. Hohn (1996). Translation in plants—rules and exceptions. Plant Mol. Biol. 32: 59-189
- Graham, L. S. and M. B. Sticklen (1994). Plant chitinases. Can. J. Bot. 72: 1057-1083
- Harman, G. E., C. K. Hayes, M. Lorito, R. M. Broadway, A. Di Pietro, C. Peterbauer, and Tronsmo A (1993). Chitinolytic enzymes ofTrichoderma harzianum: purification of chitobiosidase and endochitinase. Phytopathology 83: 313-318
- Hu, X. and A. S. N. Reddy (1997). Cloning and expression of a PR5-like protein from Arabidopsis: inhibition of fungal growth by bacterially expressed protein. Plant Mol. Biol. 34: 949-959
- Huynh, T. V., R. A. Young, and R. W. Davis. Constructing and screening cDNA libraries in Lambda-gt10 and Lambda-gt11. In:DNA Cloning Techniques: A Practical Approach, edited by D. Glover, IRL Press: Oxford, 1984, p. 1-62.
- Jach, G., B. Gornhardt, J. Mundy, J. Logemann, E. Pinsdorf, R. Leah, J. Schell and C. Maas (1995). Enhanced quantitative resistance against fungal disease by combinatorial expression of different barley antifungal proteins in transgenic tobacco. Plant J. 8: 97-109
- Jensen, L. G., O. Politz, O. Olsen, K. K. Thomsen and D. Von Wettstein (1998). Inheritance of a codon-optimized transgene expressing heat stable (1,3-1,4)—glucanase in scutellum and aleurone of germinating barley. Hereditas 129: 215-225
- Jondedijk, E., H. Tigelaar, J. S. C. van Roekel, S. A. Bres-Vloemans, I. Dekker, P. J. M. van den Elzen, B. J. C. Cornelissen, and L. S. Melchers (1995). Synergistic activity of chitinases and β-1,3-glucanases enhances fungal resistance in transgenic tomato plants. Euphytica 85: 173-180
- Jutidamrongphan, W., J. B. Anderson, C. Mackinnon, J. M. Manners, R. S. Simpson and K. J. Scott (1991). Induction of β-1,3-glucanase in barley in response to infection by fungal pathogens. Mol. Plant-Microbe Inter. 4: 234-238
- Keen N. T. and M..Yoshikawa M (1983). β-1,3-endoglucanase from soybean releases elicitor-active carbohydrates from fungus cell walls. Plant Physiol 71: 460-465
- Klebl, F. and W. Tanner (1989). Molecular cloning of a cell wall exo-β-1,3-glucanase fromSaccharomyces cerevisiae. J. Bact. 171: 6259-6264
- Kozak, M. (1987). An analysis of 5′-noncoding sequences from 699 vertebrate messenger RNAs. Nucl. Acids Res. 15: 8125-8148
- Krishnaveni, S., S. Muthukrishnan, G. H. Liang, G. Wilde and A. Manickam (1999a). Induction of chitinases and β-1,3-glucanases in resistant and susceptible cultivars of sorghum in response to insect attack, fungal infection and wounding. Plant Sci 144: 9-16
- Krishnaveni, S., G. H. Liang, S. Muthukrishnan and A. Manickam (1999b). Purification and partial characterization of chitinases from sorghum seeds. Plant Sci. 144: 1-7
- Linthorst, H. J. M. (1991). Pathogenesis-related proteins of plants. Crit. Rev. Plant Sci. 10: 123-150
- Linthorst, H. J. M., L. S. Melchers, A. Mayer, J. S. C. van Roekel, B. J. C. Cornelissen and J. F. Bol (1990). Analysis of gene families encoding acidic and basic β-1,3-glucanases of tobacco. Proc. Natl. Acad. Sci. 87: 8756-8760
- Liu, L., D. S. Maillet, J. R. H. Frappier, D. B. Walden and B. G. Atkinson (1995). Characterization, chromosomal mapping, and expression of different polyubiquitin genes in tissues from control and heat-shocked maize seedlings. Biochem. Cell Biol. 73: 19-30
- Lotan, T., N. Ori and R. Fluhr (1989). Pathogenesis-related proteins are developmentally regulated in tobacco flowers. Plant Cell 1: 881-887
- Lorito, M., S. L. Woo, I. G. Fernandez, G. Colucci, G. E. Hanlan, J. A. Pintor-Toro, E. Filippone, S. Muccifora, C. B. Lawrence, A. Zoina, S. Tuzun and F. Scala (1998). Genes from the mycoparasitic fungi as a source for improving plant resistance to fungal pathogens. Proc. Natl. Acad. Sci. 95: 7860-7865
- Luehrsen, K. R. and V. Walbot (1994). The impact of AUG start codon context on maize gene expression in vivo. Plant Cell Rep. 13: 454-458
- Malehorn, D. E., K. J. Scott and D. M. Shah (1993). Structure and expression of a barley acidic β-glucanase gene. Plant Mol. Biol. 22: 347-360
- Mauch F., B. Mauch-Mani and T. Boller (1988). Antifungal hydrolases in pea tissue. II. Inhibition of fungal growth by combinations of chitinase and β-1,3-glucanase. Plant Physiol. 88: 936-942
- McCreath, K. J. and G. W. Gooday (1992). A rapid and sensitive microassay for determination of chitinolytic activity. J Microbiol Meth 14: 229-237
- McCue, K. F. and A. D. Hanson (1990). Drought and salt tolerance: towards understanding and application. Trends Biotech. 8: 358-362
- McKay, R. and J. B. Loughnane (1945), Observations on Gibberella saubinetti (Mont.) Sacc. on cereals in Ireland in 1943 and 1944. Sci. Proc.R. Dublin Soc. 24: 9-18
- Melchers, L. S., M. Apotheker-de Groot, J. A. van der Knaap, A. S. Ponstein, M. B. Sela-Buurlage, J. F. Bol, B. J. C. Cornelissen, P. J. M. van den Elzen, and H. J. M. Linthorst (1994). A new class of tobacco chitinases homologous to bacterial exo-chitinases displays antifungal activity. Plant J. 5: 469-480
- Michael, A. H. and Nelson, P. E. (1972). Antagonistic effect of soil bacteria onFusarium roseum culmorum. Phytopathology 62: 1052-1056
- Mitchell, R. and Alexander, M. (1961). The mucolytic phenomenon and biological control of Fusarium in soil. Nature 190: 109-110
- Molina, A., A. Segura and F. Garcia-Olmedo (1993). Lipid transfer proteins (nsLTPs) from barley and maize leaves are potent inhibitors of bacterial and fungal plant pathogens. FEBS Lett. 316: 119-122
- Nehra, N. S., R. N. Chibbar, N. Leung, K. Caswell, C. Mallard, L. Steinhauer, M. Baga and K. K. Kartha (1994). Self-fertile transgcnic wheat plants regenerated from isolated scutellar tissues following microprojectile bombardment with two distinct gene constructs. Plant J. 5: 285-297
- Parry, D. W., P. Jenkinson and L. McLeod (1995). Fusarium ear blight (scab) in small grain cereals—a review. Plant Pathology 44: 207-238
- Payne, G., E. Ward, T. Gaffney, P. A. Goy, M. Moyer, A. Harper. F. Meins, Jr. and J. Ryals (1990). Evidence for a third class of β-1,3-glucanase in tobacco. Plant Mol. Biol. 15: 797-808
- Pearce, R. B., R. N. Strange and H. Smith (1976). Glycinebetaine and choline in wheat: distribution and relation to infection byFusarium graminearum. Phytochemistry 15: 953-954
- Pearson, W. R. (1990). Rapid and sensitive sequence comparison with FASTP and FASTA. Methods Enzymology 183:63-98.
- Peterbauer, C. K., M. Lorito, C. K. Hayes, G. E. Harman and C. P. Kubicek (1996). Molecular cloning and expression of the nag1 gene (N-acetyl-β-D-glucosaminidase-encoding gene) fromTrichoderma harzianum P1. Curr. Genet. 30: 325-331
- Pugh, G. W., H. Johann and J. G. Dickson (1933). Factors affecting infection of wheat heads by Gibberella saubinetii. J. Agr. Res. 46: 771-797
- Sambrook, J., E. F. Fritsch and T. Maniatis (1989).Molecular Cloning, A Laboratory Manual, vol. 1-3 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).
- Sanger, F., S. Nicklen and A. R. Coulsen (1977). DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad, Sci. 74: 5463-5467
- Seetharaman, K., E. Whitehead, N. P. Keller, R. D. Wanishka, and L. W. Rooney (1997). In vitro activity of sorgum seed antifungal proteins against grain mold pathogens. J. Agric. Food Chem. 45: 3666-3671
- Sela-Buurlage, M. B., A. S. Ponstein, S. A. Bres-Vloemans, L. S. Melchers, P. J. M. van der Elzen and B. J. C. Cornelissen (1993). Only specific tobacco (Nicotiana tabacum) chitinases and β-1,3-glucanases exhibit antifungal activity. Plant Physiol. 101: 857-863
- Simmons, C. R. (1994). The physiology and molecular biology of
plant 1,3-β-D-glucanases and 1,3;1,4-β-D-glucanases. Crit. Rev. Plant Sci. 13: 325-387 - Sivan, A. and I. Chet (1989a). Cell wall composition ofFusarium oxysporum. Soil Biol. Biochem. 21: 869-871
- Sivan A and I. Chet (1989b). Degradation of fungal cell walls by lytic enzymes ofTrichoderma harzianum. J. Gen. Microbiol. 135: 675-682
- Spratt, B. G., P.J. Hedge, S. te Heesen, A. Edelman and J. K. Broome-Smith (1986). Kanamycin-resistant vectors that are analogues of plasmids pUC8, pUC9, pEMBL8 and pEMBL9. Gene 41: 337-342
- Srivastava, A. K., G. Defago, T. Boller (1985). Secretion of chitinase byAphanocladium album, a hyperparasite of wheat rust. Experientia 41: 1612-1613
- Strange, R. N., J. R. Majer and H. Smith (1974). The isolation and identification of choline and betaine as the two major components in anthers and wheat germ that stimulateFusarium graminearum in vitro. Physiol. Plant Path. 4: 277-290
- Strange, R. N. and H. Smith (1971). A fungal growth stimulant in anthers which predisposes wheat to attack byFusarium graminearum. Physiol. Plant Path. 1: 141-150
- Strange, R. N. and H. Smith (1978). Effects of choline, betaine and wheat-germ extract on growth of cereal pathogens. Trans. Br. mycol. Soc. 70: 193-199
- Suslow, T., D. Matsubara, J. Jones, R. Lee and P. Dunsmuir (1988). Effect of expression of bacterial chitinase on tobacco susceptibility to leaf brown spot. Phytopathology 78: 1556
- Tailor, R. H., D. P. Acland, S. Attenborough, B. P. A. Cammue, I. J. Evans, R. W. Osborn, J. A. Ray, S. B. Rees, and W. F. Broekaert (1997). A novel family of small cysteine-rich antimicrobial peptides from seed ofImpatiens balsamina is derived from a single precursor protein. J. Biol. Chem. 272: 24480-24487.
- Terakawa, T., N. Tanaya, H. Horiuchi, M. Koike and M. Takagi (1997). A fungal chitinase gene fromRhizopus oligosporus confers antifungal activity to transgenic tobacco. Plant Cell Rep. 16: 439-443
- Terras, F. R. G., H. M. E. Schoofs, K. Thevissen, R. W. Osborn, J. Vanderleyden, B. P. A. Cammue and W. F. Broekaert (1993). Synergistic enhancement of the antifungal activity of wheat and barley thionins by radish and oilseed rape 2S albumins and by barley trypsin inhibitor. Plant Physiol. 103: 1311-1319
- Thompson, J. D., D. G. Higgins, and T. J. Gibson (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucl Acids Res 22: 4673-4680.
- Tzafrir, I., K. A. Torbert, B. E. L. Lockhart, D. A. Somers and N. E. Olszewski (1998). The sugarcane bacilliform badnavirus promoter is active in both monocots and dicots. Plant Mol Biol 38: 347-356
- Tronsmo A. and G. E. Harman (1993). Detection and quantification of N-acetyl-β-D-glucosaminidase, chitobiosidase, and endochitinase in solutions and on gels. Anal Biochem 208: 74-79
- Van Loon, L. C. (1997) Induced resistance in plants and the role of pathogenesis-related proteins Eur. J. Plant Path. 103: 753-765
- Vasil, V., A. M. Castillo, M. E. Fromm, and I. K. Vasil (1992). Herbicide resistant fertile transgenic wheat plants obtained by microprojectile bombardment of regenerable embryogenic callus. Bio/Technology 10: 667-674
- Vasil, V., V. Srivastava, A. M. Castillo, M. E. Fromm, and I. K. Vasil (1993). Rapid production of transgenic wheat plants by direct bombardment of cultured immature embryos. Bio/Technology 11: 1553-1558
- Vázquez-Garcidueñas, S., C. A. Leal-Morales and A. Herrera-Estrella (1998). Analysis of the β-1,3-glucanolytic system of the biocontrol agentTrichoderma harzianum. Appl. Environ. Microbiol. 64: 1442-1446
- Wang, AM, M. V. Doyle, D. F. Mark (1989). Quantitation of mRNA by the polymerase chain reaction. Proc Natl Acad Sci 86: 9717-9721
- Weeks, J. T., O. D. Anderson and A. E. Blechl (1993). Rapid production of multiple independent lines of fertile transgenic wheat. Plant Physiol. 102: 1077-1084
- Wiebe, M. G., A. P. J. Trinci, B. Cunliffe, G. D. Robson, S. G. Oliver (1991). Appearance of morphological (colonial) mutants in glucose-limited continuous flow cultures ofFusarium graminearum. Mycol Res 95: 1284-1288
- Wu, S., A. L. Kriz, and J. M. Widholm (1992). Molecular analysis of two cDNA clones encoding acidic class I chitinase in maize. Plant Physiol. 105: 1097-1105
- Xu, P., J. Wang and G. B. Fincher (1992). Evolution and differential expression of the (1-3)-β-glucan endohydrolase-encoding gene family in barley,Hordeum vulgare. Gene 120: 157-165
- Yang, C., Y. Zhu, D. M. Magee and R. A. Cox (1996). Molecular cloning and characterization of theCoccidioides immitis complement fixation/chitinase antigen. Infect. Immun. 64: 1992-1997
- Yun, D-J., M. Paino D'Urzo, L. Abad, S. Takeda, R. Salzman, Z. Chen, H. Lee, P. M. Hasegawa and R. A. Bressan (1996). Novel osmotically induced antifungal chitinases and bacterial expression of an active recombinant isoform. Plant Physiol. 111: 1219-1225
- Zhang, W., D. McElroy and R. Wu (1991). Analysis of
rice Act1 5′ region activity in transgenic rice plants. Plant Cell 3: 1155-1165 - Zimmermann, C. R., S. M. Johnson, G. W. Martens, A. G. White and D. Pappagianis (1996). Cloning and expression of the complement fixation antigen-chitinase ofCoccidioides immitis. Infect. Immun. 64: 4967-4975
- Zhu, Q., E. A. Maher, S. Masoud, R. A. Dixon and C. J. Lamb (1994). Enhanced protection against fungal attack by constitutive co-expression of chitinase and glucanase genes in transgenic tobacco. Bio/Tech. 12: 807-812
-
1 82 1 1023 DNA Fusarium venenatum CDS (37)..(942) 1 cagttatctt tttttatcta ctatctttaa ttcact atg aag ttc ttc agc act 54 Met Lys Phe Phe Ser Thr 1 5 ctt agc acc ctt gcg gtg gcc ctc atg atg agt ggc gag gct ctg gct 102 Leu Ser Thr Leu Ala Val Ala Leu Met Met Ser Gly Glu Ala Leu Ala 10 15 20 ggt acc tac aag ggt ttc agc att ggc gcc aac agg gct gat ggt gcc 150 Gly Thr Tyr Lys Gly Phe Ser Ile Gly Ala Asn Arg Ala Asp Gly Ala 25 30 35 tgt aag tgg gag gcc gac tgg aag aag gat ttc cag gcc atc aag agc 198 Cys Lys Trp Glu Ala Asp Trp Lys Lys Asp Phe Gln Ala Ile Lys Ser 40 45 50 tgg aac aag ggt ttc aac gct gtt cgt ctg tac tct gcc tct gac tgt 246 Trp Asn Lys Gly Phe Asn Ala Val Arg Leu Tyr Ser Ala Ser Asp Cys 55 60 65 70 aac aca ctt gtc aag gct gtc ccc gct gcc aag gcc act ggc atg aag 294 Asn Thr Leu Val Lys Ala Val Pro Ala Ala Lys Ala Thr Gly Met Lys 75 80 85 atc ctt gtt ggc gtc tgg gcc acc gat gat gct cac ttc ggc cgc gac 342 Ile Leu Val Gly Val Trp Ala Thr Asp Asp Ala His Phe Gly Arg Asp 90 95 100 aag gcc gcc ctc ctc aag gct atc aag cag cac ggc acc ggc tgg atc 390 Lys Ala Ala Leu Leu Lys Ala Ile Lys Gln His Gly Thr Gly Trp Ile 105 110 115 gcc gcc atc agt gtc gga tcc gag gac ctc tac cgt gag gac atc tcc 438 Ala Ala Ile Ser Val Gly Ser Glu Asp Leu Tyr Arg Glu Asp Ile Ser 120 125 130 ccc cag aag ctc gca cag cag atc tac gac gtc cga ggc atg gtc cac 486 Pro Gln Lys Leu Ala Gln Gln Ile Tyr Asp Val Arg Gly Met Val His 135 140 145 150 caa tac aac aag aac ctc aag gtc gga cat acc gac acc tgg acc gct 534 Gln Tyr Asn Lys Asn Leu Lys Val Gly His Thr Asp Thr Trp Thr Ala 155 160 165 tgg gtc gac ggc cgc aac gac gtc gtc acc aag gcc tgc gat atc gcc 582 Trp Val Asp Gly Arg Asn Asp Val Val Thr Lys Ala Cys Asp Ile Ala 170 175 180 att aca aac ggt ttc ccc tac tgg cag ggt gtt ccc atc aag gat gct 630 Ile Thr Asn Gly Phe Pro Tyr Trp Gln Gly Val Pro Ile Lys Asp Ala 185 190 195 ctc cgc ctc aag acc ttc cag aac tcc ttc tgg aac gtc cag aag cac 678 Leu Arg Leu Lys Thr Phe Gln Asn Ser Phe Trp Asn Val Gln Lys His 200 205 210 gtc aag gct gtc aac tcc aag gct gct gtc tgg gtt ggc gag acc ggc 726 Val Lys Ala Val Asn Ser Lys Ala Ala Val Trp Val Gly Glu Thr Gly 215 220 225 230 tgg cct aca aag gga ccc aac tac cag aag gct gct gcc acg acc gcc 774 Trp Pro Thr Lys Gly Pro Asn Tyr Gln Lys Ala Ala Ala Thr Thr Ala 235 240 245 agt ctc cag cag ttc tac aac aat gtc ggt tgc tgg ctc tgg cag cag 822 Ser Leu Gln Gln Phe Tyr Asn Asn Val Gly Cys Trp Leu Trp Gln Gln 250 255 260 aag gag gcc agt ggt ttc tgg ttc act gct ttc gat aca cct gcg cac 870 Lys Glu Ala Ser Gly Phe Trp Phe Thr Ala Phe Asp Thr Pro Ala His 265 270 275 agc acc gag gtt gag aag tac ttc ggt att gcc aac cag gac cgc aag 918 Ser Thr Glu Val Glu Lys Tyr Phe Gly Ile Ala Asn Gln Asp Arg Lys 280 285 290 ctc aag ttc agc ctt act tgc taa gcgttgtagc gggttttcga tgtataaatt 972 Leu Lys Phe Ser Leu Thr Cys 295 300 taatttacca tatactgtta gcgcttgtca acttgatagt atttccattc a 1023 2 301 PRT Fusarium venenatum 2 Met Lys Phe Phe Ser Thr Leu Ser Thr Leu Ala Val Ala Leu Met Met 1 5 10 15 Ser Gly Glu Ala Leu Ala Gly Thr Tyr Lys Gly Phe Ser Ile Gly Ala 20 25 30 Asn Arg Ala Asp Gly Ala Cys Lys Trp Glu Ala Asp Trp Lys Lys Asp 35 40 45 Phe Gln Ala Ile Lys Ser Trp Asn Lys Gly Phe Asn Ala Val Arg Leu 50 55 60 Tyr Ser Ala Ser Asp Cys Asn Thr Leu Val Lys Ala Val Pro Ala Ala 65 70 75 80 Lys Ala Thr Gly Met Lys Ile Leu Val Gly Val Trp Ala Thr Asp Asp 85 90 95 Ala His Phe Gly Arg Asp Lys Ala Ala Leu Leu Lys Ala Ile Lys Gln 100 105 110 His Gly Thr Gly Trp Ile Ala Ala Ile Ser Val Gly Ser Glu Asp Leu 115 120 125 Tyr Arg Glu Asp Ile Ser Pro Gln Lys Leu Ala Gln Gln Ile Tyr Asp 130 135 140 Val Arg Gly Met Val His Gln Tyr Asn Lys Asn Leu Lys Val Gly His 145 150 155 160 Thr Asp Thr Trp Thr Ala Trp Val Asp Gly Arg Asn Asp Val Val Thr 165 170 175 Lys Ala Cys Asp Ile Ala Ile Thr Asn Gly Phe Pro Tyr Trp Gln Gly 180 185 190 Val Pro Ile Lys Asp Ala Leu Arg Leu Lys Thr Phe Gln Asn Ser Phe 195 200 205 Trp Asn Val Gln Lys His Val Lys Ala Val Asn Ser Lys Ala Ala Val 210 215 220 Trp Val Gly Glu Thr Gly Trp Pro Thr Lys Gly Pro Asn Tyr Gln Lys 225 230 235 240 Ala Ala Ala Thr Thr Ala Ser Leu Gln Gln Phe Tyr Asn Asn Val Gly 245 250 255 Cys Trp Leu Trp Gln Gln Lys Glu Ala Ser Gly Phe Trp Phe Thr Ala 260 265 270 Phe Asp Thr Pro Ala His Ser Thr Glu Val Glu Lys Tyr Phe Gly Ile 275 280 285 Ala Asn Gln Asp Arg Lys Leu Lys Phe Ser Leu Thr Cys 290 295 300 3 3622 DNA Fusarium sporotrichioides CDS (1801)..(2014) CDS (2070)..(2761) 3 attgaatcgt aacgatagcc tcatgcttgg cattttgtgc cttgacatta tcgagcgcgg 60 caccccacag gccttggtct ctgaaagaaa cggtggtggc gccgtttttt gcaaggttaa 120 tcccacttcc aggcttcttg gtgacgttga gaagtgcatc gccccagcct ccgcccgtag 180 caacagttga gtctcctgta aggatgaaaa agggcgattt cgcagcctga acaccaccaa 240 agatgctcag ggcagcagaa gcaatggcca agaatctcat gctgagtaaa gatgattcag 300 atatgattct cgaaaaaaat gacttcaatc gcgtcgctac ttgaaagtat catcttcttc 360 tatatattga ccacggaccc caacatctgc cgaacactca taagtcgttt gcctcttcat 420 gattgagaaa gtggaatatt tccaccaata aacatcatgc attgactttg ggggcataaa 480 tgtcaaggtt tgcagggact gtattcatgt tcagaatccg aaccatctga ccttatgtta 540 ctcccccatg ccatgcggag aatcactgat cgtcaggctt agaaaggcga ctaagtggtt 600 gaactgtacc tcggctttca cattcggtct tgaatagccc gagtttctcg ctcgacctcg 660 ccgttagtaa cagggacagg cagtgggttg gactacctac cagactacct actgtgtttc 720 cccttgatct gatggcttta tcgataggca tatctaatct aatctcgatt tgtgctatct 780 aatctcactt tatcgtttct tatctgagta ataaccttac tcctatttta tccagtattg 840 aggtattaaa tgtcttgtgt aaatgaaaga ataaggggat aaaacgtagt atagagacct 900 attcagtgtt tgtatgaatg cttataatcc tgaactgagt tgaggacagt agtgggttgg 960 tttcttttga tcttttggtt gacttactca gctgaatttg cttaactcaa aacaacaacg 1020 ctctacgcag gtgacctatt ttcagcacaa gatcattacc attgggataa tgttccatta 1080 gcctgcagtt ctgatagact aataagctct catatgacca ggtttggcca tgttgaaggt 1140 ctgattgatg aaattgatga gtgttgtagg gtagatcaag gtagactaat ggagaattga 1200 gaaacctggc gaaggtgaaa agcggaggat ccggtgcttc cgaccaataa tcggacagca 1260 tcgcgtggac aatcgatagc caaggaagat ctcttagcct ggttccatta ttctttgatc 1320 ttgtggttgg ctaaccatct tgatttaacc aagatagcac agaacagagc agacatcgtt 1380 ttgcatcatg gatccaactc aggccgacta acccgtagtg aggcgtttcg aatttctcca 1440 gatacccaaa gatggattaa gcatgaaaac caatctgctt taattgcccg tagattgaat 1500 ccattctggc gaagccgatg ttccgcgcaa caccgtcctt tctagcgcca ttccaagacc 1560 aggccaagca taaaaatagc accacacacg gcaaaaaagg tctagaacag ctcacgctcc 1620 agggtgaata cgagcccaaa ggggcaaaaa gcaacaagaa aaaaggggca taatttccat 1680 catcccataa taaaaccaac tttccacccc aactttcttg cctttttgtc ctcttggctc 1740 tttaactgtc actcgcttat taatcagtta ttttctttca tttactatcg ttaattcact 1800 atg aag ttc ttc agc act ctc agc acc ctt gcg gtg gct ctc atg atg 1848 Met Lys Phe Phe Ser Thr Leu Ser Thr Leu Ala Val Ala Leu Met Met 1 5 10 15 agt ggc gag gcc cta gcc ggt acc tac aag ggt ttc agc att ggc gcc 1896 Ser Gly Glu Ala Leu Ala Gly Thr Tyr Lys Gly Phe Ser Ile Gly Ala 20 25 30 aac agg gct gat ggc gcc tgc aag tgg gag gcc gac tgg aag aag gat 1944 Asn Arg Ala Asp Gly Ala Cys Lys Trp Glu Ala Asp Trp Lys Lys Asp 35 40 45 ttc cag gcc atc aag agc tgg aac aag ggt ttc aac gct gtt cgt ctg 1992 Phe Gln Ala Ile Lys Ser Trp Asn Lys Gly Phe Asn Ala Val Arg Leu 50 55 60 tac tct gct agt gat tgc aac a gtaagcgttc cgcattctga acaacaataa 2044 Tyr Ser Ala Ser Asp Cys Asn 65 70 gctcttttac ttacgttctt cttag ca ctt gtc aag gcc gtc ccc gct gct 2095 Thr Leu Val Lys Ala Val Pro Ala Ala 75 80 aag gcc act ggc atg aag atc ctt gtc ggc gtt tgg gcc acc gac gat 2143 Lys Ala Thr Gly Met Lys Ile Leu Val Gly Val Trp Ala Thr Asp Asp 85 90 95 gct cac ttc ggc cgc gat aag gcc gct ctt ctc aag gct att aag cag 2191 Ala His Phe Gly Arg Asp Lys Ala Ala Leu Leu Lys Ala Ile Lys Gln 100 105 110 cac ggc acg ggc tgg atc gcc gcc atc agc gtc gga tcc gaa gac ctc 2239 His Gly Thr Gly Trp Ile Ala Ala Ile Ser Val Gly Ser Glu Asp Leu 115 120 125 tac cgt gag gat atc tcc ccc cag aag ctc gcc cag cag atc tac gat 2287 Tyr Arg Glu Asp Ile Ser Pro Gln Lys Leu Ala Gln Gln Ile Tyr Asp 130 135 140 gtc cga ggc atg gtc cac caa tac aac aag aat ctc aag gtc gga cac 2335 Val Arg Gly Met Val His Gln Tyr Asn Lys Asn Leu Lys Val Gly His 145 150 155 160 acc gac acc tgg acc gct tgg gtc gac ggc cgc aat gac gtc gtc acc 2383 Thr Asp Thr Trp Thr Ala Trp Val Asp Gly Arg Asn Asp Val Val Thr 165 170 175 aag gcc tgc gac att gcc atc aca aac ggt ttc ccc tac tgg cag ggt 2431 Lys Ala Cys Asp Ile Ala Ile Thr Asn Gly Phe Pro Tyr Trp Gln Gly 180 185 190 gtc ccc atc aag gat gct ctc cgt ctc aag act ttc cag aac tcg tac 2479 Val Pro Ile Lys Asp Ala Leu Arg Leu Lys Thr Phe Gln Asn Ser Tyr 195 200 205 tgg aac gtc aag aag cac gtc aat gct gtc aac tcc aag gct gct gtc 2527 Trp Asn Val Lys Lys His Val Asn Ala Val Asn Ser Lys Ala Ala Val 210 215 220 tgg gtt ggt gag acc ggc tgg cct acc aag gga ccc aac tac cag aag 2575 Trp Val Gly Glu Thr Gly Trp Pro Thr Lys Gly Pro Asn Tyr Gln Lys 225 230 235 240 gct gct gcc acg acc gcc agt ctg cag cag ttc tac aac aat gtc ggt 2623 Ala Ala Ala Thr Thr Ala Ser Leu Gln Gln Phe Tyr Asn Asn Val Gly 245 250 255 tgc tgg ctc tgg cag cag aag gat gcc agt ggt ttc tgg ttc act gct 2671 Cys Trp Leu Trp Gln Gln Lys Asp Ala Ser Gly Phe Trp Phe Thr Ala 260 265 270 ttc gat act cct gcc cac agc act gaa gtt gag aag tac ttc ggt att 2719 Phe Asp Thr Pro Ala His Ser Thr Glu Val Glu Lys Tyr Phe Gly Ile 275 280 285 gct aac cag gac cgc aag ctc aag ttc ggc ctt act tgc taa 2761 Ala Asn Gln Asp Arg Lys Leu Lys Phe Gly Leu Thr Cys 290 295 300 acgttgtagc ggggttgcca tgtataaatt taattgacca tatactgttg gcgtttatca 2821 acttgatagt acttacattc attgcataaa aaagtctttc ttagatccaa tatataatat 2881 caaagaccga tcacattcaa taatcttctt gtctatctat gcaaggccat tcatttcctt 2941 aagccttggt gcttccagga ggacccaggt aactctcttg caccccaccg aagtcgacga 3001 caaccttttc caacaagatg ttcgcatggt taagtcggag cttgaggtta tgcacaccag 3061 tctttaacag ccccagctca tgcctccgta cccatacgtt gtccgacgca gcaaagaacc 3121 acccgtcagc cgacgcccat cccttgtctg cggcgttctt ctcgctctgc ggagtccttc 3181 tctgtaaatt gtacgtctga ctcgggcctt cgtcaatctg cacatcgtag gtcaggacat 3241 cctcgggtga caagtcgaga gttgttccaa agtaaagcac cagctccgtc ttgtcagttt 3301 cgctgtgcgt gaagagcggg tatttcatgt aaggaatctc gctattggtc cttgtcccgg 3361 gagcgagagt cacacttccg gtctcgagtc tgcctgtatc aggaagcatc agatacggct 3421 tctccatgtg acaatcagtt gctggtatcg agatataaca gtcttgctcc acaaagccac 3481 ggaatgaatc ttggacatgt cgcccgtcaa tgagtaggtg gacttgttca aagtctcctt 3541 cacgcgactg tacatcaata agaacctcct cttggaagtc ttgaggcact tgtgaccaat 3601 tgatagaaac ttggactcgc a 3622 4 301 PRT Fusarium sporotrichioides 4 Met Lys Phe Phe Ser Thr Leu Ser Thr Leu Ala Val Ala Leu Met Met 1 5 10 15 Ser Gly Glu Ala Leu Ala Gly Thr Tyr Lys Gly Phe Ser Ile Gly Ala 20 25 30 Asn Arg Ala Asp Gly Ala Cys Lys Trp Glu Ala Asp Trp Lys Lys Asp 35 40 45 Phe Gln Ala Ile Lys Ser Trp Asn Lys Gly Phe Asn Ala Val Arg Leu 50 55 60 Tyr Ser Ala Ser Asp Cys Asn Thr Leu Val Lys Ala Val Pro Ala Ala 65 70 75 80 Lys Ala Thr Gly Met Lys Ile Leu Val Gly Val Trp Ala Thr Asp Asp 85 90 95 Ala His Phe Gly Arg Asp Lys Ala Ala Leu Leu Lys Ala Ile Lys Gln 100 105 110 His Gly Thr Gly Trp Ile Ala Ala Ile Ser Val Gly Ser Glu Asp Leu 115 120 125 Tyr Arg Glu Asp Ile Ser Pro Gln Lys Leu Ala Gln Gln Ile Tyr Asp 130 135 140 Val Arg Gly Met Val His Gln Tyr Asn Lys Asn Leu Lys Val Gly His 145 150 155 160 Thr Asp Thr Trp Thr Ala Trp Val Asp Gly Arg Asn Asp Val Val Thr 165 170 175 Lys Ala Cys Asp Ile Ala Ile Thr Asn Gly Phe Pro Tyr Trp Gln Gly 180 185 190 Val Pro Ile Lys Asp Ala Leu Arg Leu Lys Thr Phe Gln Asn Ser Tyr 195 200 205 Trp Asn Val Lys Lys His Val Asn Ala Val Asn Ser Lys Ala Ala Val 210 215 220 Trp Val Gly Glu Thr Gly Trp Pro Thr Lys Gly Pro Asn Tyr Gln Lys 225 230 235 240 Ala Ala Ala Thr Thr Ala Ser Leu Gln Gln Phe Tyr Asn Asn Val Gly 245 250 255 Cys Trp Leu Trp Gln Gln Lys Asp Ala Ser Gly Phe Trp Phe Thr Ala 260 265 270 Phe Asp Thr Pro Ala His Ser Thr Glu Val Glu Lys Tyr Phe Gly Ile 275 280 285 Ala Asn Gln Asp Arg Lys Leu Lys Phe Gly Leu Thr Cys 290 295 300 5 301 PRT Fusarium venenatum 5 Met Lys Phe Phe Ser Thr Leu Ser Thr Leu Ala Val Ala Leu Met Met 1 5 10 15 Ser Gly Glu Ala Leu Ala Gly Thr Tyr Lys Gly Phe Ser Ile Gly Ala 20 25 30 Asn Arg Ala Asp Gly Ala Cys Lys Trp Glu Ala Asp Trp Lys Lys Asp 35 40 45 Phe Gln Ala Ile Lys Ser Trp Asn Lys Gly Phe Asn Ala Val Arg Leu 50 55 60 Tyr Ser Ala Ser Asp Cys Asn Thr Leu Val Lys Ala Val Pro Ala Ala 65 70 75 80 Lys Ala Thr Gly Met Lys Ile Leu Val Gly Val Trp Ala Thr Asp Asp 85 90 95 Ala His Phe Gly Arg Asp Lys Ala Ala Leu Leu Lys Ala Ile Lys Gln 100 105 110 His Gly Thr Gly Trp Ile Ala Ala Ile Ser Val Gly Ser Glu Asp Leu 115 120 125 Tyr Arg Glu Asp Ile Ser Pro Gln Lys Leu Ala Gln Gln Ile Tyr Asp 130 135 140 Val Arg Gly Met Val His Gln Tyr Asn Lys Asn Leu Lys Val Gly His 145 150 155 160 Thr Asp Thr Trp Thr Ala Trp Val Asp Gly Arg Asn Asp Val Val Thr 165 170 175 Lys Ala Cys Asp Ile Ala Ile Thr Asn Gly Phe Pro Tyr Trp Lys Gly 180 185 190 Val Pro Ile Lys Asp Ala Leu Arg Leu Lys Thr Phe Gln Asn Ser Phe 195 200 205 Trp Asn Val Gln Lys His Val Lys Ala Val Asn Ser Lys Ala Ala Val 210 215 220 Trp Val Gly Glu Thr Gly Trp Pro Thr Lys Gly Pro Asn Tyr Gln Lys 225 230 235 240 Ala Ala Ala Thr Thr Ala Ser Leu Gln Gln Phe Tyr Asn Asn Val Gly 245 250 255 Cys Trp Leu Trp Gln Gln Lys Glu Ala Ser Gly Phe Trp Phe Thr Ala 260 265 270 Phe Asp Thr Pro Ala His Ser Thr Glu Val Glu Lys Tyr Phe Gly Ile 275 280 285 Ala Asn Gln Asp Arg Lys Leu Lys Phe Ser Leu Thr Cys 290 295 300 6 467 DNA Fusarium venenatum CDS (186)..(467) 6 gattcattcc ccacctaaaa ccctccaagt gccctccatc atcacgaatc tccacagaga 60 cagccaagat acgccttgga aatcgtacca gggcgagcct tttacttata accgccgttt 120 tcgctgcatt agttgcagta ttcctatcaa atacatcttt atctagtccc tttacttcaa 180 tcaaa atg ggt ggt gga ccc gaa ggt ttc cgc acc gtt gcg tat ttc gtc 230 Met Gly Gly Gly Pro Glu Gly Phe Arg Thr Val Ala Tyr Phe Val 1 5 10 15 aac tgg gct atc tat gca cga aag cat cgt cct cag gat ctt ccc gtg 278 Asn Trp Ala Ile Tyr Ala Arg Lys His Arg Pro Gln Asp Leu Pro Val 20 25 30 gag aac ctg aca cat att ctt tac tcg ttc gct aat att cgt agc gac 326 Glu Asn Leu Thr His Ile Leu Tyr Ser Phe Ala Asn Ile Arg Ser Asp 35 40 45 tct ggc gaa gtc cat ctc acc gac tca tgg gcc gat acc gat att cat 374 Ser Gly Glu Val His Leu Thr Asp Ser Trp Ala Asp Thr Asp Ile His 50 55 60 tgg gat gga gat tcc tgg aat gat gtc ggt acc aac ttg tac ggt tgc 422 Trp Asp Gly Asp Ser Trp Asn Asp Val Gly Thr Asn Leu Tyr Gly Cys 65 70 75 atg aag cag ctt aac ctg ttg aaa aga cgt aac cga aac ctc aag 467 Met Lys Gln Leu Asn Leu Leu Lys Arg Arg Asn Arg Asn Leu Lys 80 85 90 7 94 PRT Fusarium venenatum 7 Met Gly Gly Gly Pro Glu Gly Phe Arg Thr Val Ala Tyr Phe Val Asn 1 5 10 15 Trp Ala Ile Tyr Ala Arg Lys His Arg Pro Gln Asp Leu Pro Val Glu 20 25 30 Asn Leu Thr His Ile Leu Tyr Ser Phe Ala Asn Ile Arg Ser Asp Ser 35 40 45 Gly Glu Val His Leu Thr Asp Ser Trp Ala Asp Thr Asp Ile His Trp 50 55 60 Asp Gly Asp Ser Trp Asn Asp Val Gly Thr Asn Leu Tyr Gly Cys Met 65 70 75 80 Lys Gln Leu Asn Leu Leu Lys Arg Arg Asn Arg Asn Leu Lys 85 90 8 995 DNA Fusarium sporotrichioides CDS (160)..(303) CDS (392)..(469) misc_difference (283) Unsure, n is A or C; Xaa at amino acid 42 is Ile or Leu 8 gattcccaag ggggcggaac tggaagatca tcgccggtct tattaaacgg gnactggtac 60 tttggacact tttgtctctt ccttaaagat tagcctccct cgctcagctt ctatctacca 120 ttgttagcaa ttatctcact cacctcacct ctaggcgta atg tgg tcc aag gct 174 Met Trp Ser Lys Ala 1 5 ctt ctg gcc gtt gcc gcc ttt gcc ttc aca ccc gcc aat gct ata tgg 222 Leu Leu Ala Val Ala Ala Phe Ala Phe Thr Pro Ala Asn Ala Ile Trp 10 15 20 cca gtg cca aag aag atc tct act gga gac aag gcc ctc ttc atc gat 270 Pro Val Pro Lys Lys Ile Ser Thr Gly Asp Lys Ala Leu Phe Ile Asp 25 30 35 caa acg att gac ntc acc tac aat gga gac ttt gtacgggact ctccccggtt 323 Gln Thr Ile Asp Xaa Thr Tyr Asn Gly Asp Phe 40 45 ctgattccga ttctggtgct tgtaaccata ccgcgcagct caatactgaa actttgcttc 383 acaaacag atc ccc tac act tac aat tac caa ccc gat gct ggc tcc aag 433 Ile Pro Tyr Thr Tyr Asn Tyr Gln Pro Asp Ala Gly Ser Lys 50 55 60 ttc agt agc aag caa atc atc caa gcc ggc gtc tct cgtgccctcc 479 Phe Ser Ser Lys Gln Ile Ile Gln Ala Gly Val Ser 65 70 aaggcgtctt ccaggacaac tttgtcccat ggatgctccg cgaacgcgac tccgattttg 539 agcctgacct gcaaaagaag cagtgggtga agtcgctaaa gattatccag accgaggagg 599 atgacgagag caccttcaag cctctcaatg gtgaggttga cgagtcgtac tccctctcac 659 tttctgagaa gggcgaggct tccatcaagg ccaagtcctc tacaggtgtc ctgcacggac 719 ttgagacctt tgtccaactt ttcttcaagc acagctctgg cacttcctgg tacacgccgc 779 acgcgcctgt ctcgatccag gacgagcccg agtaccctca tcgaggtatc cttctcgatg 839 ttgcccgtag cttttttgaa gtcaagcaca tcaagcgcac aatcgatgcc atgtcgtgga 899 gcaagttgaa tcgccttcac ctccacatca ctgactcgca gtcctggcct ctcgagatcc 959 cagccctgcc caagctggcc gaaaagggcg cgtacc 995 9 74 PRT Fusarium sporotrichioides UNSURE (42) Xaa is Ile or Leu 9 Met Trp Ser Lys Ala Leu Leu Ala Val Ala Ala Phe Ala Phe Thr Pro 1 5 10 15 Ala Asn Ala Ile Trp Pro Val Pro Lys Lys Ile Ser Thr Gly Asp Lys 20 25 30 Ala Leu Phe Ile Asp Gln Thr Ile Asp Xaa Thr Tyr Asn Gly Asp Phe 35 40 45 Ile Pro Tyr Thr Tyr Asn Tyr Gln Pro Asp Ala Gly Ser Lys Phe Ser 50 55 60 Ser Lys Gln Ile Ile Gln Ala Gly Val Ser 65 70 10 1494 DNA Fusarium venenatum CDS (186)..(1385) 10 gattcattcc ccacctaaaa ccctccaagt gccctccatc atcacgaatc tccacagaga 60 cagccaagat acgccttgga aatcgtacca gggcgagcct tttacttata accgccgttt 120 tcgctgcatt agttgcagta ttcctatcaa atacatcttt atctagtccc tttacttcaa 180 tcaaa atg ggt ggt gga ccc gaa ggt ttc cgc acc gtt gcg tat ttc gtc 230 Met Gly Gly Gly Pro Glu Gly Phe Arg Thr Val Ala Tyr Phe Val 1 5 10 15 aac tgg gct atc tat gca cga aag cat cgt cct cag gat ctt ccc gtg 278 Asn Trp Ala Ile Tyr Ala Arg Lys His Arg Pro Gln Asp Leu Pro Val 20 25 30 gag aac ctg aca cat att ctt tac tcg ttc gct aat att cgt agc gac 326 Glu Asn Leu Thr His Ile Leu Tyr Ser Phe Ala Asn Ile Arg Ser Asp 35 40 45 tct ggc gaa gtc cat ctc acc gac tca tgg gcc gat acc gat att cat 374 Ser Gly Glu Val His Leu Thr Asp Ser Trp Ala Asp Thr Asp Ile His 50 55 60 tgg gat gga gat tcc tgg aat gat gtc ggt acc aac ttg tac ggt tgc 422 Trp Asp Gly Asp Ser Trp Asn Asp Val Gly Thr Asn Leu Tyr Gly Cys 65 70 75 atg aag cag ctt aac ctg ttg aaa aga cgt aac cga aac ctc aag gtt 470 Met Lys Gln Leu Asn Leu Leu Lys Arg Arg Asn Arg Asn Leu Lys Val 80 85 90 95 ctt ctc agt att gga ggt tgg acc ttc agt agc aac ttc aag ggc ccc 518 Leu Leu Ser Ile Gly Gly Trp Thr Phe Ser Ser Asn Phe Lys Gly Pro 100 105 110 gct agc aca ccc caa gga cgt gac aca ttc gcc aag agc tgt gtc gat 566 Ala Ser Thr Pro Gln Gly Arg Asp Thr Phe Ala Lys Ser Cys Val Asp 115 120 125 ctg atc aag aac ctc ggt ttt gac ggt ata gat atc gat tgg gag tac 614 Leu Ile Lys Asn Leu Gly Phe Asp Gly Ile Asp Ile Asp Trp Glu Tyr 130 135 140 ccc cag gat gcc aac gag gct aga aac tat gtc gaa ctt ctg ggc gcc 662 Pro Gln Asp Ala Asn Glu Ala Arg Asn Tyr Val Glu Leu Leu Gly Ala 145 150 155 gtg cgt cat gag atg gat gca tat gca cag aca ttg agc caa cct tat 710 Val Arg His Glu Met Asp Ala Tyr Ala Gln Thr Leu Ser Gln Pro Tyr 160 165 170 175 cac ttt gag ttg act gtg gcc tgt cca gcc ggt gcg aca aac ttc cag 758 His Phe Glu Leu Thr Val Ala Cys Pro Ala Gly Ala Thr Asn Phe Gln 180 185 190 aag ctc gat atc cgt gga atg gat caa tac ctc gac ttc tgg aac ctc 806 Lys Leu Asp Ile Arg Gly Met Asp Gln Tyr Leu Asp Phe Trp Asn Leu 195 200 205 atg gct tac gac tat gct ggt tct tgg gac caa act gcg ggt cat cag 854 Met Ala Tyr Asp Tyr Ala Gly Ser Trp Asp Gln Thr Ala Gly His Gln 210 215 220 gcc aac ctg tac cca tct cac gac aac cca gta tca acc cca ttc tct 902 Ala Asn Leu Tyr Pro Ser His Asp Asn Pro Val Ser Thr Pro Phe Ser 225 230 235 acc tct gct gcc atc gac ttt tac gtc cgc agc ggt gtg aac cct tca 950 Thr Ser Ala Ala Ile Asp Phe Tyr Val Arg Ser Gly Val Asn Pro Ser 240 245 250 255 aag ata gtt ctc ggc atg cca ctc tac ggc cga gcc ttt gag aac acc 998 Lys Ile Val Leu Gly Met Pro Leu Tyr Gly Arg Ala Phe Glu Asn Thr 260 265 270 gac ggt ccc ggc cgc ccc tac caa ggc gtt gga caa ggt tcc tgg gaa 1046 Asp Gly Pro Gly Arg Pro Tyr Gln Gly Val Gly Gln Gly Ser Trp Glu 275 280 285 cag gga gtc tac gat tac aag gcg ctt ccc cta gag ggc gcg caa gag 1094 Gln Gly Val Tyr Asp Tyr Lys Ala Leu Pro Leu Glu Gly Ala Gln Glu 290 295 300 tac gga gat aga gga tgc tgt gcc agc tac tgc tac aac cct cag tca 1142 Tyr Gly Asp Arg Gly Cys Cys Ala Ser Tyr Cys Tyr Asn Pro Gln Ser 305 310 315 cgt acc atg gtc acc tac gac acg ccg cgg gtc gcc tgg gat aag gcc 1190 Arg Thr Met Val Thr Tyr Asp Thr Pro Arg Val Ala Trp Asp Lys Ala 320 325 330 335 gag tat gtg aag agg tgg aag ctg gga ggc gct atg tgg tgg gag agc 1238 Glu Tyr Val Lys Arg Trp Lys Leu Gly Gly Ala Met Trp Trp Glu Ser 340 345 350 agc gcg gat aag cag ggc gag cag agt ttg atc aca acg gtt gtg aac 1286 Ser Ala Asp Lys Gln Gly Glu Gln Ser Leu Ile Thr Thr Val Val Asn 355 360 365 gga ttc gga ggt cag gga gcg ctc atg aga cag gac aac tgt att gag 1334 Gly Phe Gly Gly Gln Gly Ala Leu Met Arg Gln Asp Asn Cys Ile Glu 370 375 380 tat ccc gcg acc aag tac gat aac ttg cga aat ggg ttc ccg gac aat 1382 Tyr Pro Ala Thr Lys Tyr Asp Asn Leu Arg Asn Gly Phe Pro Asp Asn 385 390 395 tga ggcgctaaag tggagttggt gggtcagagg aagcagaaac attgatgata 1435 gtttgcaacg actcatagtg ataacatgaa taaatgaata gtaaaaatat ccattacga 1494 11 399 PRT Fusarium venenatum 11 Met Gly Gly Gly Pro Glu Gly Phe Arg Thr Val Ala Tyr Phe Val Asn 1 5 10 15 Trp Ala Ile Tyr Ala Arg Lys His Arg Pro Gln Asp Leu Pro Val Glu 20 25 30 Asn Leu Thr His Ile Leu Tyr Ser Phe Ala Asn Ile Arg Ser Asp Ser 35 40 45 Gly Glu Val His Leu Thr Asp Ser Trp Ala Asp Thr Asp Ile His Trp 50 55 60 Asp Gly Asp Ser Trp Asn Asp Val Gly Thr Asn Leu Tyr Gly Cys Met 65 70 75 80 Lys Gln Leu Asn Leu Leu Lys Arg Arg Asn Arg Asn Leu Lys Val Leu 85 90 95 Leu Ser Ile Gly Gly Trp Thr Phe Ser Ser Asn Phe Lys Gly Pro Ala 100 105 110 Ser Thr Pro Gln Gly Arg Asp Thr Phe Ala Lys Ser Cys Val Asp Leu 115 120 125 Ile Lys Asn Leu Gly Phe Asp Gly Ile Asp Ile Asp Trp Glu Tyr Pro 130 135 140 Gln Asp Ala Asn Glu Ala Arg Asn Tyr Val Glu Leu Leu Gly Ala Val 145 150 155 160 Arg His Glu Met Asp Ala Tyr Ala Gln Thr Leu Ser Gln Pro Tyr His 165 170 175 Phe Glu Leu Thr Val Ala Cys Pro Ala Gly Ala Thr Asn Phe Gln Lys 180 185 190 Leu Asp Ile Arg Gly Met Asp Gln Tyr Leu Asp Phe Trp Asn Leu Met 195 200 205 Ala Tyr Asp Tyr Ala Gly Ser Trp Asp Gln Thr Ala Gly His Gln Ala 210 215 220 Asn Leu Tyr Pro Ser His Asp Asn Pro Val Ser Thr Pro Phe Ser Thr 225 230 235 240 Ser Ala Ala Ile Asp Phe Tyr Val Arg Ser Gly Val Asn Pro Ser Lys 245 250 255 Ile Val Leu Gly Met Pro Leu Tyr Gly Arg Ala Phe Glu Asn Thr Asp 260 265 270 Gly Pro Gly Arg Pro Tyr Gln Gly Val Gly Gln Gly Ser Trp Glu Gln 275 280 285 Gly Val Tyr Asp Tyr Lys Ala Leu Pro Leu Glu Gly Ala Gln Glu Tyr 290 295 300 Gly Asp Arg Gly Cys Cys Ala Ser Tyr Cys Tyr Asn Pro Gln Ser Arg 305 310 315 320 Thr Met Val Thr Tyr Asp Thr Pro Arg Val Ala Trp Asp Lys Ala Glu 325 330 335 Tyr Val Lys Arg Trp Lys Leu Gly Gly Ala Met Trp Trp Glu Ser Ser 340 345 350 Ala Asp Lys Gln Gly Glu Gln Ser Leu Ile Thr Thr Val Val Asn Gly 355 360 365 Phe Gly Gly Gln Gly Ala Leu Met Arg Gln Asp Asn Cys Ile Glu Tyr 370 375 380 Pro Ala Thr Lys Tyr Asp Asn Leu Arg Asn Gly Phe Pro Asp Asn 385 390 395 12 1949 DNA Fusarium sp. CDS (108)..(1853) misc_difference (231) Unsure, n is A or C; Xaa at amino acid 42 is Ile or Leu 12 actggtactt tggacacttt tgtctcttcc ttaaagatta gcctccctcg ctcagcttct 60 atctaccatt gttagcaatt atctcactca cctcacctct aggcgta atg tgg tcc 116 Met Trp Ser 1 aag gct ctt ctg gcc gtt gcc gcc ttt gcc ttc aca ccc gcc aat gct 164 Lys Ala Leu Leu Ala Val Ala Ala Phe Ala Phe Thr Pro Ala Asn Ala 5 10 15 ata tgg cca gtg cca aag aag atc tct act gga gac aag gcc ctc ttc 212 Ile Trp Pro Val Pro Lys Lys Ile Ser Thr Gly Asp Lys Ala Leu Phe 20 25 30 35 atc gat caa acg att gac ntc acc tac aat gga gac ttt atc ccc tac 260 Ile Asp Gln Thr Ile Asp Xaa Thr Tyr Asn Gly Asp Phe Ile Pro Tyr 40 45 50 act tac aat tac caa ccc gat gct ggc tcc aag ttc agt agc aag caa 308 Thr Tyr Asn Tyr Gln Pro Asp Ala Gly Ser Lys Phe Ser Ser Lys Gln 55 60 65 atc atc caa gcc ggc gtc tct cgt gcc ctc caa ggc gtc ttc cag gac 356 Ile Ile Gln Ala Gly Val Ser Arg Ala Leu Gln Gly Val Phe Gln Asp 70 75 80 aac ttt gtc cca tgg atg ctc cgc gaa cgc gac tcc gat ttt gag cct 404 Asn Phe Val Pro Trp Met Leu Arg Glu Arg Asp Ser Asp Phe Glu Pro 85 90 95 gac ctg caa aag aag cag tgg gtg aag tcg cta aag att atc cag acc 452 Asp Leu Gln Lys Lys Gln Trp Val Lys Ser Leu Lys Ile Ile Gln Thr 100 105 110 115 gag gag gat gac gag agc acc ttc aag cct ctc aat ggt gag gtt gac 500 Glu Glu Asp Asp Glu Ser Thr Phe Lys Pro Leu Asn Gly Glu Val Asp 120 125 130 gag tcg tac tcc ctc tca ctt tct gag aag ggc gag gct tcc atc aag 548 Glu Ser Tyr Ser Leu Ser Leu Ser Glu Lys Gly Glu Ala Ser Ile Lys 135 140 145 gcc aag tcc tct aca ggt gtc ctg cac gga ctt gag acc ttt gtc caa 596 Ala Lys Ser Ser Thr Gly Val Leu His Gly Leu Glu Thr Phe Val Gln 150 155 160 ctt ttc ttc aag cac agc tct ggc act tcc tgg tac acg ccg cac gcg 644 Leu Phe Phe Lys His Ser Ser Gly Thr Ser Trp Tyr Thr Pro His Ala 165 170 175 cct gtc tcg atc cag gat gag ccc gag tac cct cat cga ggc att ctt 692 Pro Val Ser Ile Gln Asp Glu Pro Glu Tyr Pro His Arg Gly Ile Leu 180 185 190 195 ctc gat gtt gcc cgt agc ttt ttt gaa gtc aag cac atc aag cgc aca 740 Leu Asp Val Ala Arg Ser Phe Phe Glu Val Lys His Ile Lys Arg Thr 200 205 210 atc gac gcc atg tcg tgg agc aag ctg aat cgc ctt cac ctt cac atc 788 Ile Asp Ala Met Ser Trp Ser Lys Leu Asn Arg Leu His Leu His Ile 215 220 225 act gac tcg cag tcc tgg cct ctc gag atc cca gcc cta ccc aaa ctg 836 Thr Asp Ser Gln Ser Trp Pro Leu Glu Ile Pro Ala Leu Pro Lys Leu 230 235 240 gcc gaa aag ggt gca tac cgc aaa ggc ctg acc tac tct cct gag gat 884 Ala Glu Lys Gly Ala Tyr Arg Lys Gly Leu Thr Tyr Ser Pro Glu Asp 245 250 255 ctt gcc ggt att tat gag tat ggt atc cac cgc gga gtc gag gtc atc 932 Leu Ala Gly Ile Tyr Glu Tyr Gly Ile His Arg Gly Val Glu Val Ile 260 265 270 275 atg gag att gac atg ccc ggc cat atc ggt gtc gtt gag ctt gcc tat 980 Met Glu Ile Asp Met Pro Gly His Ile Gly Val Val Glu Leu Ala Tyr 280 285 290 aag gat ctc att gtc gcg tac aat gag aag cct tat caa tgg tgg tgt 1028 Lys Asp Leu Ile Val Ala Tyr Asn Glu Lys Pro Tyr Gln Trp Trp Cys 295 300 305 aag gag cca ccc tgt ggt gcg ttc cgc atg aac agc tct gat gtt tat 1076 Lys Glu Pro Pro Cys Gly Ala Phe Arg Met Asn Ser Ser Asp Val Tyr 310 315 320 gac ttt ctc gac act ctt ttt gat gac ctc ttc cct cgc att tcc aag 1124 Asp Phe Leu Asp Thr Leu Phe Asp Asp Leu Phe Pro Arg Ile Ser Lys 325 330 335 tac agt cct tac ttc cac ctt ggt gga gac gag ctc aac cac aac gat 1172 Tyr Ser Pro Tyr Phe His Leu Gly Gly Asp Glu Leu Asn His Asn Asp 340 345 350 355 tcc aga ctt gac cct gat gtg cgc tct aac gag acc gag gtt ctg gcg 1220 Ser Arg Leu Asp Pro Asp Val Arg Ser Asn Glu Thr Glu Val Leu Ala 360 365 370 cct ctt ttg caa aag ttc gtc gat tac act cac ggc aag gtt cga gat 1268 Pro Leu Leu Gln Lys Phe Val Asp Tyr Thr His Gly Lys Val Arg Asp 375 380 385 gcc ggc atg act ccg ttc gtc tgg gag gag atg att acc gaa tgg aac 1316 Ala Gly Met Thr Pro Phe Val Trp Glu Glu Met Ile Thr Glu Trp Asn 390 395 400 atg act ctg ggt aaa gac gtt gtg att cag tcc tgg ctc ggt ggc ggt 1364 Met Thr Leu Gly Lys Asp Val Val Ile Gln Ser Trp Leu Gly Gly Gly 405 410 415 gct atc aag acc ctg gct gag gct ggt cac aag gta atc gat agt gat 1412 Ala Ile Lys Thr Leu Ala Glu Ala Gly His Lys Val Ile Asp Ser Asp 420 425 430 435 tac aac ttc tgg tac ctt gac tgt ggg cgt gga cag tgg ctc aac ttt 1460 Tyr Asn Phe Trp Tyr Leu Asp Cys Gly Arg Gly Gln Trp Leu Asn Phe 440 445 450 gac aac ggc gat gcc ttt caa aca tac tac ccc ttc aac gac tgg tgc 1508 Asp Asn Gly Asp Ala Phe Gln Thr Tyr Tyr Pro Phe Asn Asp Trp Cys 455 460 465 ggt cct acc aag agc tgg cgg ctc atc tac tcc cac gat cct cgg gcc 1556 Gly Pro Thr Lys Ser Trp Arg Leu Ile Tyr Ser His Asp Pro Arg Ala 470 475 480 ggt cta tcc gag gaa gca gcc aag cgc gtg ctt ggt ggt gag gcg gcc 1604 Gly Leu Ser Glu Glu Ala Ala Lys Arg Val Leu Gly Gly Glu Ala Ala 485 490 495 gta tgg act gag act atc gac agt gtt aac ctc gat acc att gtg tgg 1652 Val Trp Thr Glu Thr Ile Asp Ser Val Asn Leu Asp Thr Ile Val Trp 500 505 510 515 ccc cgc gct gca gtg atg gga gaa gtt ctc tgg tca ggc cga act gac 1700 Pro Arg Ala Ala Val Met Gly Glu Val Leu Trp Ser Gly Arg Thr Asp 520 525 530 gcc tca ggc cag aac aga tcg cag tat gat gct gca ccg cga ctg gct 1748 Ala Ser Gly Gln Asn Arg Ser Gln Tyr Asp Ala Ala Pro Arg Leu Ala 535 540 545 gag atg cgc gag cgt atg gtg gct cga gga gtg agt gct tca cca att 1796 Glu Met Arg Glu Arg Met Val Ala Arg Gly Val Ser Ala Ser Pro Ile 550 555 560 cag atg ccc ttc tgt aca cag ggc aat gcc acc gag tgt gcg caa gtc 1844 Gln Met Pro Phe Cys Thr Gln Gly Asn Ala Thr Glu Cys Ala Gln Val 565 570 575 gag gga tga taaatttgac gcgtggtgca tctgtactta ttacgaatta 1893 Glu Gly 580 ctttgtacat agtttgttct cagcattttg aatagagaat tatttcccct ctttca 1949 13 581 PRT Fusarium sp. UNSURE (42) Xaa is Ile or Leu 13 Met Trp Ser Lys Ala Leu Leu Ala Val Ala Ala Phe Ala Phe Thr Pro 1 5 10 15 Ala Asn Ala Ile Trp Pro Val Pro Lys Lys Ile Ser Thr Gly Asp Lys 20 25 30 Ala Leu Phe Ile Asp Gln Thr Ile Asp Xaa Thr Tyr Asn Gly Asp Phe 35 40 45 Ile Pro Tyr Thr Tyr Asn Tyr Gln Pro Asp Ala Gly Ser Lys Phe Ser 50 55 60 Ser Lys Gln Ile Ile Gln Ala Gly Val Ser Arg Ala Leu Gln Gly Val 65 70 75 80 Phe Gln Asp Asn Phe Val Pro Trp Met Leu Arg Glu Arg Asp Ser Asp 85 90 95 Phe Glu Pro Asp Leu Gln Lys Lys Gln Trp Val Lys Ser Leu Lys Ile 100 105 110 Ile Gln Thr Glu Glu Asp Asp Glu Ser Thr Phe Lys Pro Leu Asn Gly 115 120 125 Glu Val Asp Glu Ser Tyr Ser Leu Ser Leu Ser Glu Lys Gly Glu Ala 130 135 140 Ser Ile Lys Ala Lys Ser Ser Thr Gly Val Leu His Gly Leu Glu Thr 145 150 155 160 Phe Val Gln Leu Phe Phe Lys His Ser Ser Gly Thr Ser Trp Tyr Thr 165 170 175 Pro His Ala Pro Val Ser Ile Gln Asp Glu Pro Glu Tyr Pro His Arg 180 185 190 Gly Ile Leu Leu Asp Val Ala Arg Ser Phe Phe Glu Val Lys His Ile 195 200 205 Lys Arg Thr Ile Asp Ala Met Ser Trp Ser Lys Leu Asn Arg Leu His 210 215 220 Leu His Ile Thr Asp Ser Gln Ser Trp Pro Leu Glu Ile Pro Ala Leu 225 230 235 240 Pro Lys Leu Ala Glu Lys Gly Ala Tyr Arg Lys Gly Leu Thr Tyr Ser 245 250 255 Pro Glu Asp Leu Ala Gly Ile Tyr Glu Tyr Gly Ile His Arg Gly Val 260 265 270 Glu Val Ile Met Glu Ile Asp Met Pro Gly His Ile Gly Val Val Glu 275 280 285 Leu Ala Tyr Lys Asp Leu Ile Val Ala Tyr Asn Glu Lys Pro Tyr Gln 290 295 300 Trp Trp Cys Lys Glu Pro Pro Cys Gly Ala Phe Arg Met Asn Ser Ser 305 310 315 320 Asp Val Tyr Asp Phe Leu Asp Thr Leu Phe Asp Asp Leu Phe Pro Arg 325 330 335 Ile Ser Lys Tyr Ser Pro Tyr Phe His Leu Gly Gly Asp Glu Leu Asn 340 345 350 His Asn Asp Ser Arg Leu Asp Pro Asp Val Arg Ser Asn Glu Thr Glu 355 360 365 Val Leu Ala Pro Leu Leu Gln Lys Phe Val Asp Tyr Thr His Gly Lys 370 375 380 Val Arg Asp Ala Gly Met Thr Pro Phe Val Trp Glu Glu Met Ile Thr 385 390 395 400 Glu Trp Asn Met Thr Leu Gly Lys Asp Val Val Ile Gln Ser Trp Leu 405 410 415 Gly Gly Gly Ala Ile Lys Thr Leu Ala Glu Ala Gly His Lys Val Ile 420 425 430 Asp Ser Asp Tyr Asn Phe Trp Tyr Leu Asp Cys Gly Arg Gly Gln Trp 435 440 445 Leu Asn Phe Asp Asn Gly Asp Ala Phe Gln Thr Tyr Tyr Pro Phe Asn 450 455 460 Asp Trp Cys Gly Pro Thr Lys Ser Trp Arg Leu Ile Tyr Ser His Asp 465 470 475 480 Pro Arg Ala Gly Leu Ser Glu Glu Ala Ala Lys Arg Val Leu Gly Gly 485 490 495 Glu Ala Ala Val Trp Thr Glu Thr Ile Asp Ser Val Asn Leu Asp Thr 500 505 510 Ile Val Trp Pro Arg Ala Ala Val Met Gly Glu Val Leu Trp Ser Gly 515 520 525 Arg Thr Asp Ala Ser Gly Gln Asn Arg Ser Gln Tyr Asp Ala Ala Pro 530 535 540 Arg Leu Ala Glu Met Arg Glu Arg Met Val Ala Arg Gly Val Ser Ala 545 550 555 560 Ser Pro Ile Gln Met Pro Phe Cys Thr Gln Gly Asn Ala Thr Glu Cys 565 570 575 Ala Gln Val Glu Gly 580 14 249 PRT Fusarium venenatum 14 Met Trp Ser Lys Ala Leu Leu Ala Val Ala Ala Phe Ala Phe Thr Pro 1 5 10 15 Ala Asn Ala Ile Trp Pro Val Pro Lys Lys Ile Ser Thr Gly Asp Lys 20 25 30 Ala Phe Phe Ile Asp Gln Thr Ile Asp Ile Thr Tyr Asn Gly Gly Phe 35 40 45 Ile Pro Tyr Thr Tyr Asn Tyr Gln Pro Asp Ala Gly Ser Lys Phe Ser 50 55 60 Ser Lys Gln Ile Val Gln Ala Gly Val Ser Arg Ala Leu Gln Gly Ile 65 70 75 80 Phe Gln Asp Asn Phe Val Pro Trp Met Leu Arg Glu Arg Asp Ser Asp 85 90 95 Phe Glu Pro Asp Leu Gln Lys Lys Gln Trp Val Lys Ser Leu Lys Ile 100 105 110 Val Gln Thr Glu Glu Asp Asp Glu Ser Thr Phe Lys Pro Leu Asn Gly 115 120 125 Glu Val Asp Glu Ser Tyr Ser Leu Ser Leu Ser Glu Lys Gly Glu Ala 130 135 140 Ser Ile Lys Ala Lys Ser Ser Thr Gly Val Leu His Gly Leu Glu Thr 145 150 155 160 Phe Val Gln Leu Phe Phe Lys His Ser Ser Gly Thr Ser Trp Tyr Thr 165 170 175 Pro His Ala Pro Val Ser Ile Gln Asp Glu Pro Glu Tyr Pro His Arg 180 185 190 Gly Ile Leu Leu Asp Val Ala Arg Ser Phe Phe Glu Val Lys His Ile 195 200 205 Lys Arg Thr Ile Asp Ala Met Ser Trp Ser Lys Leu Asn Arg Leu His 210 215 220 Leu His Ile Thr Asp Ser Gln Ser Trp Pro Leu Glu Ile Pro Ala Leu 225 230 235 240 Pro Lys Leu Ala Glu Lys Gly Ala Tyr 245 15 249 PRT Fusarium sporotrichiodes UNSURE (42) Xaa is Ile or Leu 15 Met Trp Ser Lys Ala Leu Leu Ala Val Ala Ala Phe Ala Phe Thr Pro 1 5 10 15 Ala Asn Ala Ile Trp Pro Val Pro Lys Lys Ile Ser Thr Gly Asp Lys 20 25 30 Ala Leu Phe Ile Asp Gln Thr Ile Asp Xaa Thr Tyr Asn Gly Asp Phe 35 40 45 Ile Pro Tyr Thr Tyr Asn Tyr Gln Pro Asp Ala Gly Ser Lys Phe Ser 50 55 60 Ser Lys Gln Ile Ile Gln Ala Gly Val Ser Arg Ala Leu Gln Gly Val 65 70 75 80 Phe Gln Asp Asn Phe Val Pro Trp Met Leu Arg Glu Arg Asp Ser Asp 85 90 95 Phe Glu Pro Asp Leu Gln Lys Lys Gln Trp Val Lys Ser Leu Lys Ile 100 105 110 Ile Gln Thr Glu Glu Asp Asp Glu Ser Thr Phe Lys Pro Leu Asn Gly 115 120 125 Glu Val Asp Glu Ser Tyr Ser Leu Ser Leu Ser Glu Lys Gly Glu Ala 130 135 140 Ser Ile Lys Ala Lys Ser Ser Thr Gly Val Leu His Gly Leu Glu Thr 145 150 155 160 Phe Val Gln Leu Phe Phe Lys His Ser Ser Gly Thr Ser Trp Tyr Thr 165 170 175 Pro His Ala Pro Val Ser Ile Gln Asp Glu Pro Glu Tyr Pro His Arg 180 185 190 Gly Ile Leu Leu Asp Val Ala Arg Ser Phe Phe Glu Val Lys His Ile 195 200 205 Lys Arg Thr Ile Asp Ala Met Ser Trp Ser Lys Leu Asn Arg Leu His 210 215 220 Leu His Ile Thr Asp Ser Gln Ser Trp Pro Leu Glu Ile Pro Ala Leu 225 230 235 240 Pro Lys Leu Ala Glu Lys Gly Ala Tyr 245 16 932 DNA Fusarium venenatum CDS (18)..(923) 16 ggatccacca accagcg atg aag ttc ttc agc act ctt agc acc ctt gcg 50 Met Lys Phe Phe Ser Thr Leu Ser Thr Leu Ala 1 5 10 gtg gcc ctc atg atg agt ggc gag gct ctg gct ggt acc tac aag ggt 98 Val Ala Leu Met Met Ser Gly Glu Ala Leu Ala Gly Thr Tyr Lys Gly 15 20 25 ttc agc att ggc gcc aac agg gct gat ggt gcc tgt aag tgg gag gcc 146 Phe Ser Ile Gly Ala Asn Arg Ala Asp Gly Ala Cys Lys Trp Glu Ala 30 35 40 gac tgg aag aag gat ttc cag gcc atc aag agc tgg aac aag ggt ttc 194 Asp Trp Lys Lys Asp Phe Gln Ala Ile Lys Ser Trp Asn Lys Gly Phe 45 50 55 aac gct gtt cgt ctg tac tct gcc tct gac tgt aac aca ctt gtc aag 242 Asn Ala Val Arg Leu Tyr Ser Ala Ser Asp Cys Asn Thr Leu Val Lys 60 65 70 75 gct gtc ccc gct gcc aag gcc act ggc atg aag atc ctt gtt ggc gtc 290 Ala Val Pro Ala Ala Lys Ala Thr Gly Met Lys Ile Leu Val Gly Val 80 85 90 tgg gcc acc gat gat gct cac ttc ggc cgc gac aag gcc gcc ctc ctc 338 Trp Ala Thr Asp Asp Ala His Phe Gly Arg Asp Lys Ala Ala Leu Leu 95 100 105 aag gct atc aag cag cac ggc acc ggc tgg atc gcc gcc atc agt gtc 386 Lys Ala Ile Lys Gln His Gly Thr Gly Trp Ile Ala Ala Ile Ser Val 110 115 120 gga tcc gag gac ctc tac cgt gag gac atc tcc ccc cag aag ctc gca 434 Gly Ser Glu Asp Leu Tyr Arg Glu Asp Ile Ser Pro Gln Lys Leu Ala 125 130 135 cag cag atc tac gac gtc cga ggc atg gtc cac caa tac aac aag aac 482 Gln Gln Ile Tyr Asp Val Arg Gly Met Val His Gln Tyr Asn Lys Asn 140 145 150 155 ctc aag gtc gga cat acc gac acc tgg acc gct tgg gtc gac ggc cgc 530 Leu Lys Val Gly His Thr Asp Thr Trp Thr Ala Trp Val Asp Gly Arg 160 165 170 aac gac gtc gtc acc aag gcc tgc gat atc gcc att aca aac ggt ttc 578 Asn Asp Val Val Thr Lys Ala Cys Asp Ile Ala Ile Thr Asn Gly Phe 175 180 185 ccc tac tgg aag ggt gtt ccc atc aag gat gct ctc cgc ctc aag acc 626 Pro Tyr Trp Lys Gly Val Pro Ile Lys Asp Ala Leu Arg Leu Lys Thr 190 195 200 ttc cag aac tcc ttc tgg aac gtc cag aag cac gtc aag gct gtc aac 674 Phe Gln Asn Ser Phe Trp Asn Val Gln Lys His Val Lys Ala Val Asn 205 210 215 tcc aag gct gct gtc tgg gtt ggc gag acc ggc tgg cct aca aag gga 722 Ser Lys Ala Ala Val Trp Val Gly Glu Thr Gly Trp Pro Thr Lys Gly 220 225 230 235 ccc aac tac cag aag gct gct gcc acg acc gcc agt ctc cag cag ttc 770 Pro Asn Tyr Gln Lys Ala Ala Ala Thr Thr Ala Ser Leu Gln Gln Phe 240 245 250 tac aac aat gtc ggt tgc tgg ctc tgg cag cag aag gag gcc agt ggt 818 Tyr Asn Asn Val Gly Cys Trp Leu Trp Gln Gln Lys Glu Ala Ser Gly 255 260 265 ttc tgg ttc act gct ttc gat aca cct gcg cac agc acc gag gtt gag 866 Phe Trp Phe Thr Ala Phe Asp Thr Pro Ala His Ser Thr Glu Val Glu 270 275 280 aag tac ttc ggt att gcc aac cag gac cgc aag ctc aag ttc agc ctt 914 Lys Tyr Phe Gly Ile Ala Asn Gln Asp Arg Lys Leu Lys Phe Ser Leu 285 290 295 act tgc taa gccagatct 932 Thr Cys 300 17 301 PRT Fusarium venenatum 17 Met Lys Phe Phe Ser Thr Leu Ser Thr Leu Ala Val Ala Leu Met Met 1 5 10 15 Ser Gly Glu Ala Leu Ala Gly Thr Tyr Lys Gly Phe Ser Ile Gly Ala 20 25 30 Asn Arg Ala Asp Gly Ala Cys Lys Trp Glu Ala Asp Trp Lys Lys Asp 35 40 45 Phe Gln Ala Ile Lys Ser Trp Asn Lys Gly Phe Asn Ala Val Arg Leu 50 55 60 Tyr Ser Ala Ser Asp Cys Asn Thr Leu Val Lys Ala Val Pro Ala Ala 65 70 75 80 Lys Ala Thr Gly Met Lys Ile Leu Val Gly Val Trp Ala Thr Asp Asp 85 90 95 Ala His Phe Gly Arg Asp Lys Ala Ala Leu Leu Lys Ala Ile Lys Gln 100 105 110 His Gly Thr Gly Trp Ile Ala Ala Ile Ser Val Gly Ser Glu Asp Leu 115 120 125 Tyr Arg Glu Asp Ile Ser Pro Gln Lys Leu Ala Gln Gln Ile Tyr Asp 130 135 140 Val Arg Gly Met Val His Gln Tyr Asn Lys Asn Leu Lys Val Gly His 145 150 155 160 Thr Asp Thr Trp Thr Ala Trp Val Asp Gly Arg Asn Asp Val Val Thr 165 170 175 Lys Ala Cys Asp Ile Ala Ile Thr Asn Gly Phe Pro Tyr Trp Lys Gly 180 185 190 Val Pro Ile Lys Asp Ala Leu Arg Leu Lys Thr Phe Gln Asn Ser Phe 195 200 205 Trp Asn Val Gln Lys His Val Lys Ala Val Asn Ser Lys Ala Ala Val 210 215 220 Trp Val Gly Glu Thr Gly Trp Pro Thr Lys Gly Pro Asn Tyr Gln Lys 225 230 235 240 Ala Ala Ala Thr Thr Ala Ser Leu Gln Gln Phe Tyr Asn Asn Val Gly 245 250 255 Cys Trp Leu Trp Gln Gln Lys Glu Ala Ser Gly Phe Trp Phe Thr Ala 260 265 270 Phe Asp Thr Pro Ala His Ser Thr Glu Val Glu Lys Tyr Phe Gly Ile 275 280 285 Ala Asn Gln Asp Arg Lys Leu Lys Phe Ser Leu Thr Cys 290 295 300 18 1227 DNA Fusarium venenatum CDS (18)..(1217) 18 ggatccaaca ccacgcg atg ggt ggt gga ccc gaa ggt ttc cgc acc gtt 50 Met Gly Gly Gly Pro Glu Gly Phe Arg Thr Val 1 5 10 gcg tat ttc gtc aac tgg gct atc tat gca cga aag cat cgt cct cag 98 Ala Tyr Phe Val Asn Trp Ala Ile Tyr Ala Arg Lys His Arg Pro Gln 15 20 25 gat ctt ccc gtg gag aac ctg aca cat att ctt tac tcg ttc gct aat 146 Asp Leu Pro Val Glu Asn Leu Thr His Ile Leu Tyr Ser Phe Ala Asn 30 35 40 att cgt agc gac tct ggc gaa gtc cat ctc acc gac tca tgg gcc gat 194 Ile Arg Ser Asp Ser Gly Glu Val His Leu Thr Asp Ser Trp Ala Asp 45 50 55 acc gat att cat tgg gat gga gat tcc tgg aat gat gtc ggt acc aac 242 Thr Asp Ile His Trp Asp Gly Asp Ser Trp Asn Asp Val Gly Thr Asn 60 65 70 75 ttg tac ggt tgc atg aag cag ctt aac ctg ttg aaa aga cgt aac cga 290 Leu Tyr Gly Cys Met Lys Gln Leu Asn Leu Leu Lys Arg Arg Asn Arg 80 85 90 aac ctc aag gtt ctt ctc agt att gga ggt tgg acc ttc agt agc aac 338 Asn Leu Lys Val Leu Leu Ser Ile Gly Gly Trp Thr Phe Ser Ser Asn 95 100 105 ttc aag ggc ccc gct agc aca ccc caa gga cgt gac aca ttc gcc aag 386 Phe Lys Gly Pro Ala Ser Thr Pro Gln Gly Arg Asp Thr Phe Ala Lys 110 115 120 agc tgt gtc gat ctg atc aag aac ctc ggt ttt gac ggt ata gat atc 434 Ser Cys Val Asp Leu Ile Lys Asn Leu Gly Phe Asp Gly Ile Asp Ile 125 130 135 gat tgg gag tac ccc cag gat gcc aac gag gct aga aac tat gtc gaa 482 Asp Trp Glu Tyr Pro Gln Asp Ala Asn Glu Ala Arg Asn Tyr Val Glu 140 145 150 155 ctt ctg ggc gcc gtg cgt cat gag atg gat gca tat gca cag aca ttg 530 Leu Leu Gly Ala Val Arg His Glu Met Asp Ala Tyr Ala Gln Thr Leu 160 165 170 agc caa cct tat cac ttt gag ttg act gtg gcc tgt cca gcc ggt gcg 578 Ser Gln Pro Tyr His Phe Glu Leu Thr Val Ala Cys Pro Ala Gly Ala 175 180 185 aca aac ttc cag aag ctc gat atc cgt gga atg gat caa tac ctc gac 626 Thr Asn Phe Gln Lys Leu Asp Ile Arg Gly Met Asp Gln Tyr Leu Asp 190 195 200 ttc tgg aac ctc atg gct tac gac tat gct ggt tct tgg gac caa act 674 Phe Trp Asn Leu Met Ala Tyr Asp Tyr Ala Gly Ser Trp Asp Gln Thr 205 210 215 gcg ggt cat cag gcc aac ctg tac cca tct cac gac aac cca gta tca 722 Ala Gly His Gln Ala Asn Leu Tyr Pro Ser His Asp Asn Pro Val Ser 220 225 230 235 acc cca ttc tct acc tct gct gcc atc gac ttt tac gtc cgc agc ggt 770 Thr Pro Phe Ser Thr Ser Ala Ala Ile Asp Phe Tyr Val Arg Ser Gly 240 245 250 gtg aac cct tca aag ata gtt ctc ggc atg cca ctc tac ggc cga gcc 818 Val Asn Pro Ser Lys Ile Val Leu Gly Met Pro Leu Tyr Gly Arg Ala 255 260 265 ttt gag aac acc gac ggt ccc ggc cgc ccc tac caa ggc gtt gga caa 866 Phe Glu Asn Thr Asp Gly Pro Gly Arg Pro Tyr Gln Gly Val Gly Gln 270 275 280 ggt tcc tgg gaa cag gga gtc tac gat tac aag gcg ctt ccc cta gag 914 Gly Ser Trp Glu Gln Gly Val Tyr Asp Tyr Lys Ala Leu Pro Leu Glu 285 290 295 ggc gcg caa gag tac gga gat aga gga tgc tgt gcc agc tac tgc tac 962 Gly Ala Gln Glu Tyr Gly Asp Arg Gly Cys Cys Ala Ser Tyr Cys Tyr 300 305 310 315 aac cct cag tca cgt acc atg gtc acc tac gac acg ccg cgg gtc gcc 1010 Asn Pro Gln Ser Arg Thr Met Val Thr Tyr Asp Thr Pro Arg Val Ala 320 325 330 tgg gat aag gcc gag tat gtg aag agg tgg aag ctg gga ggc gct atg 1058 Trp Asp Lys Ala Glu Tyr Val Lys Arg Trp Lys Leu Gly Gly Ala Met 335 340 345 tgg tgg gag agc agc gcg gat aag cag ggc gag cag agt ttg atc aca 1106 Trp Trp Glu Ser Ser Ala Asp Lys Gln Gly Glu Gln Ser Leu Ile Thr 350 355 360 acg gtt gtg aac gga ttc gga ggt cag gga gcg ctc atg aga cag gac 1154 Thr Val Val Asn Gly Phe Gly Gly Gln Gly Ala Leu Met Arg Gln Asp 365 370 375 aac tgt att gag tat ccc gcg acc aag tac gat aac ttg cga aat ggg 1202 Asn Cys Ile Glu Tyr Pro Ala Thr Lys Tyr Asp Asn Leu Arg Asn Gly 380 385 390 395 ttc ccg gac aat tga ggcgagatct 1227 Phe Pro Asp Asn 19 399 PRT Fusarium venenatum 19 Met Gly Gly Gly Pro Glu Gly Phe Arg Thr Val Ala Tyr Phe Val Asn 1 5 10 15 Trp Ala Ile Tyr Ala Arg Lys His Arg Pro Gln Asp Leu Pro Val Glu 20 25 30 Asn Leu Thr His Ile Leu Tyr Ser Phe Ala Asn Ile Arg Ser Asp Ser 35 40 45 Gly Glu Val His Leu Thr Asp Ser Trp Ala Asp Thr Asp Ile His Trp 50 55 60 Asp Gly Asp Ser Trp Asn Asp Val Gly Thr Asn Leu Tyr Gly Cys Met 65 70 75 80 Lys Gln Leu Asn Leu Leu Lys Arg Arg Asn Arg Asn Leu Lys Val Leu 85 90 95 Leu Ser Ile Gly Gly Trp Thr Phe Ser Ser Asn Phe Lys Gly Pro Ala 100 105 110 Ser Thr Pro Gln Gly Arg Asp Thr Phe Ala Lys Ser Cys Val Asp Leu 115 120 125 Ile Lys Asn Leu Gly Phe Asp Gly Ile Asp Ile Asp Trp Glu Tyr Pro 130 135 140 Gln Asp Ala Asn Glu Ala Arg Asn Tyr Val Glu Leu Leu Gly Ala Val 145 150 155 160 Arg His Glu Met Asp Ala Tyr Ala Gln Thr Leu Ser Gln Pro Tyr His 165 170 175 Phe Glu Leu Thr Val Ala Cys Pro Ala Gly Ala Thr Asn Phe Gln Lys 180 185 190 Leu Asp Ile Arg Gly Met Asp Gln Tyr Leu Asp Phe Trp Asn Leu Met 195 200 205 Ala Tyr Asp Tyr Ala Gly Ser Trp Asp Gln Thr Ala Gly His Gln Ala 210 215 220 Asn Leu Tyr Pro Ser His Asp Asn Pro Val Ser Thr Pro Phe Ser Thr 225 230 235 240 Ser Ala Ala Ile Asp Phe Tyr Val Arg Ser Gly Val Asn Pro Ser Lys 245 250 255 Ile Val Leu Gly Met Pro Leu Tyr Gly Arg Ala Phe Glu Asn Thr Asp 260 265 270 Gly Pro Gly Arg Pro Tyr Gln Gly Val Gly Gln Gly Ser Trp Glu Gln 275 280 285 Gly Val Tyr Asp Tyr Lys Ala Leu Pro Leu Glu Gly Ala Gln Glu Tyr 290 295 300 Gly Asp Arg Gly Cys Cys Ala Ser Tyr Cys Tyr Asn Pro Gln Ser Arg 305 310 315 320 Thr Met Val Thr Tyr Asp Thr Pro Arg Val Ala Trp Asp Lys Ala Glu 325 330 335 Tyr Val Lys Arg Trp Lys Leu Gly Gly Ala Met Trp Trp Glu Ser Ser 340 345 350 Ala Asp Lys Gln Gly Glu Gln Ser Leu Ile Thr Thr Val Val Asn Gly 355 360 365 Phe Gly Gly Gln Gly Ala Leu Met Arg Gln Asp Asn Cys Ile Glu Tyr 370 375 380 Pro Ala Thr Lys Tyr Asp Asn Leu Arg Asn Gly Phe Pro Asp Asn 385 390 395 20 1781 DNA Fusarium venenatum CDS (18)..(1763) 20 ggatccacca accagcg atg tgg tcc aag gct ctt ctg gcc gtt gcc gcc 50 Met Trp Ser Lys Ala Leu Leu Ala Val Ala Ala 1 5 10 ttc gcc ttt acc ccc gcc aat gcc ata tgg cca gtg cca aag aag atc 98 Phe Ala Phe Thr Pro Ala Asn Ala Ile Trp Pro Val Pro Lys Lys Ile 15 20 25 tct act gga gac aag gcc ttc ttc atc gat caa aca att gat atc acc 146 Ser Thr Gly Asp Lys Ala Phe Phe Ile Asp Gln Thr Ile Asp Ile Thr 30 35 40 tat aat gga ggc ttt atc cct tac act tac aac tac cag ccc gat gct 194 Tyr Asn Gly Gly Phe Ile Pro Tyr Thr Tyr Asn Tyr Gln Pro Asp Ala 45 50 55 ggc tcc aag ttc agc agt aag caa atc gtc caa gcc ggc gtc tct cgt 242 Gly Ser Lys Phe Ser Ser Lys Gln Ile Val Gln Ala Gly Val Ser Arg 60 65 70 75 gcc ctc cag ggc atc ttc cag gac aac ttt gtc cca tgg atg ctt cgc 290 Ala Leu Gln Gly Ile Phe Gln Asp Asn Phe Val Pro Trp Met Leu Arg 80 85 90 gaa cgc gat tcc gat ttc gag cct gac ctg caa aag aag cag tgg gtg 338 Glu Arg Asp Ser Asp Phe Glu Pro Asp Leu Gln Lys Lys Gln Trp Val 95 100 105 aag tcg cta aag att gtc cag acc gag gag gat gac gag agt acc ttc 386 Lys Ser Leu Lys Ile Val Gln Thr Glu Glu Asp Asp Glu Ser Thr Phe 110 115 120 aag cct ctc aat ggt gag gtt gac gag tcg tac tct ctc tca ctt tct 434 Lys Pro Leu Asn Gly Glu Val Asp Glu Ser Tyr Ser Leu Ser Leu Ser 125 130 135 gaa aag ggc gag gct tcc atc aag gcc aag tct tct aca ggc gtc ctg 482 Glu Lys Gly Glu Ala Ser Ile Lys Ala Lys Ser Ser Thr Gly Val Leu 140 145 150 155 cat gga ctt gag acc ttt gtc caa ctt ttc ttc aag cac agc tct ggc 530 His Gly Leu Glu Thr Phe Val Gln Leu Phe Phe Lys His Ser Ser Gly 160 165 170 act tcc tgg tac acg ccg cac gcg cct gtc tcg atc cag gat gag ccc 578 Thr Ser Trp Tyr Thr Pro His Ala Pro Val Ser Ile Gln Asp Glu Pro 175 180 185 gag tac cct cat cga ggc att ctt ctc gat gtt gcc cgt agc ttt ttt 626 Glu Tyr Pro His Arg Gly Ile Leu Leu Asp Val Ala Arg Ser Phe Phe 190 195 200 gaa gtc aag cac atc aag cgc aca atc gac gcc atg tcg tgg agc aag 674 Glu Val Lys His Ile Lys Arg Thr Ile Asp Ala Met Ser Trp Ser Lys 205 210 215 ctg aat cgc ctt cac ctt cac atc act gac tcg cag tcc tgg cct ctc 722 Leu Asn Arg Leu His Leu His Ile Thr Asp Ser Gln Ser Trp Pro Leu 220 225 230 235 gag atc cca gcc cta ccc aaa ctg gcc gaa aag ggt gca tac cgc aaa 770 Glu Ile Pro Ala Leu Pro Lys Leu Ala Glu Lys Gly Ala Tyr Arg Lys 240 245 250 ggc ctg acc tac tct cct gag gat ctt gcc ggt att tat gag tat ggt 818 Gly Leu Thr Tyr Ser Pro Glu Asp Leu Ala Gly Ile Tyr Glu Tyr Gly 255 260 265 atc cac cgc gga gtc gag gtc atc atg gag att gac atg ccc ggc cat 866 Ile His Arg Gly Val Glu Val Ile Met Glu Ile Asp Met Pro Gly His 270 275 280 atc ggt gtc gtt gag ctt gcc tat aag gat ctc att gtc gcg tac aat 914 Ile Gly Val Val Glu Leu Ala Tyr Lys Asp Leu Ile Val Ala Tyr Asn 285 290 295 gag aag cct tat caa tgg tgg tgt aag gag cca ccc tgt ggt gcg ttc 962 Glu Lys Pro Tyr Gln Trp Trp Cys Lys Glu Pro Pro Cys Gly Ala Phe 300 305 310 315 cgc atg aac agc tct gat gtt tat gac ttt ctc gac act ctt ttt gat 1010 Arg Met Asn Ser Ser Asp Val Tyr Asp Phe Leu Asp Thr Leu Phe Asp 320 325 330 gac ctc ttc cct cgc att tcc aag tac agt cct tac ttc cac ctt ggt 1058 Asp Leu Phe Pro Arg Ile Ser Lys Tyr Ser Pro Tyr Phe His Leu Gly 335 340 345 gga gac gag ctc aac cac aac gat tcc aga ctt gac cct gat gtg cgc 1106 Gly Asp Glu Leu Asn His Asn Asp Ser Arg Leu Asp Pro Asp Val Arg 350 355 360 tct aac gag acc gag gtt ctg gcg cct ctt ttg caa aag ttc gtc gat 1154 Ser Asn Glu Thr Glu Val Leu Ala Pro Leu Leu Gln Lys Phe Val Asp 365 370 375 tac act cac ggc aag gtt cga gat gcc ggc atg act ccg ttc gtc tgg 1202 Tyr Thr His Gly Lys Val Arg Asp Ala Gly Met Thr Pro Phe Val Trp 380 385 390 395 gag gag atg att acc gaa tgg aac atg act ctg ggt aaa gac gtt gtg 1250 Glu Glu Met Ile Thr Glu Trp Asn Met Thr Leu Gly Lys Asp Val Val 400 405 410 att cag tcc tgg ctc ggt ggc ggt gct atc aag acc ctg gct gag gct 1298 Ile Gln Ser Trp Leu Gly Gly Gly Ala Ile Lys Thr Leu Ala Glu Ala 415 420 425 ggt cac aag gta atc gat agt gat tac aac ttc tgg tac ctt gac tgt 1346 Gly His Lys Val Ile Asp Ser Asp Tyr Asn Phe Trp Tyr Leu Asp Cys 430 435 440 ggg cgt gga cag tgg ctc aac ttt gac aac ggc gat gcc ttt caa aca 1394 Gly Arg Gly Gln Trp Leu Asn Phe Asp Asn Gly Asp Ala Phe Gln Thr 445 450 455 tac tac ccc ttc aac gac tgg tgc ggt cct acc aag agc tgg cgg ctc 1442 Tyr Tyr Pro Phe Asn Asp Trp Cys Gly Pro Thr Lys Ser Trp Arg Leu 460 465 470 475 atc tac tcc cac gat cct cgg gcc ggt cta tcc gag gaa gca gcc aag 1490 Ile Tyr Ser His Asp Pro Arg Ala Gly Leu Ser Glu Glu Ala Ala Lys 480 485 490 cgc gtg ctt ggt ggt gag gcg gcc gta tgg act gag act atc gac agt 1538 Arg Val Leu Gly Gly Glu Ala Ala Val Trp Thr Glu Thr Ile Asp Ser 495 500 505 gtt aac ctc gat acc att gtg tgg ccc cgc gct gca gtg atg gga gaa 1586 Val Asn Leu Asp Thr Ile Val Trp Pro Arg Ala Ala Val Met Gly Glu 510 515 520 gtt ctc tgg tca ggc cga act gac gcc tca ggc cag aac aga tcg cag 1634 Val Leu Trp Ser Gly Arg Thr Asp Ala Ser Gly Gln Asn Arg Ser Gln 525 530 535 tat gat gct gca ccg cga ctg gct gag atg cgc gag cgt atg gtg gct 1682 Tyr Asp Ala Ala Pro Arg Leu Ala Glu Met Arg Glu Arg Met Val Ala 540 545 550 555 cga gga gtg agt gct tca cca att cag atg ccc ttc tgt aca cag ggc 1730 Arg Gly Val Ser Ala Ser Pro Ile Gln Met Pro Phe Cys Thr Gln Gly 560 565 570 aat gcc acc gag tgt gcg caa gtc gag gga tga taaatttgac gcagatct 1781 Asn Ala Thr Glu Cys Ala Gln Val Glu Gly 575 580 21 581 PRT Fusarium venenatum 21 Met Trp Ser Lys Ala Leu Leu Ala Val Ala Ala Phe Ala Phe Thr Pro 1 5 10 15 Ala Asn Ala Ile Trp Pro Val Pro Lys Lys Ile Ser Thr Gly Asp Lys 20 25 30 Ala Phe Phe Ile Asp Gln Thr Ile Asp Ile Thr Tyr Asn Gly Gly Phe 35 40 45 Ile Pro Tyr Thr Tyr Asn Tyr Gln Pro Asp Ala Gly Ser Lys Phe Ser 50 55 60 Ser Lys Gln Ile Val Gln Ala Gly Val Ser Arg Ala Leu Gln Gly Ile 65 70 75 80 Phe Gln Asp Asn Phe Val Pro Trp Met Leu Arg Glu Arg Asp Ser Asp 85 90 95 Phe Glu Pro Asp Leu Gln Lys Lys Gln Trp Val Lys Ser Leu Lys Ile 100 105 110 Val Gln Thr Glu Glu Asp Asp Glu Ser Thr Phe Lys Pro Leu Asn Gly 115 120 125 Glu Val Asp Glu Ser Tyr Ser Leu Ser Leu Ser Glu Lys Gly Glu Ala 130 135 140 Ser Ile Lys Ala Lys Ser Ser Thr Gly Val Leu His Gly Leu Glu Thr 145 150 155 160 Phe Val Gln Leu Phe Phe Lys His Ser Ser Gly Thr Ser Trp Tyr Thr 165 170 175 Pro His Ala Pro Val Ser Ile Gln Asp Glu Pro Glu Tyr Pro His Arg 180 185 190 Gly Ile Leu Leu Asp Val Ala Arg Ser Phe Phe Glu Val Lys His Ile 195 200 205 Lys Arg Thr Ile Asp Ala Met Ser Trp Ser Lys Leu Asn Arg Leu His 210 215 220 Leu His Ile Thr Asp Ser Gln Ser Trp Pro Leu Glu Ile Pro Ala Leu 225 230 235 240 Pro Lys Leu Ala Glu Lys Gly Ala Tyr Arg Lys Gly Leu Thr Tyr Ser 245 250 255 Pro Glu Asp Leu Ala Gly Ile Tyr Glu Tyr Gly Ile His Arg Gly Val 260 265 270 Glu Val Ile Met Glu Ile Asp Met Pro Gly His Ile Gly Val Val Glu 275 280 285 Leu Ala Tyr Lys Asp Leu Ile Val Ala Tyr Asn Glu Lys Pro Tyr Gln 290 295 300 Trp Trp Cys Lys Glu Pro Pro Cys Gly Ala Phe Arg Met Asn Ser Ser 305 310 315 320 Asp Val Tyr Asp Phe Leu Asp Thr Leu Phe Asp Asp Leu Phe Pro Arg 325 330 335 Ile Ser Lys Tyr Ser Pro Tyr Phe His Leu Gly Gly Asp Glu Leu Asn 340 345 350 His Asn Asp Ser Arg Leu Asp Pro Asp Val Arg Ser Asn Glu Thr Glu 355 360 365 Val Leu Ala Pro Leu Leu Gln Lys Phe Val Asp Tyr Thr His Gly Lys 370 375 380 Val Arg Asp Ala Gly Met Thr Pro Phe Val Trp Glu Glu Met Ile Thr 385 390 395 400 Glu Trp Asn Met Thr Leu Gly Lys Asp Val Val Ile Gln Ser Trp Leu 405 410 415 Gly Gly Gly Ala Ile Lys Thr Leu Ala Glu Ala Gly His Lys Val Ile 420 425 430 Asp Ser Asp Tyr Asn Phe Trp Tyr Leu Asp Cys Gly Arg Gly Gln Trp 435 440 445 Leu Asn Phe Asp Asn Gly Asp Ala Phe Gln Thr Tyr Tyr Pro Phe Asn 450 455 460 Asp Trp Cys Gly Pro Thr Lys Ser Trp Arg Leu Ile Tyr Ser His Asp 465 470 475 480 Pro Arg Ala Gly Leu Ser Glu Glu Ala Ala Lys Arg Val Leu Gly Gly 485 490 495 Glu Ala Ala Val Trp Thr Glu Thr Ile Asp Ser Val Asn Leu Asp Thr 500 505 510 Ile Val Trp Pro Arg Ala Ala Val Met Gly Glu Val Leu Trp Ser Gly 515 520 525 Arg Thr Asp Ala Ser Gly Gln Asn Arg Ser Gln Tyr Asp Ala Ala Pro 530 535 540 Arg Leu Ala Glu Met Arg Glu Arg Met Val Ala Arg Gly Val Ser Ala 545 550 555 560 Ser Pro Ile Gln Met Pro Phe Cys Thr Gln Gly Asn Ala Thr Glu Cys 565 570 575 Ala Gln Val Glu Gly 580 22 33 DNA Zea mays 22 cacacacaac agatctcccc caaatccacc cgt 33 23 33 DNA Artificial Sequence Description of Artificial Sequence MODIFIED UBI-1 23 cacacacaac agatttcccc caaatccacc cgt 33 24 29 DNA Artificial Sequence Description of Artificial Sequence MODIFIED UBI-1 24 cagatttccc ccaaatccca cacacaacc 29 25 17 DNA Artificial Sequence Description of Artificial Sequence LEADER SEQUENCE 25 ggatccacca accagcg 17 26 16 DNA Artificial Sequence Description of Artificial Sequence M13F primer 26 gtaaaacgac ggccag 16 27 24 DNA Artificial Sequence Description of Artificial Sequence M13R primer 27 agcgaataac aatttcacac agga 24 28 22 DNA Artificial Sequence Description of Artificial SequencePrimer 28 tcctggagcc cgtcagtatc gg 22 29 22 DNA Artificial Sequence Description of Artificial SequencePrimer 29 tggctgaata tcgacggttt cc 22 30 18 DNA Artificial Sequence Description of Artificial SequencePrimer 30 atttaggtga cactatag 18 31 23 DNA Artificial Sequence Description of Artificial SequencePrimer 31 caggacagga aacagctatg acc 23 32 16 DNA Artificial Sequence Description of Artificial SequencePrimer 32 ctatcaagca gcacgg 16 33 17 DNA Artificial Sequence Description of Artificial SequencePrimer 33 ctggaaggtc ttgaggc 17 34 19 DNA Artificial Sequence Description of Artificial SequencePrimer 34 gatatcgatt gggagtacc 19 35 18 DNA Artificial Sequence Description of Artificial SequencePrimer 35 catggtacgt gactgagg 18 36 18 DNA Artificial Sequence Description of Artificial SequencePrimer 36 caaccctcag tcacgtac 18 37 16 DNA Artificial Sequence Description of Artificial SequencePrimer 37 cctcgttggc atcctg 16 38 30 DNA Artificial Sequence Description of Artificial SequencePrimer 38 agatccttat aggcaagctc aacgacaccg 30 39 20 DNA Artificial Sequence Description of Artificial SequencePrimer 39 cataaatacc ggcaagatcc 20 40 25 DNA Artificial Sequence Description of Artificial SequencePrimer 40 atcgatatct ataccgtcaa aaccg 25 41 25 DNA Artificial Sequence Description of Artificial SequencePrimer 41 atcgatatct ataccgtcaa aaccg 25 42 18 DNA Artificial Sequence Description of Artificial SequencePrimer 42 atctgctggg cgagcttc 18 43 18 DNA Artificial Sequence Description of Artificial SequencePrimer 43 cagaaaccac tggcatcc 18 44 18 DNA Artificial Sequence Description of Artificial SequencePrimer 44 aagttcggcc ttacttgc 18 45 18 DNA Artificial Sequence Description of Artificial SequencePrimer 45 ctttgggctc gtattcac 18 46 18 DNA Artificial Sequence Description of Artificial SequencePrimer 46 cttcagcact ctcagcac 18 47 21 DNA Artificial Sequence Description of Artificial SequencePrimer 47 ggactgaggg agtgttggtt c 21 48 20 DNA Artificial Sequence Description of Artificial SequencePrimer 48 tctaccctac aacactcatc 20 49 18 DNA Artificial Sequence Description of Artificial SequencePrimer 49 gtcgatcatg gtggaaag 18 50 18 DNA Artificial Sequence Description of Artificial SequencePrimer 50 tgtccctgtt actaacgg 18 51 18 DNA Artificial Sequence Description of Artificial SequencePrimer 51 tagtcgcctt tctaagcc 18 52 25 DNA Artificial Sequence Description of Artificial SequencePrimer 52 aagaatgcct cgatgagggt actcg 25 53 19 DNA Artificial Sequence Description of Artificial SequencePrimer 53 atctcactca cctcacctc 19 54 19 DNA Artificial Sequence Description of Artificial SequencePrimer 54 ccggtattta tgagtatgg 19 55 20 DNA Artificial Sequence Description of Artificial SequencePrimer 55 gatagtctca gtccatacgg 20 56 17 DNA Artificial Sequence Description of Artificial SequencePrimer 56 gtaccttgac tgtgggc 17 57 16 DNA Artificial Sequence Description of Artificial SequencePrimer 57 ggaaatgcga gggaag 16 58 27 DNA Artificial Sequence Description of Artificial SequencePrimer 58 agatctcgcc tcaattgtcc gggaacc 27 59 37 DNA Artificial Sequence Description of Artificial SequencePrimer 59 ggatccaaca ccacgcgatg ggtggtggac ccgaagg 37 60 28 DNA Artificial Sequence Description of Artificial SequencePrimer 60 agatctgcgt caaatttatc atccctcg 28 61 45 DNA Artificial Sequence Description of Artificial SequencePrimer 61 ggatccacca accagcgatg tggtccaagg ctcttctggc cgttg 45 62 24 DNA Artificial Sequence Description of Artificial SequencePrimer 62 gcttcatgca accgtacaag ttgg 24 63 25 DNA Artificial Sequence Description of Artificial SequencePrimer 63 cgacttcacc cactgcttct tttgc 25 64 23 DNA Artificial Sequence Description of Artificial SequencePrimer 64 cccagacgcc aacaaggatc ttc 23 65 28 DNA Artificial Sequence Description of Artificial SequencePrimer 65 agatctggct tagcaagtaa ggctgaac 28 66 41 DNA Artificial Sequence Description of Artificial SequencePrimer 66 ggatccacca accagcgatg aagttcttca gcactcttag c 41 67 18 DNA Artificial Sequence Description of Artificial SequencePrimer 67 gtactgggtt ggtgagac 18 68 17 DNA Artificial Sequence Description of Artificial SequencePrimer 68 ccattccaag accaggc 17 69 20 DNA Artificial Sequence Description of Artificial SequencePrimer 69 cccatctcat aaataacgtc 20 70 18 DNA Artificial Sequence Description of Artificial SequencePrimer 70 cctgccttca tacgctat 18 71 26 DNA Artificial Sequence Description of Artificial SequencePrimer 71 cctgccttca tacgctattt atttgc 26 72 19 DNA Artificial Sequence Description of Artificial SequencePrimer 72 gacaccaacc agcgaacca 19 73 31 DNA Artificial Sequence Description of Artificial SequencePrimer 73 cacacacaac cagatttccc ccaaatccac c 31 74 31 DNA Artificial Sequence Description of Artificial SequencePrimer 74 ggtggatttg ggggaaatct ggttgtgtgt g 31 75 19 DNA Artificial Sequence Description of Artificial Sequence RT-PCR Primer 75 ctggaagttt gtcgcaccg 19 76 20 DNA Artificial Sequence Description of Artificial Sequence RT-PCR Primer 76 gagatagagg atgctgtgcc 20 77 21 DNA Artificial Sequence Description of Artificial Sequence RT-PCR Primer 77 cagtgatgtg aaggtgaagg c 21 78 22 DNA Artificial Sequence Description of Artificial Sequence RT-PCR Primer 78 cgtatggact gagactatcg ac 22 79 21 DNA Artificial Sequence Description of Artificial Sequence RT-PCR Primer 79 gagcatcctt gatgggaaca c 21 80 21 DNA Artificial Sequence Description of Artificial Sequence RT-PCR Primer 80 gtcaactcca aggctgtcgt c 21 81 22 DNA Artificial Sequence Description of Artificial Sequence RT-PCR Primer 81 gccaaatgtt tgaacgatct gc 22 82 20 DNA Artificial Sequence Description of Artificial Sequence RT-PCR Primer 82 caacctcgtg ttgttcggag 20
Claims (39)
1. An isolated nucleic acid molecule encoding a polypeptide having glucanase activity, selected from the group consisting of:
(a) a nucleic acid sequence having at least 70% nucleotide sequence identity with SEQ ID NO:1 from nucleotide 37 through nucleotide 942, SEQ ID NO:3 from nucleotide 1801 through nucleotide 2761 or SEQ ID NO:16 from nucleotide 18 to nucleotide 923;
(b) a nucleic acid sequence encoding a polypeptide having glucanase activity, said polypeptide having an amino acid sequence which has at least 80% identity with amino acids 1 to 301 of SEQ ID NO:2, 4, 5 or 17.
(c) a nucleic acid sequence which hybridizes under medium stringency or high stringency conditions with (i) SEQ ID NO:1 from nucleotide 37 through nucleotide 942, SEQ ID NO:3 from nucleotide 1801 through nucleotide 2761 or SEQ ID NO:16 from nucleotide 18 to nucleotide 923; (ii) a subsequence of (i) of at least 100 contiguous nucleotides, or (iii) a complementary strand of (i) or (ii); and
(d) a subsequence of (a), (b) or (c) wherein the subsequence encodes a polypeptide fragment which has glucanase activity.
2. The nucleic acid molecule of claim 1 as shown in SEQ ID NO:1, 3 or 16.
3. The nucleic acid molecule of claim 1 which is contained in plasmid Glu2, FvGluS or FvGluAS.
4. A nucleic acid construct comprising a nucleic acid molecule of claim 1 operably linked to one or more control sequences which direct the production of a polypeptide having glucanase activity in an expression host.
5. A cell transformed with the isolated nucleic acid molecule of claim 1 .
6. A plant transformed with the isolated nucleic acid molecule of claim 1 .
7. A seed of the plant according to claim 6 .
8. The plant of claim 6 wherein the plant is a monocot.
9. The plant of claim 8 wherein said monocotyledonous plant is wheat.
10. Sexually or asexually derived progeny of the plant of claim 6 .
11. A glucanase polypeptide encoded by the nucleic acid molecule of claim 1 .
12. An isolated polypeptide having glucanase activity, selected from the group consisting of:
(a) a polypeptide having an amino acid sequence which has at least 80% identity with amino acids 1 to 301 of SEQ ID NO:2, 4, 5 or 17;
(b) a polypeptide encoded by a nucleic acid sequence which hybridizes under medium stringency or high stringency conditions with (i) SEQ ID NO:1 from nucleotide 37 through nucleotide 942, SEQ ID NO:3 from nucleotide 1801 through nucleotide 2761 or SEQ ID NO:16 from nucleotide 18 to nucleotide 923; (ii) a subsequence of (i) of at least 100 nucleotides, or (iii) a complementary strand of (i) or (ii); and
(c) a fragment of (a), or (b) that has glucanase activity.
13. An isolated nucleic acid molecule encoding a polypeptide having endochitinase activity, selected from the group consisting of:
(a) a nucleic acid sequence having at least 75% nucleotide sequence identity with SEQ ID NO:10 from nucleotide 186 through nucleotide 1385 or SEQ ID NO:18 from nucleotide 18 to nucleotide 1217;
(b) a nucleic acid sequence encoding a polypeptide having endochitinase activity, said polypeptide having an amino acid sequence which has at least 85% identity with amino acids 1 to 399 of SEQ ID NO:11 or 19;
(c) a nucleic acid sequence which hybridizes under medium stringency or high stringency conditions with (i) SEQ ID NO:10 from nucleotide 186 through nucleotide 1385 or SEQ ID NO:18 from nucleotide 18 to nucleotide 1217; (ii) a subsequence of (i) of at least 100 contiguous nucleotides, or (iii) a complementary strand of (i) or (ii); and
(d) a subsequence of (a), (b) or (c) wherein the subsequence encodes a polypeptide fragment which has endochitinase activity.
14. The nucleic acid molecule of claim 13 as shown in SEQ ID NO:10 or 18.
15. The nucleic acid molecule of claim 13 which is contained in plasmid Endo176, FvEndoS or FvEndoAS.
16. A nucleic acid construct comprising a nucleic acid molecule of claim 13 operably linked to one or more control sequences which direct the production of a polypeptide having endochitinase activity in an expression host.
17. A cell transformed with the isolated nucleic acid molecule of claim 13 .
18. A plant transformed with the isolated nucleic acid molecule of claim 13 .
19. A seed of the plant according to claim 18 .
20. The plant of claim 18 wherein the plant is a monocot.
21. The plant of claim 20 wherein said monocotyledonous plant is wheat.
22. Sexually or asexually derived progeny of the plant of claim 18 .
23. An endochitinase polypeptide encoded by the nucleic acid molecule of claim 13 .
24. An isolated polypeptide having endochitinase activity, selected from the group consisting of:
(a) a polypeptide having an amino acid sequence which has at least 85% identity with amino acids 1 to 399 of SEQ ID NO:11 or 19;
(b) a polypeptide encoded by a nucleic acid sequence which hybridizes under medium stringency or high stringency conditions with (i) SEQ ID NO:10 from nucleotide 186 through nucleotide 1385 or SEQ ID NO:18 from nucleotide 18 to nucleotide 1217; (ii) a subsequence of (i) of at least 100 contiguous nucleotides, or (iii) a complementary strand of (i) or (ii); and
(c) a fragment of (a), or (b) that has endochitinase activity.
25. An isolated nucleic acid molecule encoding a polypeptide having exochitinase activity, selected from the group consisting of:
(a) a nucleic acid sequence having at least 75% nucleotide sequence identity with SEQ ID NO:12 from nucleotide 108 to 1853 or SEQ ID NO:20 from nucleotide 18 to 1763;
(b) a nucleic acid sequence encoding a polypeptide having exochitinase activity, said polypeptide having an amino acid sequence which has at least 85% identity with amino acids 1 to 581 of SEQ ID NO:13 or 21;
(c) a nucleic acid sequence which hybridizes under medium stringency or high stringency conditions with (i) SEQ ID NO:12 from nucleotide 108 to 1853 or SEQ ID NO:20 from nucleotide 18 to 1763; (ii) a subsequence of (i) of at least 100 contiguous nucleotides, or (iii) a complementary strand of (i) or (ii); and
(d) a subsequence of (a), (b) or (c) wherein the subsequence encodes a polypeptide fragment which has exochitinase activity.
26. The nucleic acid molecule of claim 25 as shown in SEQ ID NO:8, 12 or 20.
27. The nucleic acid molecule of claim 25 which is contained in plasmid Exo9, FvExoS or FvExoAS.
28. A nucleic acid construct comprising a nucleic acid molecule of claim 25 operably linked to one or more control sequences which direct the production of a polypeptide having exochitinase activity in an expression host.
29. A cell transformed with the isolated nucleic acid molecule of claim 25 .
30. A plant transformed with the isolated nucleic acid molecule of claim 25 .
31. A seed of the plant according to claim 30 .
32. The plant of claim 30 wherein the plant is a monocot.
33. The plant of claim 30 wherein said monocotyledonous plant is wheat.
34. Sexually or asexually derived progeny of claim 30 .
35. An exochitinase polypeptide encoded by the nucleic acid molecule of claim 25 .
36. An isolated polypeptide having exochitinase activity, selected from the group consisting of:
(a) a polypeptide having an amino acid sequence which has at least 85% identity with amino acids 1 to 581 of SEQ ID NO:13 or 21;
(b) a polypeptide encoded by a nucleic acid sequence which hybridizes under medium stringency or high stringency conditions with (i) SEQ ID NO:12 from nucleotide 108 to 1853 or SEQ ID NO:20 from nucleotide 18 to 1763; (ii) a subsequence of (i) of at least 100 contiguous nucleotides, or (iii) a complementary strand of (i) or (ii); and
(c) a fragment of (a), or (b) that has exochitinase activity.
37. A plant transformed with one or more isolated nucleic acid molecules of claims 1, 13, or 25.
38. A method of conferring or enhancing a plant's resistance to a fungal pathogen, which comprises transforming said plant with one or more isolated nucleic acid molecules which encode a polypeptide having glucanase activity, a polypeptide having endochitinase activity or a polypeptide having exochitinase activity.
39. A method for producing a polypeptide having glucanase, endochitinase or exochitinase activity, which comprises cultivating a recombinant host cell comprising a transformed cell having a nucleic acid molecule which encodes a polypeptide having glucanase, endochitinase or exochitinase activity under conditions suitable for production of the polypeptide; and recovering the polypeptide.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US10/316,754 US20030131376A1 (en) | 1999-08-30 | 2002-12-10 | Nucleic acid sequences encoding cell wall-degrading enzymes and use to engineer resistance to fusarium and other pathogens |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US15158299P | 1999-08-30 | 1999-08-30 | |
US22494600P | 2000-08-11 | 2000-08-11 | |
US09/649,747 US6521435B1 (en) | 1999-08-30 | 2000-08-28 | Nucleic acid sequences encoding cell wall-degrading enzymes and use to engineer resistance to Fusarium and other pathogens |
US10/316,754 US20030131376A1 (en) | 1999-08-30 | 2002-12-10 | Nucleic acid sequences encoding cell wall-degrading enzymes and use to engineer resistance to fusarium and other pathogens |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US09/649,747 Division US6521435B1 (en) | 1999-08-30 | 2000-08-28 | Nucleic acid sequences encoding cell wall-degrading enzymes and use to engineer resistance to Fusarium and other pathogens |
Publications (1)
Publication Number | Publication Date |
---|---|
US20030131376A1 true US20030131376A1 (en) | 2003-07-10 |
Family
ID=27387147
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US09/649,747 Expired - Fee Related US6521435B1 (en) | 1999-08-30 | 2000-08-28 | Nucleic acid sequences encoding cell wall-degrading enzymes and use to engineer resistance to Fusarium and other pathogens |
US10/316,754 Abandoned US20030131376A1 (en) | 1999-08-30 | 2002-12-10 | Nucleic acid sequences encoding cell wall-degrading enzymes and use to engineer resistance to fusarium and other pathogens |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US09/649,747 Expired - Fee Related US6521435B1 (en) | 1999-08-30 | 2000-08-28 | Nucleic acid sequences encoding cell wall-degrading enzymes and use to engineer resistance to Fusarium and other pathogens |
Country Status (6)
Country | Link |
---|---|
US (2) | US6521435B1 (en) |
EP (1) | EP1220941A4 (en) |
CN (1) | CN1382220A (en) |
AU (1) | AU7337600A (en) |
CA (1) | CA2383516A1 (en) |
WO (1) | WO2001016353A1 (en) |
Cited By (14)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20030215464A1 (en) * | 2002-03-21 | 2003-11-20 | Klimpel Gary R. | Methods and compositions for vaccination against or involving enterobacteriaceae bacteria |
US20040058872A1 (en) * | 2002-09-24 | 2004-03-25 | Wisconsin Alumni Research Foundation | Global regulator of secondary metabolite biosynthesis and methods of use |
US9486513B1 (en) | 2010-02-09 | 2016-11-08 | David Gordon Bermudes | Immunization and/or treatment of parasites and infectious agents by live bacteria |
US9737592B1 (en) | 2014-02-14 | 2017-08-22 | David Gordon Bermudes | Topical and orally administered protease inhibitors and bacterial vectors for the treatment of disorders and methods of treatment |
US9878023B1 (en) | 2010-02-09 | 2018-01-30 | David Gordon Bermudes | Protease inhibitor: protease sensitive expression system composition and methods improving the therapeutic activity and specificity of proteins delivered by bacteria |
US10006038B2 (en) | 2009-11-06 | 2018-06-26 | Agrivida, Inc. | Consolidated pretreatment and hydrolysis of plant biomass expressing cell wall degrading enzymes |
US10194463B2 (en) | 2004-07-21 | 2019-01-29 | Qualcomm Incorporated | Efficient signaling over access channel |
US10196623B2 (en) | 2009-11-06 | 2019-02-05 | Agrivida, Inc. | Intein-modified enzymes, their production and industrial application |
US10407742B2 (en) | 2009-11-06 | 2019-09-10 | Agrivida, Inc. | Intein-modified enzymes, their production and industrial applications |
US10857233B1 (en) | 2010-02-09 | 2020-12-08 | David Gordon Bermudes | Protease inhibitor combination with therapeutic proteins including antibodies |
US10988788B2 (en) | 2009-11-06 | 2021-04-27 | Agrivida, Inc. | Plants expressing cell wall degrading enzymes and expression vectors |
US11129906B1 (en) | 2016-12-07 | 2021-09-28 | David Gordon Bermudes | Chimeric protein toxins for expression by therapeutic bacteria |
US11180535B1 (en) | 2016-12-07 | 2021-11-23 | David Gordon Bermudes | Saccharide binding, tumor penetration, and cytotoxic antitumor chimeric peptides from therapeutic bacteria |
WO2023145948A1 (en) * | 2022-01-31 | 2023-08-03 | 国立研究開発法人農業・食品産業技術総合研究機構 | Wheat or the like having short anther trait, and method for producing same |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
UA117654C2 (en) * | 2009-11-06 | 2018-09-10 | Агрівіда, Інк. | A TRANSGENIC PLANT EXPRESSING AN ENZYME THAT DEGRADES A CELL WALL AND AN EXPRESSION VECTOR |
CN113980918B (en) * | 2021-10-18 | 2023-11-24 | 自然资源部第一海洋研究所 | Antarctic ice algae MAAs synthetase, and coding gene and application thereof |
Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5290687A (en) * | 1984-03-26 | 1994-03-01 | Dna Plant Technology Corporation | Chitinase-producing bacteria and plants |
US5378821A (en) * | 1991-06-17 | 1995-01-03 | Cornell Research Foundation, Inc. | Gene encoding for endochitinase |
US5446138A (en) * | 1991-09-06 | 1995-08-29 | Elf Sanofi | Recombinant DNA coding for a protein with endochitinase activity |
US5554521A (en) * | 1984-03-26 | 1996-09-10 | Dna Plant Technology Corporation | Chitinase-producing plants |
US5670706A (en) * | 1990-01-30 | 1997-09-23 | Mogen International, N.V. | Fungal resistant plants, process for obtaining fungal resistant plants and recombinant polynucleotides for use therein |
US5804184A (en) * | 1992-10-09 | 1998-09-08 | Max Planck Gesellschaft Zur Furderung Der Wissen Schaften, E.V. | Transgenic pathogen-resistant organism |
US5919688A (en) * | 1994-10-14 | 1999-07-06 | Novo Nordisk A/S | Enzyme with B-1, 3-glucanase activity |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AU5685396A (en) * | 1995-05-16 | 1996-11-29 | Novo Nordisk A/S | An enzyme with exochitinase activity |
WO2000056762A2 (en) * | 1999-03-22 | 2000-09-28 | Novozymes Biotech, Inc. | Methods for monitoring multiple gene expression |
-
2000
- 2000-08-28 US US09/649,747 patent/US6521435B1/en not_active Expired - Fee Related
- 2000-08-30 CN CN00813628A patent/CN1382220A/en active Pending
- 2000-08-30 EP EP00961421A patent/EP1220941A4/en not_active Withdrawn
- 2000-08-30 AU AU73376/00A patent/AU7337600A/en not_active Abandoned
- 2000-08-30 WO PCT/US2000/023802 patent/WO2001016353A1/en not_active Application Discontinuation
- 2000-08-30 CA CA002383516A patent/CA2383516A1/en not_active Abandoned
-
2002
- 2002-12-10 US US10/316,754 patent/US20030131376A1/en not_active Abandoned
Patent Citations (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5290687A (en) * | 1984-03-26 | 1994-03-01 | Dna Plant Technology Corporation | Chitinase-producing bacteria and plants |
US5374540A (en) * | 1984-03-26 | 1994-12-20 | Dna Plant Technology Corporation | Chitinase-producing bacteria and plants |
US5554521A (en) * | 1984-03-26 | 1996-09-10 | Dna Plant Technology Corporation | Chitinase-producing plants |
US5633450A (en) * | 1984-03-26 | 1997-05-27 | Dna Plant Technology Corporation | Chitinase-producing plants |
US5670706A (en) * | 1990-01-30 | 1997-09-23 | Mogen International, N.V. | Fungal resistant plants, process for obtaining fungal resistant plants and recombinant polynucleotides for use therein |
US5378821A (en) * | 1991-06-17 | 1995-01-03 | Cornell Research Foundation, Inc. | Gene encoding for endochitinase |
US5446138A (en) * | 1991-09-06 | 1995-08-29 | Elf Sanofi | Recombinant DNA coding for a protein with endochitinase activity |
US5804184A (en) * | 1992-10-09 | 1998-09-08 | Max Planck Gesellschaft Zur Furderung Der Wissen Schaften, E.V. | Transgenic pathogen-resistant organism |
US5919688A (en) * | 1994-10-14 | 1999-07-06 | Novo Nordisk A/S | Enzyme with B-1, 3-glucanase activity |
Cited By (26)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7655241B2 (en) * | 2002-03-21 | 2010-02-02 | The Board Of Regents Of The University Of Texas System | Methods and compositions for vaccination against or involving enterobacteriaceae bacteria |
US20030215464A1 (en) * | 2002-03-21 | 2003-11-20 | Klimpel Gary R. | Methods and compositions for vaccination against or involving enterobacteriaceae bacteria |
US20070003999A1 (en) * | 2002-09-24 | 2007-01-04 | Wisconsin Alumni Research Foundation | Global regulator of secondary metabolite biosynthesis and methods of use |
US7427484B2 (en) | 2002-09-24 | 2008-09-23 | Wisconsin Alumni Research Foundation | Global regulator of secondary metabolite biosynthesis and methods of use |
US7053204B2 (en) | 2002-09-24 | 2006-05-30 | Wisconsin Alumni Research Foundation | Global regulator of secondary metabolite biosynthesis and methods of use |
US20040058872A1 (en) * | 2002-09-24 | 2004-03-25 | Wisconsin Alumni Research Foundation | Global regulator of secondary metabolite biosynthesis and methods of use |
US10517114B2 (en) | 2004-07-21 | 2019-12-24 | Qualcomm Incorporated | Efficient signaling over access channel |
US11039468B2 (en) | 2004-07-21 | 2021-06-15 | Qualcomm Incorporated | Efficient signaling over access channel |
US10194463B2 (en) | 2004-07-21 | 2019-01-29 | Qualcomm Incorporated | Efficient signaling over access channel |
US10237892B2 (en) | 2004-07-21 | 2019-03-19 | Qualcomm Incorporated | Efficient signaling over access channel |
US10849156B2 (en) | 2004-07-21 | 2020-11-24 | Qualcomm Incorporated | Efficient signaling over access channel |
US10988788B2 (en) | 2009-11-06 | 2021-04-27 | Agrivida, Inc. | Plants expressing cell wall degrading enzymes and expression vectors |
US10006038B2 (en) | 2009-11-06 | 2018-06-26 | Agrivida, Inc. | Consolidated pretreatment and hydrolysis of plant biomass expressing cell wall degrading enzymes |
US10196623B2 (en) | 2009-11-06 | 2019-02-05 | Agrivida, Inc. | Intein-modified enzymes, their production and industrial application |
US10407742B2 (en) | 2009-11-06 | 2019-09-10 | Agrivida, Inc. | Intein-modified enzymes, their production and industrial applications |
US9486513B1 (en) | 2010-02-09 | 2016-11-08 | David Gordon Bermudes | Immunization and/or treatment of parasites and infectious agents by live bacteria |
US10364435B1 (en) | 2010-02-09 | 2019-07-30 | David Gordon Bermudes | Immunization and/or treatment of parasites and infectious agents by live bacteria |
US10857233B1 (en) | 2010-02-09 | 2020-12-08 | David Gordon Bermudes | Protease inhibitor combination with therapeutic proteins including antibodies |
US10954521B1 (en) | 2010-02-09 | 2021-03-23 | David Gordon Bermudes | Immunization and/or treatment of parasites and infectious agents by live bacteria |
US9878023B1 (en) | 2010-02-09 | 2018-01-30 | David Gordon Bermudes | Protease inhibitor: protease sensitive expression system composition and methods improving the therapeutic activity and specificity of proteins delivered by bacteria |
US11219671B1 (en) | 2010-02-09 | 2022-01-11 | David Gordon Bermudes | Protease inhibitor:protease sensitive expression system, composition and methods for improving the therapeutic activity and specificity of proteins delivered by bacteria |
US10828350B1 (en) | 2014-02-14 | 2020-11-10 | David Gordon Bermudes | Topical and orally administered protease inhibitors and bacterial vectors for the treatment of disorders and methods of treatment |
US9737592B1 (en) | 2014-02-14 | 2017-08-22 | David Gordon Bermudes | Topical and orally administered protease inhibitors and bacterial vectors for the treatment of disorders and methods of treatment |
US11129906B1 (en) | 2016-12-07 | 2021-09-28 | David Gordon Bermudes | Chimeric protein toxins for expression by therapeutic bacteria |
US11180535B1 (en) | 2016-12-07 | 2021-11-23 | David Gordon Bermudes | Saccharide binding, tumor penetration, and cytotoxic antitumor chimeric peptides from therapeutic bacteria |
WO2023145948A1 (en) * | 2022-01-31 | 2023-08-03 | 国立研究開発法人農業・食品産業技術総合研究機構 | Wheat or the like having short anther trait, and method for producing same |
Also Published As
Publication number | Publication date |
---|---|
EP1220941A4 (en) | 2005-09-07 |
AU7337600A (en) | 2001-03-26 |
CN1382220A (en) | 2002-11-27 |
US6521435B1 (en) | 2003-02-18 |
EP1220941A1 (en) | 2002-07-10 |
WO2001016353A1 (en) | 2001-03-08 |
CA2383516A1 (en) | 2001-03-08 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US6521435B1 (en) | Nucleic acid sequences encoding cell wall-degrading enzymes and use to engineer resistance to Fusarium and other pathogens | |
US5614395A (en) | Chemically regulatable and anti-pathogenic DNA sequences and uses thereof | |
US5789214A (en) | Method of inducing gene transcription in a plant | |
AU659455B2 (en) | A plant chitinase gene and use thereof | |
US5993808A (en) | Chitinase, DNA coding therefor and plants containing same | |
US5554521A (en) | Chitinase-producing plants | |
WO1995005467A9 (en) | Chitinase, dna coding therefor and plants containing same | |
IE912039A1 (en) | Novel signal sequences | |
EP0939798B1 (en) | Antifungal proteins, dna coding therefore, and hosts incorporating same | |
Liao et al. | Characterization of a wheat class Ib chitinase gene differentially induced in isogenic lines by infection with Puccinia graminis | |
US6271442B1 (en) | Method of producing pathogen-resistant plants | |
MXPA00009573A (en) | Salicylic acid pathway genes and their use for the induction of resistance in plants. | |
US5851766A (en) | Process for isolating chemically regulatable DNA sequences | |
Zhang | Induction, purification and characterization of chitinases in cucumber (Cucumis sativus L.) and carrot (Daucus carota L.) | |
Zou | Characterization of chitinase activity and gene expression in muskmelon seeds | |
Mei | Maize transformation for resistance to Aspergillus flavus | |
Leubner-Metzger et al. | 3 Functions and Regulation of Plant β-(PR-2) |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AS | Assignment |
Owner name: NOVOZYMES, INC., CALIFORNIA Free format text: CHANGE OF NAME;ASSIGNOR:NOVOZYMES, INC.;REEL/FRAME:016937/0522 Effective date: 20050829 |
|
STCB | Information on status: application discontinuation |
Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION |