US20020068822A1 - Dwf7 mutants - Google Patents
Dwf7 mutants Download PDFInfo
- Publication number
- US20020068822A1 US20020068822A1 US09/775,879 US77587901A US2002068822A1 US 20020068822 A1 US20020068822 A1 US 20020068822A1 US 77587901 A US77587901 A US 77587901A US 2002068822 A1 US2002068822 A1 US 2002068822A1
- Authority
- US
- United States
- Prior art keywords
- dwf7
- polynucleotide
- plant
- leu
- sequence
- 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
- 102000040430 polynucleotide Human genes 0.000 claims abstract description 114
- 108091033319 polynucleotide Proteins 0.000 claims abstract description 114
- 239000002157 polynucleotide Substances 0.000 claims abstract description 114
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 89
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 82
- 229920001184 polypeptide Polymers 0.000 claims abstract description 81
- 238000000034 method Methods 0.000 claims abstract description 78
- 230000009261 transgenic effect Effects 0.000 claims abstract description 39
- 238000004519 manufacturing process Methods 0.000 claims abstract description 14
- 229930182558 Sterol Natural products 0.000 claims description 52
- 239000002773 nucleotide Substances 0.000 claims description 52
- 235000003702 sterols Nutrition 0.000 claims description 52
- 125000003729 nucleotide group Chemical group 0.000 claims description 51
- 150000003432 sterols Chemical class 0.000 claims description 50
- 101100364962 Arabidopsis thaliana STE1 gene Proteins 0.000 claims description 45
- 108091026890 Coding region Proteins 0.000 claims description 42
- 239000013598 vector Substances 0.000 claims description 36
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 25
- 239000012634 fragment Substances 0.000 claims description 24
- 230000001965 increasing effect Effects 0.000 claims description 18
- 239000000203 mixture Substances 0.000 claims description 15
- 230000000295 complement effect Effects 0.000 claims description 11
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 claims description 8
- 238000012258 culturing Methods 0.000 claims description 6
- 235000012000 cholesterol Nutrition 0.000 claims description 4
- 241000196324 Embryophyta Species 0.000 description 251
- 108090000623 proteins and genes Proteins 0.000 description 130
- 210000004027 cell Anatomy 0.000 description 124
- IXVMHGVQKLDRKH-VRESXRICSA-N Brassinolide Natural products O=C1OC[C@@H]2[C@@H]3[C@@](C)([C@H]([C@@H]([C@@H](O)[C@H](O)[C@H](C(C)C)C)C)CC3)CC[C@@H]2[C@]2(C)[C@@H]1C[C@H](O)[C@H](O)C2 IXVMHGVQKLDRKH-VRESXRICSA-N 0.000 description 90
- 108020004414 DNA Proteins 0.000 description 71
- IXVMHGVQKLDRKH-KNBKMWSGSA-N brassinolide Chemical compound C1OC(=O)[C@H]2C[C@H](O)[C@H](O)C[C@]2(C)[C@H]2CC[C@]3(C)[C@@H]([C@H](C)[C@@H](O)[C@H](O)[C@@H](C)C(C)C)CC[C@H]3[C@@H]21 IXVMHGVQKLDRKH-KNBKMWSGSA-N 0.000 description 46
- 230000014509 gene expression Effects 0.000 description 45
- IXVMHGVQKLDRKH-YEJCTVDLSA-N (22s,23s)-epibrassinolide Chemical compound C1OC(=O)[C@H]2C[C@H](O)[C@H](O)C[C@]2(C)[C@H]2CC[C@]3(C)[C@@H]([C@H](C)[C@H](O)[C@@H](O)[C@H](C)C(C)C)CC[C@H]3[C@@H]21 IXVMHGVQKLDRKH-YEJCTVDLSA-N 0.000 description 44
- 150000007523 nucleic acids Chemical class 0.000 description 43
- 235000018102 proteins Nutrition 0.000 description 35
- 102000004169 proteins and genes Human genes 0.000 description 35
- 102000039446 nucleic acids Human genes 0.000 description 30
- 108020004707 nucleic acids Proteins 0.000 description 30
- 235000001014 amino acid Nutrition 0.000 description 29
- 229940024606 amino acid Drugs 0.000 description 27
- 230000035772 mutation Effects 0.000 description 26
- 150000001413 amino acids Chemical class 0.000 description 25
- 230000001851 biosynthetic effect Effects 0.000 description 23
- 102000004190 Enzymes Human genes 0.000 description 22
- 108090000790 Enzymes Proteins 0.000 description 22
- 241000219194 Arabidopsis Species 0.000 description 21
- 210000001519 tissue Anatomy 0.000 description 21
- 230000000694 effects Effects 0.000 description 20
- 239000002299 complementary DNA Substances 0.000 description 19
- 101100364963 Arabidopsis thaliana HDF7 gene Proteins 0.000 description 18
- 230000015572 biosynthetic process Effects 0.000 description 18
- 108020004999 messenger RNA Proteins 0.000 description 18
- 239000000047 product Substances 0.000 description 18
- 108700028369 Alleles Proteins 0.000 description 17
- SGNBVLSWZMBQTH-FGAXOLDCSA-N Campesterol Natural products O[C@@H]1CC=2[C@@](C)([C@@H]3[C@H]([C@H]4[C@@](C)([C@H]([C@H](CC[C@H](C(C)C)C)C)CC4)CC3)CC=2)CC1 SGNBVLSWZMBQTH-FGAXOLDCSA-N 0.000 description 17
- BTEISVKTSQLKST-UHFFFAOYSA-N Haliclonasterol Natural products CC(C=CC(C)C(C)(C)C)C1CCC2C3=CC=C4CC(O)CCC4(C)C3CCC12C BTEISVKTSQLKST-UHFFFAOYSA-N 0.000 description 17
- 108091028043 Nucleic acid sequence Proteins 0.000 description 17
- SGNBVLSWZMBQTH-PODYLUTMSA-N campesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CC[C@@H](C)C(C)C)[C@@]1(C)CC2 SGNBVLSWZMBQTH-PODYLUTMSA-N 0.000 description 17
- 235000000431 campesterol Nutrition 0.000 description 17
- 230000006870 function Effects 0.000 description 17
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 16
- 101710133654 C-5 sterol desaturase Proteins 0.000 description 16
- 101710111046 Delta(7)-sterol 5(6)-desaturase Proteins 0.000 description 16
- 101710137448 Lathosterol oxidase Proteins 0.000 description 16
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 16
- 230000012010 growth Effects 0.000 description 16
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 15
- 230000002950 deficient Effects 0.000 description 15
- 238000009396 hybridization Methods 0.000 description 15
- 239000000543 intermediate Substances 0.000 description 15
- 238000003752 polymerase chain reaction Methods 0.000 description 15
- 238000013518 transcription Methods 0.000 description 15
- 230000035897 transcription Effects 0.000 description 15
- 230000002792 vascular Effects 0.000 description 15
- 230000002829 reductive effect Effects 0.000 description 14
- 102100037199 Lathosterol oxidase Human genes 0.000 description 13
- 230000006696 biosynthetic metabolic pathway Effects 0.000 description 13
- 230000007547 defect Effects 0.000 description 13
- 230000009467 reduction Effects 0.000 description 13
- INDVLXYUCBVVKW-RNWIMVDMSA-N 24-Methylene cholesterol Natural products O[C@@H]1CC=2[C@@](C)([C@H]3[C@H]([C@H]4[C@@](C)([C@@H]([C@@H](CCC(C(C)C)=C)C)CC4)CC3)CC=2)CC1 INDVLXYUCBVVKW-RNWIMVDMSA-N 0.000 description 12
- INDVLXYUCBVVKW-PXBBAZSNSA-N 24-methylenecholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCC(=C)C(C)C)[C@@]1(C)CC2 INDVLXYUCBVVKW-PXBBAZSNSA-N 0.000 description 12
- 206010013883 Dwarfism Diseases 0.000 description 12
- 108700024394 Exon Proteins 0.000 description 12
- 108091092195 Intron Proteins 0.000 description 12
- XVZCXCTYGHPNEM-UHFFFAOYSA-N Leu-Leu-Pro Natural products CC(C)CC(N)C(=O)NC(CC(C)C)C(=O)N1CCCC1C(O)=O XVZCXCTYGHPNEM-UHFFFAOYSA-N 0.000 description 12
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 12
- INDVLXYUCBVVKW-UHFFFAOYSA-N Methylencholesterol Natural products C1C=C2CC(O)CCC2(C)C2C1C1CCC(C(C)CCC(=C)C(C)C)C1(C)CC2 INDVLXYUCBVVKW-UHFFFAOYSA-N 0.000 description 12
- 238000004458 analytical method Methods 0.000 description 12
- 238000003556 assay Methods 0.000 description 12
- 150000001875 compounds Chemical class 0.000 description 12
- 238000012163 sequencing technique Methods 0.000 description 12
- 238000013519 translation Methods 0.000 description 12
- 230000014616 translation Effects 0.000 description 12
- 108090000994 Catalytic RNA Proteins 0.000 description 11
- 102000053642 Catalytic RNA Human genes 0.000 description 11
- 101001077420 Homo sapiens Potassium voltage-gated channel subfamily H member 7 Proteins 0.000 description 11
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 11
- AJLFOPYRIVGYMJ-UHFFFAOYSA-N SJ000287055 Natural products C12C(OC(=O)C(C)CC)CCC=C2C=CC(C)C1CCC1CC(O)CC(=O)O1 AJLFOPYRIVGYMJ-UHFFFAOYSA-N 0.000 description 11
- 239000003550 marker Substances 0.000 description 11
- AJLFOPYRIVGYMJ-INTXDZFKSA-N mevastatin Chemical compound C([C@H]1[C@@H](C)C=CC2=CCC[C@@H]([C@H]12)OC(=O)[C@@H](C)CC)C[C@@H]1C[C@@H](O)CC(=O)O1 AJLFOPYRIVGYMJ-INTXDZFKSA-N 0.000 description 11
- BOZILQFLQYBIIY-UHFFFAOYSA-N mevastatin hydroxy acid Natural products C1=CC(C)C(CCC(O)CC(O)CC(O)=O)C2C(OC(=O)C(C)CC)CCC=C21 BOZILQFLQYBIIY-UHFFFAOYSA-N 0.000 description 11
- 108091092562 ribozyme Proteins 0.000 description 11
- 239000000523 sample Substances 0.000 description 11
- VGMFHMLQOYWYHN-UHFFFAOYSA-N Compactin Natural products OCC1OC(OC2C(O)C(O)C(CO)OC2Oc3cc(O)c4C(=O)C(=COc4c3)c5ccc(O)c(O)c5)C(O)C(O)C1O VGMFHMLQOYWYHN-UHFFFAOYSA-N 0.000 description 10
- 108020004485 Nonsense Codon Proteins 0.000 description 10
- 102100025133 Potassium voltage-gated channel subfamily H member 7 Human genes 0.000 description 10
- 210000000349 chromosome Anatomy 0.000 description 10
- 230000004048 modification Effects 0.000 description 10
- 238000012986 modification Methods 0.000 description 10
- 210000000056 organ Anatomy 0.000 description 10
- XVZCXCTYGHPNEM-IHRRRGAJSA-N (2s)-1-[(2s)-2-[[(2s)-2-amino-4-methylpentanoyl]amino]-4-methylpentanoyl]pyrrolidine-2-carboxylic acid Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N1CCC[C@H]1C(O)=O XVZCXCTYGHPNEM-IHRRRGAJSA-N 0.000 description 9
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 9
- 230000037361 pathway Effects 0.000 description 9
- 230000001105 regulatory effect Effects 0.000 description 9
- KJTLQQUUPVSXIM-ZCFIWIBFSA-N (R)-mevalonic acid Chemical compound OCC[C@](O)(C)CC(O)=O KJTLQQUUPVSXIM-ZCFIWIBFSA-N 0.000 description 8
- 101150029901 CPD gene Proteins 0.000 description 8
- 108020004705 Codon Proteins 0.000 description 8
- KJTLQQUUPVSXIM-UHFFFAOYSA-N DL-mevalonic acid Natural products OCCC(O)(C)CC(O)=O KJTLQQUUPVSXIM-UHFFFAOYSA-N 0.000 description 8
- 230000000692 anti-sense effect Effects 0.000 description 8
- 230000027455 binding Effects 0.000 description 8
- 230000033228 biological regulation Effects 0.000 description 8
- 238000006243 chemical reaction Methods 0.000 description 8
- 238000006467 substitution reaction Methods 0.000 description 8
- 229930192334 Auxin Natural products 0.000 description 7
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 7
- 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 7
- 230000003321 amplification Effects 0.000 description 7
- 239000002363 auxin Substances 0.000 description 7
- 238000012217 deletion Methods 0.000 description 7
- 230000037430 deletion Effects 0.000 description 7
- 238000001514 detection method Methods 0.000 description 7
- 108010044374 isoleucyl-tyrosine Proteins 0.000 description 7
- 108010044056 leucyl-phenylalanine Proteins 0.000 description 7
- 238000002844 melting Methods 0.000 description 7
- 230000008018 melting Effects 0.000 description 7
- 238000003199 nucleic acid amplification method Methods 0.000 description 7
- 230000009466 transformation Effects 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- ARYTXMNEANMLMU-UHFFFAOYSA-N 24alpha-methylcholestanol Natural products C1CC2CC(O)CCC2(C)C2C1C1CCC(C(C)CCC(C)C(C)C)C1(C)CC2 ARYTXMNEANMLMU-UHFFFAOYSA-N 0.000 description 6
- 108020005345 3' Untranslated Regions Proteins 0.000 description 6
- 108020003589 5' Untranslated Regions Proteins 0.000 description 6
- -1 C29 sterols Chemical class 0.000 description 6
- 102000053602 DNA Human genes 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
- 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 6
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 6
- 108700019146 Transgenes Proteins 0.000 description 6
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 6
- 238000009825 accumulation Methods 0.000 description 6
- 108010070783 alanyltyrosine Proteins 0.000 description 6
- 108010047857 aspartylglycine Proteins 0.000 description 6
- ARYTXMNEANMLMU-ATEDBJNTSA-N campestanol Chemical compound C([C@@H]1CC2)[C@@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@H](C)CC[C@@H](C)C(C)C)[C@@]2(C)CC1 ARYTXMNEANMLMU-ATEDBJNTSA-N 0.000 description 6
- 238000010367 cloning Methods 0.000 description 6
- 230000007812 deficiency Effects 0.000 description 6
- 235000013399 edible fruits Nutrition 0.000 description 6
- BTCAEOLDEYPGGE-JVAZTMFWSA-N episterol Chemical compound C1[C@@H](O)CC[C@]2(C)[C@@H](CC[C@@]3([C@@H]([C@H](C)CCC(=C)C(C)C)CC[C@H]33)C)C3=CC[C@H]21 BTCAEOLDEYPGGE-JVAZTMFWSA-N 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 6
- 230000002068 genetic effect Effects 0.000 description 6
- XBGGUPMXALFZOT-UHFFFAOYSA-N glycyl-L-tyrosine hemihydrate Natural products NCC(=O)NC(C(O)=O)CC1=CC=C(O)C=C1 XBGGUPMXALFZOT-UHFFFAOYSA-N 0.000 description 6
- 125000000487 histidyl group Chemical group [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C([H])=N1 0.000 description 6
- 230000014759 maintenance of location Effects 0.000 description 6
- 230000001404 mediated effect Effects 0.000 description 6
- BTCAEOLDEYPGGE-UHFFFAOYSA-N methylene-24 cholesten-7 ol-3 beta Natural products C1C(O)CCC2(C)C(CCC3(C(C(C)CCC(=C)C(C)C)CCC33)C)C3=CCC21 BTCAEOLDEYPGGE-UHFFFAOYSA-N 0.000 description 6
- 230000000877 morphologic effect Effects 0.000 description 6
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 description 6
- 210000001938 protoplast Anatomy 0.000 description 6
- 230000008929 regeneration Effects 0.000 description 6
- 238000011069 regeneration method Methods 0.000 description 6
- 230000004044 response Effects 0.000 description 6
- 108091008146 restriction endonucleases Proteins 0.000 description 6
- YYGNTYWPHWGJRM-UHFFFAOYSA-N (6E,10E,14E,18E)-2,6,10,15,19,23-hexamethyltetracosa-2,6,10,14,18,22-hexaene Chemical compound CC(C)=CCCC(C)=CCCC(C)=CCCC=C(C)CCC=C(C)CCC=C(C)C YYGNTYWPHWGJRM-UHFFFAOYSA-N 0.000 description 5
- OKIKVSXTXVVFDV-MMWGEVLESA-N Ala-Ile-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](C)N OKIKVSXTXVVFDV-MMWGEVLESA-N 0.000 description 5
- HYIDEIQUCBKIPL-CQDKDKBSSA-N Ala-Phe-His Chemical compound C[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)N HYIDEIQUCBKIPL-CQDKDKBSSA-N 0.000 description 5
- PLUBXMRUUVWRLT-UHFFFAOYSA-N Ethyl methanesulfonate Chemical compound CCOS(C)(=O)=O PLUBXMRUUVWRLT-UHFFFAOYSA-N 0.000 description 5
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 5
- XVYKMNXXJXQKME-XEGUGMAKSA-N Gly-Ile-Tyr Chemical compound NCC(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 XVYKMNXXJXQKME-XEGUGMAKSA-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
- YRAWWKUTNBILNT-FXQIFTODSA-N Met-Ala-Ala Chemical compound CSCC[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(O)=O YRAWWKUTNBILNT-FXQIFTODSA-N 0.000 description 5
- 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 5
- 108020004711 Nucleic Acid Probes Proteins 0.000 description 5
- 102000004316 Oxidoreductases Human genes 0.000 description 5
- 108090000854 Oxidoreductases Proteins 0.000 description 5
- BHEOSNUKNHRBNM-UHFFFAOYSA-N Tetramethylsqualene Natural products CC(=C)C(C)CCC(=C)C(C)CCC(C)=CCCC=C(C)CCC(C)C(=C)CCC(C)C(C)=C BHEOSNUKNHRBNM-UHFFFAOYSA-N 0.000 description 5
- JXWGBRRVTRAZQA-ULQDDVLXSA-N Val-Tyr-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)NC(=O)[C@H](C(C)C)N JXWGBRRVTRAZQA-ULQDDVLXSA-N 0.000 description 5
- 241000700605 Viruses Species 0.000 description 5
- 230000008859 change Effects 0.000 description 5
- PRAKJMSDJKAYCZ-UHFFFAOYSA-N dodecahydrosqualene Natural products CC(C)CCCC(C)CCCC(C)CCCCC(C)CCCC(C)CCCC(C)C PRAKJMSDJKAYCZ-UHFFFAOYSA-N 0.000 description 5
- 230000007613 environmental effect Effects 0.000 description 5
- 230000002255 enzymatic effect Effects 0.000 description 5
- 150000002410 histidine derivatives Chemical class 0.000 description 5
- 238000000338 in vitro Methods 0.000 description 5
- 230000005764 inhibitory process Effects 0.000 description 5
- 238000002955 isolation Methods 0.000 description 5
- 108010034529 leucyl-lysine Proteins 0.000 description 5
- 108010057821 leucylproline Proteins 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- 239000002853 nucleic acid probe Substances 0.000 description 5
- 230000008635 plant growth Effects 0.000 description 5
- 239000013612 plasmid Substances 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 108010090894 prolylleucine Proteins 0.000 description 5
- 230000010076 replication Effects 0.000 description 5
- 229940031439 squalene Drugs 0.000 description 5
- TUHBEKDERLKLEC-UHFFFAOYSA-N squalene Natural products CC(=CCCC(=CCCC(=CCCC=C(/C)CCC=C(/C)CC=C(C)C)C)C)C TUHBEKDERLKLEC-UHFFFAOYSA-N 0.000 description 5
- 238000011144 upstream manufacturing Methods 0.000 description 5
- 241000589158 Agrobacterium Species 0.000 description 4
- 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 4
- 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 4
- HOVPGJUNRLMIOZ-CIUDSAMLSA-N Ala-Ser-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@H](C)N HOVPGJUNRLMIOZ-CIUDSAMLSA-N 0.000 description 4
- 101100165817 Arabidopsis thaliana CYP90B1 gene Proteins 0.000 description 4
- LLUGJARLJCGLAR-CYDGBPFRSA-N Arg-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](CCCN=C(N)N)N LLUGJARLJCGLAR-CYDGBPFRSA-N 0.000 description 4
- DATSKXOXPUAOLK-KKUMJFAQSA-N Asn-Tyr-Leu Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(C)C)C(O)=O DATSKXOXPUAOLK-KKUMJFAQSA-N 0.000 description 4
- YNQIDCRRTWGHJD-ZLUOBGJFSA-N Asp-Asn-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](N)CC(O)=O YNQIDCRRTWGHJD-ZLUOBGJFSA-N 0.000 description 4
- XDGBFDYXZCMYEX-NUMRIWBASA-N Asp-Glu-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CC(=O)O)N)O XDGBFDYXZCMYEX-NUMRIWBASA-N 0.000 description 4
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- 108010015742 Cytochrome P-450 Enzyme System Proteins 0.000 description 4
- 102000002004 Cytochrome P-450 Enzyme System Human genes 0.000 description 4
- 208000034454 F12-related hereditary angioedema with normal C1Inh Diseases 0.000 description 4
- QFXNFFZTMFHPST-DZKIICNBSA-N Gln-Phe-Val Chemical compound CC(C)[C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)NC(=O)[C@H](CCC(=O)N)N QFXNFFZTMFHPST-DZKIICNBSA-N 0.000 description 4
- SYWCGQOIIARSIX-SRVKXCTJSA-N Glu-Pro-Leu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(C)C)C(O)=O SYWCGQOIIARSIX-SRVKXCTJSA-N 0.000 description 4
- 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 4
- ZZWUYQXMIFTIIY-WEDXCCLWSA-N Gly-Thr-Leu Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(O)=O ZZWUYQXMIFTIIY-WEDXCCLWSA-N 0.000 description 4
- 239000004471 Glycine Substances 0.000 description 4
- 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 4
- UIEZQYNXCYHMQS-BJDJZHNGSA-N Ile-Lys-Ala Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)O)N UIEZQYNXCYHMQS-BJDJZHNGSA-N 0.000 description 4
- ZLFNNVATRMCAKN-ZKWXMUAHSA-N Ile-Ser-Gly Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)NCC(=O)O)N ZLFNNVATRMCAKN-ZKWXMUAHSA-N 0.000 description 4
- RQZFWBLDTBDEOF-RNJOBUHISA-N Ile-Val-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C(C)C)C(=O)N1CCC[C@@H]1C(=O)O)N RQZFWBLDTBDEOF-RNJOBUHISA-N 0.000 description 4
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 4
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 4
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 4
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 4
- CIVKXGPFXDIQBV-WDCWCFNPSA-N Leu-Gln-Thr Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O CIVKXGPFXDIQBV-WDCWCFNPSA-N 0.000 description 4
- ADJWHHZETYAAAX-SRVKXCTJSA-N Leu-Ser-His Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N ADJWHHZETYAAAX-SRVKXCTJSA-N 0.000 description 4
- SBANPBVRHYIMRR-UHFFFAOYSA-N Leu-Ser-Pro Natural products CC(C)CC(N)C(=O)NC(CO)C(=O)N1CCCC1C(O)=O SBANPBVRHYIMRR-UHFFFAOYSA-N 0.000 description 4
- RDFIVFHPOSOXMW-ACRUOGEOSA-N Leu-Tyr-Phe Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O RDFIVFHPOSOXMW-ACRUOGEOSA-N 0.000 description 4
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 4
- HQPWNHXERZCIHP-PMVMPFDFSA-N Phe-Leu-Trp Chemical compound C([C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(O)=O)C1=CC=CC=C1 HQPWNHXERZCIHP-PMVMPFDFSA-N 0.000 description 4
- RTUWVJVJSMOGPL-KKUMJFAQSA-N Phe-Met-Glu Chemical compound CSCC[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)N RTUWVJVJSMOGPL-KKUMJFAQSA-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
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 4
- XQHGISDMVBTGAL-ULQDDVLXSA-N Pro-His-Phe Chemical compound C([C@@H](C(=O)[O-])NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H]1[NH2+]CCC1)C1=CC=CC=C1 XQHGISDMVBTGAL-ULQDDVLXSA-N 0.000 description 4
- 108020004511 Recombinant DNA Proteins 0.000 description 4
- 101150059968 STE1 gene Proteins 0.000 description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 4
- AIISTODACBDQLW-WDSOQIARSA-N Trp-Leu-Arg Chemical compound C1=CC=C2C(C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O)=CNC2=C1 AIISTODACBDQLW-WDSOQIARSA-N 0.000 description 4
- DAOREBHZAKCOEN-ULQDDVLXSA-N Tyr-Leu-Met 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](CCSC)C(O)=O DAOREBHZAKCOEN-ULQDDVLXSA-N 0.000 description 4
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 4
- 239000011543 agarose gel Substances 0.000 description 4
- 230000004075 alteration Effects 0.000 description 4
- 125000000539 amino acid group Chemical group 0.000 description 4
- 108010038633 aspartylglutamate Proteins 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 4
- 230000003115 biocidal effect Effects 0.000 description 4
- 230000001419 dependent effect Effects 0.000 description 4
- 238000013461 design Methods 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 238000004520 electroporation Methods 0.000 description 4
- 239000003623 enhancer Substances 0.000 description 4
- 231100000502 fertility decrease Toxicity 0.000 description 4
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 description 4
- 108010081551 glycylphenylalanine Proteins 0.000 description 4
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 4
- 208000016861 hereditary angioedema type 3 Diseases 0.000 description 4
- 239000005556 hormone Substances 0.000 description 4
- 229940088597 hormone Drugs 0.000 description 4
- 238000005805 hydroxylation reaction Methods 0.000 description 4
- SEOVTRFCIGRIMH-UHFFFAOYSA-N indole-3-acetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CNC2=C1 SEOVTRFCIGRIMH-UHFFFAOYSA-N 0.000 description 4
- 230000002401 inhibitory effect Effects 0.000 description 4
- 108010009298 lysylglutamic acid Proteins 0.000 description 4
- 108010054155 lysyllysine Proteins 0.000 description 4
- 238000013507 mapping Methods 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- 229910052751 metal Inorganic materials 0.000 description 4
- 239000002184 metal Substances 0.000 description 4
- 230000008520 organization Effects 0.000 description 4
- 230000000243 photosynthetic effect Effects 0.000 description 4
- 108010015796 prolylisoleucine Proteins 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 238000012216 screening Methods 0.000 description 4
- 238000002741 site-directed mutagenesis Methods 0.000 description 4
- 230000001629 suppression Effects 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 4
- 238000012546 transfer Methods 0.000 description 4
- 108010035534 tyrosyl-leucyl-alanine Proteins 0.000 description 4
- 230000017260 vegetative to reproductive phase transition of meristem Effects 0.000 description 4
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 3
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 3
- 241000589155 Agrobacterium tumefaciens Species 0.000 description 3
- PCIFXPRIFWKWLK-YUMQZZPRSA-N Ala-Gly-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)CNC(=O)[C@H](C)N PCIFXPRIFWKWLK-YUMQZZPRSA-N 0.000 description 3
- 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 3
- GKAZXNDATBWNBI-DCAQKATOSA-N Ala-Met-Lys Chemical compound C[C@@H](C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCCN)C(=O)O)N GKAZXNDATBWNBI-DCAQKATOSA-N 0.000 description 3
- DEWWPUNXRNGMQN-LPEHRKFASA-N Ala-Met-Pro Chemical compound C[C@@H](C(=O)N[C@@H](CCSC)C(=O)N1CCC[C@@H]1C(=O)O)N DEWWPUNXRNGMQN-LPEHRKFASA-N 0.000 description 3
- OVVUNXXROOFSIM-SDDRHHMPSA-N Arg-Arg-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CCCN=C(N)N)N)C(=O)O OVVUNXXROOFSIM-SDDRHHMPSA-N 0.000 description 3
- NKBQZKVMKJJDLX-SRVKXCTJSA-N Arg-Glu-Leu Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O NKBQZKVMKJJDLX-SRVKXCTJSA-N 0.000 description 3
- GOKCTAJWRPSCHP-VHWLVUOQSA-N Asn-Ile-Trp Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)O)NC(=O)[C@H](CC(=O)N)N GOKCTAJWRPSCHP-VHWLVUOQSA-N 0.000 description 3
- HNXWVVHIGTZTBO-LKXGYXEUSA-N Asn-Ser-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O HNXWVVHIGTZTBO-LKXGYXEUSA-N 0.000 description 3
- 241000701489 Cauliflower mosaic virus Species 0.000 description 3
- UIKLEGZPIOXFHJ-DLOVCJGASA-N Cys-Phe-Ala Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](C)C(O)=O UIKLEGZPIOXFHJ-DLOVCJGASA-N 0.000 description 3
- JEKIARHEWURQRJ-BZSNNMDCSA-N Cys-Phe-Tyr Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CC2=CC=C(C=C2)O)C(=O)O)NC(=O)[C@H](CS)N JEKIARHEWURQRJ-BZSNNMDCSA-N 0.000 description 3
- 101150066284 DET2 gene Proteins 0.000 description 3
- 102000052510 DNA-Binding Proteins Human genes 0.000 description 3
- 108700020911 DNA-Binding Proteins Proteins 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 108091029865 Exogenous DNA Proteins 0.000 description 3
- 108700028146 Genetic Enhancer Elements Proteins 0.000 description 3
- JSYULGSPLTZDHM-NRPADANISA-N Gln-Ala-Val Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](C(C)C)C(O)=O JSYULGSPLTZDHM-NRPADANISA-N 0.000 description 3
- GMGKDVVBSVVKCT-NUMRIWBASA-N Gln-Asn-Thr Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O GMGKDVVBSVVKCT-NUMRIWBASA-N 0.000 description 3
- DFRYZTUPVZNRLG-KKUMJFAQSA-N Gln-Met-Phe Chemical compound CSCC[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)NC(=O)[C@H](CCC(=O)N)N DFRYZTUPVZNRLG-KKUMJFAQSA-N 0.000 description 3
- YRMZCZIRHYCNHX-RYUDHWBXSA-N Glu-Phe-Gly Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)NCC(O)=O YRMZCZIRHYCNHX-RYUDHWBXSA-N 0.000 description 3
- GUOWMVFLAJNPDY-CIUDSAMLSA-N Glu-Ser-Met Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCSC)C(O)=O GUOWMVFLAJNPDY-CIUDSAMLSA-N 0.000 description 3
- UHPAZODVFFYEEL-QWRGUYRKSA-N Gly-Leu-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)CN UHPAZODVFFYEEL-QWRGUYRKSA-N 0.000 description 3
- AWASVTXPTOLPPP-MBLNEYKQSA-N His-Ala-Thr Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O AWASVTXPTOLPPP-MBLNEYKQSA-N 0.000 description 3
- ZJSMFRTVYSLKQU-DJFWLOJKSA-N His-Asp-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CC1=CN=CN1)N ZJSMFRTVYSLKQU-DJFWLOJKSA-N 0.000 description 3
- SYIPVNMWBZXKMU-HJPIBITLSA-N His-His-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC1=CN=CN1)NC(=O)[C@H](CC2=CN=CN2)N SYIPVNMWBZXKMU-HJPIBITLSA-N 0.000 description 3
- QLBXWYXMLHAREM-PYJNHQTQSA-N His-Val-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC1=CN=CN1)N QLBXWYXMLHAREM-PYJNHQTQSA-N 0.000 description 3
- 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 3
- JDAWAWXGAUZPNJ-ZPFDUUQYSA-N Ile-Glu-Arg Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)N JDAWAWXGAUZPNJ-ZPFDUUQYSA-N 0.000 description 3
- UQXADIGYEYBJEI-DJFWLOJKSA-N Ile-His-Asp Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)N[C@@H](CC(=O)O)C(=O)O)N UQXADIGYEYBJEI-DJFWLOJKSA-N 0.000 description 3
- UASTVUQJMLZWGG-PEXQALLHSA-N Ile-His-Gly Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)NCC(=O)O)N UASTVUQJMLZWGG-PEXQALLHSA-N 0.000 description 3
- CCYGNFBYUNHFSC-MGHWNKPDSA-N Ile-His-Phe Chemical compound [H]N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O CCYGNFBYUNHFSC-MGHWNKPDSA-N 0.000 description 3
- NSPNUMNLZNOPAQ-SJWGOKEGSA-N Ile-Tyr-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N2CCC[C@@H]2C(=O)O)N NSPNUMNLZNOPAQ-SJWGOKEGSA-N 0.000 description 3
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 3
- HGCNKOLVKRAVHD-UHFFFAOYSA-N L-Met-L-Phe Natural products CSCCC(N)C(=O)NC(C(O)=O)CC1=CC=CC=C1 HGCNKOLVKRAVHD-UHFFFAOYSA-N 0.000 description 3
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 3
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 3
- LHSGPCFBGJHPCY-UHFFFAOYSA-N L-leucine-L-tyrosine Natural products CC(C)CC(N)C(=O)NC(C(O)=O)CC1=CC=C(O)C=C1 LHSGPCFBGJHPCY-UHFFFAOYSA-N 0.000 description 3
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 3
- POJPZSMTTMLSTG-SRVKXCTJSA-N Leu-Asn-Lys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CCCCN)C(=O)O)N POJPZSMTTMLSTG-SRVKXCTJSA-N 0.000 description 3
- UCNNZELZXFXXJQ-BZSNNMDCSA-N Leu-Leu-Tyr Chemical compound CC(C)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 UCNNZELZXFXXJQ-BZSNNMDCSA-N 0.000 description 3
- SBANPBVRHYIMRR-GARJFASQSA-N Leu-Ser-Pro Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CO)C(=O)N1CCC[C@@H]1C(=O)O)N SBANPBVRHYIMRR-GARJFASQSA-N 0.000 description 3
- XOEDPXDZJHBQIX-ULQDDVLXSA-N Leu-Val-Phe Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 XOEDPXDZJHBQIX-ULQDDVLXSA-N 0.000 description 3
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 3
- 241000227653 Lycopersicon Species 0.000 description 3
- KYNNSEJZFVCDIV-ZPFDUUQYSA-N Lys-Ile-Asn Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(N)=O)C(O)=O KYNNSEJZFVCDIV-ZPFDUUQYSA-N 0.000 description 3
- WDTLNWHPIPCMMP-AVGNSLFASA-N Met-Arg-Leu Chemical compound [H]N[C@@H](CCSC)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(O)=O WDTLNWHPIPCMMP-AVGNSLFASA-N 0.000 description 3
- OSOLWRWQADPDIQ-DCAQKATOSA-N Met-Asp-Leu Chemical compound [H]N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O OSOLWRWQADPDIQ-DCAQKATOSA-N 0.000 description 3
- 101001034830 Mus musculus Interferon-induced transmembrane protein 5 Proteins 0.000 description 3
- 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 3
- 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 3
- 238000000636 Northern blotting Methods 0.000 description 3
- 241000209094 Oryza Species 0.000 description 3
- GAMLAXHLYGLQBJ-UFYCRDLUSA-N Phe-Val-Tyr Chemical compound N[C@H](C(=O)N[C@H](C(=O)N[C@H](C(=O)O)CC1=CC=C(C=C1)O)C(C)C)CC1=CC=CC=C1 GAMLAXHLYGLQBJ-UFYCRDLUSA-N 0.000 description 3
- 235000008331 Pinus X rigitaeda Nutrition 0.000 description 3
- 241000018646 Pinus brutia Species 0.000 description 3
- 235000011613 Pinus brutia Nutrition 0.000 description 3
- 239000002202 Polyethylene glycol Substances 0.000 description 3
- ZLXKLMHAMDENIO-DCAQKATOSA-N Pro-Lys-Asp Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(O)=O ZLXKLMHAMDENIO-DCAQKATOSA-N 0.000 description 3
- OOZJHTXCLJUODH-QXEWZRGKSA-N Pro-Val-Asp Chemical compound OC(=O)C[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H]1CCCN1 OOZJHTXCLJUODH-QXEWZRGKSA-N 0.000 description 3
- 241000235347 Schizosaccharomyces pombe Species 0.000 description 3
- BKZYBLLIBOBOOW-GHCJXIJMSA-N Ser-Ile-Asp Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(O)=O)C(O)=O BKZYBLLIBOBOOW-GHCJXIJMSA-N 0.000 description 3
- XVWDJUROVRQKAE-KKUMJFAQSA-N Ser-Phe-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)CO)CC1=CC=CC=C1 XVWDJUROVRQKAE-KKUMJFAQSA-N 0.000 description 3
- 244000062793 Sorghum vulgare Species 0.000 description 3
- 108700026226 TATA Box Proteins 0.000 description 3
- 108010006785 Taq Polymerase Proteins 0.000 description 3
- DFTCYYILCSQGIZ-GCJQMDKQSA-N Thr-Ala-Asn Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(N)=O)C(O)=O DFTCYYILCSQGIZ-GCJQMDKQSA-N 0.000 description 3
- VGNLMPBYWWNQFS-ZEILLAHLSA-N Thr-Thr-His Chemical compound C[C@H]([C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N)O VGNLMPBYWWNQFS-ZEILLAHLSA-N 0.000 description 3
- MNYNCKZAEIAONY-XGEHTFHBSA-N Thr-Val-Ser Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(O)=O MNYNCKZAEIAONY-XGEHTFHBSA-N 0.000 description 3
- KULBQAVOXHQLIY-HSCHXYMDSA-N Trp-Ile-Leu Chemical compound C1=CC=C2C(C[C@H](N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(C)C)C(O)=O)=CNC2=C1 KULBQAVOXHQLIY-HSCHXYMDSA-N 0.000 description 3
- KCZGSXPFPNKGLE-WDSOQIARSA-N Trp-Met-His Chemical compound CSCC[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@H](CC2=CNC3=CC=CC=C32)N KCZGSXPFPNKGLE-WDSOQIARSA-N 0.000 description 3
- DYIXEGROAOVQPK-VFAJRCTISA-N Trp-Thr-Lys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)N)O DYIXEGROAOVQPK-VFAJRCTISA-N 0.000 description 3
- YTHWAWACWGWBLE-MNSWYVGCSA-N Trp-Tyr-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=C(O)C=C1 YTHWAWACWGWBLE-MNSWYVGCSA-N 0.000 description 3
- 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 3
- HZDQUVQEVVYDDA-ACRUOGEOSA-N Tyr-Tyr-Leu Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(O)=O)NC(=O)[C@@H](N)CC=1C=CC(O)=CC=1)C1=CC=C(O)C=C1 HZDQUVQEVVYDDA-ACRUOGEOSA-N 0.000 description 3
- FOADDSDHGRFUOC-DZKIICNBSA-N Val-Glu-Phe Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)N FOADDSDHGRFUOC-DZKIICNBSA-N 0.000 description 3
- LYERIXUFCYVFFX-GVXVVHGQSA-N Val-Leu-Glu Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)O)NC(=O)[C@H](C(C)C)N LYERIXUFCYVFFX-GVXVVHGQSA-N 0.000 description 3
- 108010043240 arginyl-leucyl-glycine Proteins 0.000 description 3
- 108010092854 aspartyllysine Proteins 0.000 description 3
- 230000002520 cambial effect Effects 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 230000007423 decrease Effects 0.000 description 3
- 230000023753 dehiscence Effects 0.000 description 3
- 239000012153 distilled water Substances 0.000 description 3
- 230000002708 enhancing effect Effects 0.000 description 3
- 230000001747 exhibiting effect Effects 0.000 description 3
- 108020001507 fusion proteins Proteins 0.000 description 3
- 102000037865 fusion proteins Human genes 0.000 description 3
- 108010010147 glycylglutamine Proteins 0.000 description 3
- 108010084389 glycyltryptophan Proteins 0.000 description 3
- 230000002363 herbicidal effect Effects 0.000 description 3
- 239000004009 herbicide Substances 0.000 description 3
- 108010028295 histidylhistidine Proteins 0.000 description 3
- 108010085325 histidylproline Proteins 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 230000001939 inductive effect Effects 0.000 description 3
- 229960000310 isoleucine Drugs 0.000 description 3
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 3
- 108010012058 leucyltyrosine Proteins 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 150000002739 metals Chemical class 0.000 description 3
- 108010068488 methionylphenylalanine Proteins 0.000 description 3
- 238000010369 molecular cloning Methods 0.000 description 3
- 238000002887 multiple sequence alignment Methods 0.000 description 3
- 230000003647 oxidation Effects 0.000 description 3
- 238000007254 oxidation reaction Methods 0.000 description 3
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 3
- 108010073025 phenylalanylphenylalanine Proteins 0.000 description 3
- 230000008488 polyadenylation Effects 0.000 description 3
- 229920001223 polyethylene glycol Polymers 0.000 description 3
- 102000054765 polymorphisms of proteins Human genes 0.000 description 3
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 3
- 230000002035 prolonged effect Effects 0.000 description 3
- 238000005215 recombination Methods 0.000 description 3
- 230000006798 recombination Effects 0.000 description 3
- 230000002441 reversible effect Effects 0.000 description 3
- 230000001568 sexual effect Effects 0.000 description 3
- 241000894007 species Species 0.000 description 3
- 150000003431 steroids Chemical class 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 230000005026 transcription initiation Effects 0.000 description 3
- 230000002103 transcriptional effect Effects 0.000 description 3
- 230000001131 transforming effect Effects 0.000 description 3
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 3
- 108010051110 tyrosyl-lysine Proteins 0.000 description 3
- 239000004474 valine Substances 0.000 description 3
- 230000003612 virological effect Effects 0.000 description 3
- QYIMSPSDBYKPPY-RSKUXYSASA-N (S)-2,3-epoxysqualene Chemical compound CC(C)=CCC\C(C)=C\CC\C(C)=C\CC\C=C(/C)CC\C=C(/C)CC[C@@H]1OC1(C)C QYIMSPSDBYKPPY-RSKUXYSASA-N 0.000 description 2
- 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 2
- XZEUYTKSAYNYPK-UHFFFAOYSA-N 3beta-29-Norcycloart-24-en-3-ol Natural products C1CC2(C)C(C(CCC=C(C)C)C)CCC2(C)C2CCC3C(C)C(O)CCC33C21C3 XZEUYTKSAYNYPK-UHFFFAOYSA-N 0.000 description 2
- WPHVOXMMNSLJSF-PDQCMOIVSA-N 6-deoxotyphasterol Natural products O[C@@H]([C@H](O)[C@@H](C)[C@@H]1[C@@]2(C)[C@@H]([C@H]3[C@H]([C@@]4(C)[C@H](C[C@H](O)CC4)CC3)CC2)CC1)[C@H](C(C)C)C WPHVOXMMNSLJSF-PDQCMOIVSA-N 0.000 description 2
- 229930024421 Adenine Natural products 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 2
- LZRNYBIJOSKKRJ-XVYDVKMFSA-N Ala-Asp-His Chemical compound C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N LZRNYBIJOSKKRJ-XVYDVKMFSA-N 0.000 description 2
- ZPXCNXMJEZKRLU-LSJOCFKGSA-N Ala-His-Arg Chemical compound NC(N)=NCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)C)CC1=CN=CN1 ZPXCNXMJEZKRLU-LSJOCFKGSA-N 0.000 description 2
- DPNZTBKGAUAZQU-DLOVCJGASA-N Ala-Leu-His Chemical compound C[C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N DPNZTBKGAUAZQU-DLOVCJGASA-N 0.000 description 2
- 108020005544 Antisense RNA Proteins 0.000 description 2
- USNSOPDIZILSJP-FXQIFTODSA-N Arg-Asn-Asn Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O USNSOPDIZILSJP-FXQIFTODSA-N 0.000 description 2
- MFAMTAVAFBPXDC-LPEHRKFASA-N Arg-Asp-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC(=O)O)NC(=O)[C@H](CCCN=C(N)N)N)C(=O)O MFAMTAVAFBPXDC-LPEHRKFASA-N 0.000 description 2
- CLICCYPMVFGUOF-IHRRRGAJSA-N Arg-Lys-Leu Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(O)=O CLICCYPMVFGUOF-IHRRRGAJSA-N 0.000 description 2
- 239000004475 Arginine Substances 0.000 description 2
- FTSAJSADJCMDHH-CIUDSAMLSA-N Asn-Lys-Asp Chemical compound C(CCN)C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)O)NC(=O)[C@H](CC(=O)N)N FTSAJSADJCMDHH-CIUDSAMLSA-N 0.000 description 2
- YSYTWUMRHSFODC-QWRGUYRKSA-N Asn-Tyr-Gly Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)NCC(O)=O YSYTWUMRHSFODC-QWRGUYRKSA-N 0.000 description 2
- IXIWEFWRKIUMQX-DCAQKATOSA-N Asp-Arg-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@@H](N)CC(O)=O IXIWEFWRKIUMQX-DCAQKATOSA-N 0.000 description 2
- HAFCJCDJGIOYPW-WDSKDSINSA-N Asp-Gly-Gln Chemical compound OC(=O)C[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCC(N)=O HAFCJCDJGIOYPW-WDSKDSINSA-N 0.000 description 2
- GWOVSEVNXNVMMY-BPUTZDHNSA-N Asp-Trp-Met Chemical compound CSCC[C@@H](C(=O)O)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)NC(=O)[C@H](CC(=O)O)N GWOVSEVNXNVMMY-BPUTZDHNSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 244000197813 Camelina sativa Species 0.000 description 2
- 101710132601 Capsid protein Proteins 0.000 description 2
- 101710094648 Coat protein Proteins 0.000 description 2
- 108020004635 Complementary DNA Proteins 0.000 description 2
- 108091035707 Consensus sequence Proteins 0.000 description 2
- RRTBTJPVUGMUNR-UHFFFAOYSA-N Cycloartanol Natural products C12CCC(C(C(O)CC3)(C)C)C3C2(CC)CCC2(C)C1(C)CCC2C(C)CCCC(C)C RRTBTJPVUGMUNR-UHFFFAOYSA-N 0.000 description 2
- 108010076161 Cycloartenol synthase Proteins 0.000 description 2
- 108010017826 DNA Polymerase I Proteins 0.000 description 2
- 102000004594 DNA Polymerase I Human genes 0.000 description 2
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 2
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- PPFRJNLKWADOTL-SSIUHVNYSA-N Dolicholide Chemical compound C[C@@H]([C@H]1CCC2C1(CCC3C2COC(=O)C4C3(C[C@H]([C@H](C4)O)O)C)C)[C@H]([C@@H](C(=C)C(C)C)O)O PPFRJNLKWADOTL-SSIUHVNYSA-N 0.000 description 2
- 108700039964 Duplicate Genes Proteins 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 101150103048 ERG3 gene Proteins 0.000 description 2
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 2
- 241000233866 Fungi Species 0.000 description 2
- 229930191978 Gibberellin Natural products 0.000 description 2
- DRNMNLKUUKKPIA-HTUGSXCWSA-N Gln-Phe-Thr Chemical compound C[C@@H](O)[C@H](NC(=O)[C@H](Cc1ccccc1)NC(=O)[C@@H](N)CCC(N)=O)C(O)=O DRNMNLKUUKKPIA-HTUGSXCWSA-N 0.000 description 2
- JPHYJQHPILOKHC-ACZMJKKPSA-N Glu-Asp-Asp Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O JPHYJQHPILOKHC-ACZMJKKPSA-N 0.000 description 2
- LZEUDRYSAZAJIO-AUTRQRHGSA-N Glu-Val-Glu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O LZEUDRYSAZAJIO-AUTRQRHGSA-N 0.000 description 2
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 2
- UGVQELHRNUDMAA-BYPYZUCNSA-N Gly-Ala-Gly Chemical compound [NH3+]CC(=O)N[C@@H](C)C(=O)NCC([O-])=O UGVQELHRNUDMAA-BYPYZUCNSA-N 0.000 description 2
- FUTAPPOITCCWTH-WHFBIAKZSA-N Gly-Asp-Asp Chemical compound [H]NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O FUTAPPOITCCWTH-WHFBIAKZSA-N 0.000 description 2
- YWAQATDNEKZFFK-BYPYZUCNSA-N Gly-Gly-Ser Chemical compound NCC(=O)NCC(=O)N[C@@H](CO)C(O)=O YWAQATDNEKZFFK-BYPYZUCNSA-N 0.000 description 2
- YNIMVVJTPWCUJH-KBPBESRZSA-N Gly-His-Tyr Chemical compound [H]NCC(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O YNIMVVJTPWCUJH-KBPBESRZSA-N 0.000 description 2
- DNAZKGFYFRGZIH-QWRGUYRKSA-N Gly-Tyr-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CC1=CC=C(O)C=C1 DNAZKGFYFRGZIH-QWRGUYRKSA-N 0.000 description 2
- 244000068988 Glycine max Species 0.000 description 2
- 235000010469 Glycine max Nutrition 0.000 description 2
- 102100021181 Golgi phosphoprotein 3 Human genes 0.000 description 2
- VOKCBYNCZVSILJ-KKUMJFAQSA-N His-Asn-Tyr Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CC2=CN=CN2)N)O VOKCBYNCZVSILJ-KKUMJFAQSA-N 0.000 description 2
- PMWSGVRIMIFXQH-KKUMJFAQSA-N His-His-Leu Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(O)=O)NC(=O)[C@@H](N)CC=1NC=NC=1)C1=CN=CN1 PMWSGVRIMIFXQH-KKUMJFAQSA-N 0.000 description 2
- CNHSMSFYVARZLI-YJRXYDGGSA-N His-His-Thr Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H]([C@@H](C)O)C(O)=O CNHSMSFYVARZLI-YJRXYDGGSA-N 0.000 description 2
- MVZASEMJYJPJSI-IHPCNDPISA-N His-Lys-Trp Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC3=CN=CN3)N MVZASEMJYJPJSI-IHPCNDPISA-N 0.000 description 2
- VXZZUXWAOMWWJH-QTKMDUPCSA-N His-Thr-Val Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(O)=O VXZZUXWAOMWWJH-QTKMDUPCSA-N 0.000 description 2
- 206010020649 Hyperkeratosis Diseases 0.000 description 2
- GLYJPWIRLBAIJH-UHFFFAOYSA-N Ile-Lys-Pro Natural products CCC(C)C(N)C(=O)NC(CCCCN)C(=O)N1CCCC1C(O)=O GLYJPWIRLBAIJH-UHFFFAOYSA-N 0.000 description 2
- SIKJAQJRHWYJAI-UHFFFAOYSA-N Indole Chemical compound C1=CC=C2NC=CC2=C1 SIKJAQJRHWYJAI-UHFFFAOYSA-N 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- HVXLSFNCWWWDPA-UHFFFAOYSA-N Isocycloartenol Natural products C1CC(O)C(C)(C)C2C31CC13CCC3(C)C(C(CCCC(C)=C)C)CCC3(C)C1CC2 HVXLSFNCWWWDPA-UHFFFAOYSA-N 0.000 description 2
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 2
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 2
- KUIDCYNIEJBZBU-AJNGGQMLSA-N Leu-Ile-Leu Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(C)C)C(O)=O KUIDCYNIEJBZBU-AJNGGQMLSA-N 0.000 description 2
- QNBVTHNJGCOVFA-AVGNSLFASA-N Leu-Leu-Glu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CCC(O)=O QNBVTHNJGCOVFA-AVGNSLFASA-N 0.000 description 2
- WXUOJXIGOPMDJM-SRVKXCTJSA-N Leu-Lys-Asn Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(O)=O WXUOJXIGOPMDJM-SRVKXCTJSA-N 0.000 description 2
- ZAVCJRJOQKIOJW-KKUMJFAQSA-N Leu-Phe-Asp Chemical compound CC(C)C[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](CC(O)=O)C(O)=O)CC1=CC=CC=C1 ZAVCJRJOQKIOJW-KKUMJFAQSA-N 0.000 description 2
- DRWMRVFCKKXHCH-BZSNNMDCSA-N Leu-Phe-Leu Chemical compound CC(C)C[C@H]([NH3+])C(=O)N[C@H](C(=O)N[C@@H](CC(C)C)C([O-])=O)CC1=CC=CC=C1 DRWMRVFCKKXHCH-BZSNNMDCSA-N 0.000 description 2
- YWKNKRAKOCLOLH-OEAJRASXSA-N Leu-Phe-Thr Chemical compound CC(C)C[C@H](N)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)O)C(O)=O)CC1=CC=CC=C1 YWKNKRAKOCLOLH-OEAJRASXSA-N 0.000 description 2
- TUIOUEWKFFVNLH-DCAQKATOSA-N Leu-Val-Cys Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CS)C(O)=O TUIOUEWKFFVNLH-DCAQKATOSA-N 0.000 description 2
- 108060001084 Luciferase Proteins 0.000 description 2
- 239000005089 Luciferase Substances 0.000 description 2
- 235000007688 Lycopersicon esculentum Nutrition 0.000 description 2
- PNPYKQFJGRFYJE-GUBZILKMSA-N Lys-Ala-Glu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(O)=O PNPYKQFJGRFYJE-GUBZILKMSA-N 0.000 description 2
- YKIRNDPUWONXQN-GUBZILKMSA-N Lys-Asn-Gln Chemical compound C(CCN)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N YKIRNDPUWONXQN-GUBZILKMSA-N 0.000 description 2
- IUWMQCZOTYRXPL-ZPFDUUQYSA-N Lys-Ile-Asp Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(O)=O)C(O)=O IUWMQCZOTYRXPL-ZPFDUUQYSA-N 0.000 description 2
- RIJCHEVHFWMDKD-SRVKXCTJSA-N Lys-Lys-Asn Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(O)=O RIJCHEVHFWMDKD-SRVKXCTJSA-N 0.000 description 2
- MSSABBQOBUZFKZ-IHRRRGAJSA-N Lys-Pro-His Chemical compound C1C[C@H](N(C1)C(=O)[C@H](CCCCN)N)C(=O)N[C@@H](CC2=CN=CN2)C(=O)O MSSABBQOBUZFKZ-IHRRRGAJSA-N 0.000 description 2
- HYSVGEAWTGPMOA-IHRRRGAJSA-N Lys-Pro-Leu Chemical compound [H]N[C@@H](CCCCN)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(C)C)C(O)=O HYSVGEAWTGPMOA-IHRRRGAJSA-N 0.000 description 2
- 239000004472 Lysine Substances 0.000 description 2
- 101710125418 Major capsid protein Proteins 0.000 description 2
- 241000220225 Malus Species 0.000 description 2
- NLHSFJQUHGCWSD-PYJNHQTQSA-N Met-Ile-His Chemical compound N[C@@H](CCSC)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC1=CNC=N1)C(O)=O NLHSFJQUHGCWSD-PYJNHQTQSA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 108060004795 Methyltransferase Proteins 0.000 description 2
- 102000016397 Methyltransferase Human genes 0.000 description 2
- 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 2
- XMBSYZWANAQXEV-UHFFFAOYSA-N N-alpha-L-glutamyl-L-phenylalanine Natural products OC(=O)CCC(N)C(=O)NC(C(O)=O)CC1=CC=CC=C1 XMBSYZWANAQXEV-UHFFFAOYSA-N 0.000 description 2
- 101710163270 Nuclease Proteins 0.000 description 2
- 101710141454 Nucleoprotein Proteins 0.000 description 2
- 108091034117 Oligonucleotide Proteins 0.000 description 2
- 108020005187 Oligonucleotide Probes Proteins 0.000 description 2
- 108700026244 Open Reading Frames Proteins 0.000 description 2
- 235000007164 Oryza sativa Nutrition 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- OXUMFAOVGFODPN-KKUMJFAQSA-N Phe-Asn-His Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)N OXUMFAOVGFODPN-KKUMJFAQSA-N 0.000 description 2
- BIYWZVCPZIFGPY-QWRGUYRKSA-N Phe-Gly-Ser Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)NCC(=O)N[C@@H](CO)C(O)=O BIYWZVCPZIFGPY-QWRGUYRKSA-N 0.000 description 2
- BEEVXUYVEHXWRQ-YESZJQIVSA-N Phe-His-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC2=CN=CN2)NC(=O)[C@H](CC3=CC=CC=C3)N)C(=O)O BEEVXUYVEHXWRQ-YESZJQIVSA-N 0.000 description 2
- KZRQONDKKJCAOL-DKIMLUQUSA-N Phe-Leu-Ile Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O KZRQONDKKJCAOL-DKIMLUQUSA-N 0.000 description 2
- YCCUXNNKXDGMAM-KKUMJFAQSA-N Phe-Leu-Ser Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O YCCUXNNKXDGMAM-KKUMJFAQSA-N 0.000 description 2
- BPIMVBKDLSBKIJ-FCLVOEFKSA-N Phe-Thr-Phe Chemical compound C([C@H](N)C(=O)N[C@@H]([C@H](O)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 BPIMVBKDLSBKIJ-FCLVOEFKSA-N 0.000 description 2
- 108700001094 Plant Genes Proteins 0.000 description 2
- HXQRIQXPGMPSRW-UHZRDUGNSA-N Pollinastanol Natural products O[C@@H]1C[C@H]2[C@@]3([C@]4([C@H]([C@@]5(C)[C@@](C)([C@H]([C@H](CCCC(C)C)C)CC5)CC4)CC2)C3)CC1 HXQRIQXPGMPSRW-UHZRDUGNSA-N 0.000 description 2
- FYPGHGXAOZTOBO-IHRRRGAJSA-N Pro-Leu-His Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@@H]2CCCN2 FYPGHGXAOZTOBO-IHRRRGAJSA-N 0.000 description 2
- WCNVGGZRTNHOOS-ULQDDVLXSA-N Pro-Lys-Tyr Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC2=CC=C(C=C2)O)C(=O)O WCNVGGZRTNHOOS-ULQDDVLXSA-N 0.000 description 2
- XRGIDCGRSSWCKE-SRVKXCTJSA-N Pro-Val-Met Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCSC)C(O)=O XRGIDCGRSSWCKE-SRVKXCTJSA-N 0.000 description 2
- 101710083689 Probable capsid protein Proteins 0.000 description 2
- RJKFOVLPORLFTN-LEKSSAKUSA-N Progesterone Chemical compound C1CC2=CC(=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H](C(=O)C)[C@@]1(C)CC2 RJKFOVLPORLFTN-LEKSSAKUSA-N 0.000 description 2
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 2
- 108010076504 Protein Sorting Signals Proteins 0.000 description 2
- 241000220324 Pyrus Species 0.000 description 2
- 108700008625 Reporter Genes Proteins 0.000 description 2
- 108020005543 Satellite RNA Proteins 0.000 description 2
- 101100219191 Schizosaccharomyces pombe (strain 972 / ATCC 24843) byr1 gene Proteins 0.000 description 2
- 238000012300 Sequence Analysis Methods 0.000 description 2
- OOKCGAYXSNJBGQ-ZLUOBGJFSA-N Ser-Asn-Asn Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O OOKCGAYXSNJBGQ-ZLUOBGJFSA-N 0.000 description 2
- FYUIFUJFNCLUIX-XVYDVKMFSA-N Ser-His-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](C)C(O)=O FYUIFUJFNCLUIX-XVYDVKMFSA-N 0.000 description 2
- HXPNJVLVHKABMJ-KKUMJFAQSA-N Ser-Tyr-His Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)NC(=O)[C@H](CO)N)O HXPNJVLVHKABMJ-KKUMJFAQSA-N 0.000 description 2
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 2
- 108020004682 Single-Stranded DNA Proteins 0.000 description 2
- 238000002105 Southern blotting Methods 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- 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 2
- 239000008049 TAE buffer Substances 0.000 description 2
- YRNBANYVJJBGDI-VZFHVOOUSA-N Thr-Ala-Cys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](C)C(=O)N[C@@H](CS)C(=O)O)N)O YRNBANYVJJBGDI-VZFHVOOUSA-N 0.000 description 2
- XUGYQLFEJYZOKQ-NGTWOADLSA-N Thr-Ile-Trp Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)O)NC(=O)[C@H]([C@@H](C)O)N XUGYQLFEJYZOKQ-NGTWOADLSA-N 0.000 description 2
- 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 2
- ISLDRLHVPXABBC-IEGACIPQSA-N Thr-Leu-Trp Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O ISLDRLHVPXABBC-IEGACIPQSA-N 0.000 description 2
- 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 2
- 108091036066 Three prime untranslated region Proteins 0.000 description 2
- 108020004566 Transfer RNA Proteins 0.000 description 2
- 241000209140 Triticum Species 0.000 description 2
- 235000021307 Triticum Nutrition 0.000 description 2
- IQXWAJUIAQLZNX-IHPCNDPISA-N Trp-Leu-His Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@H](CC2=CNC3=CC=CC=C32)N IQXWAJUIAQLZNX-IHPCNDPISA-N 0.000 description 2
- HJWLQSFTGDQSRX-BPUTZDHNSA-N Trp-Met-Ser Chemical compound [H]N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CO)C(O)=O HJWLQSFTGDQSRX-BPUTZDHNSA-N 0.000 description 2
- STKZKWFOKOCSLW-UMPQAUOISA-N Trp-Thr-Val Chemical compound C1=CC=C2C(C[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](C(C)C)C(O)=O)[C@@H](C)O)=CNC2=C1 STKZKWFOKOCSLW-UMPQAUOISA-N 0.000 description 2
- ZWZOCUWOXSDYFZ-CQDKDKBSSA-N Tyr-Ala-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 ZWZOCUWOXSDYFZ-CQDKDKBSSA-N 0.000 description 2
- CVXURBLRELTJKO-BWAGICSOSA-N Tyr-His-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC1=CN=CN1)NC(=O)[C@H](CC2=CC=C(C=C2)O)N)O CVXURBLRELTJKO-BWAGICSOSA-N 0.000 description 2
- CNNVVEPJTFOGHI-ACRUOGEOSA-N Tyr-Lys-Tyr Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O CNNVVEPJTFOGHI-ACRUOGEOSA-N 0.000 description 2
- BGFCXQXETBDEHP-BZSNNMDCSA-N Tyr-Phe-Asn 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](CC(N)=O)C(O)=O BGFCXQXETBDEHP-BZSNNMDCSA-N 0.000 description 2
- NVJCMGGZHOJNBU-UFYCRDLUSA-N Tyr-Val-Phe Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)NC(=O)[C@H](CC2=CC=C(C=C2)O)N NVJCMGGZHOJNBU-UFYCRDLUSA-N 0.000 description 2
- ZEPNVCGPJXYABB-UHFFFAOYSA-N UNPD5911 Natural products C1C(O)CCC2(C)C(CCC3(C(C(C)CCC(=C)C(C)C)CCC33)C)C3=CC=C21 ZEPNVCGPJXYABB-UHFFFAOYSA-N 0.000 description 2
- 108091023045 Untranslated Region Proteins 0.000 description 2
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 2
- UDNYEPLJTRDMEJ-RCOVLWMOSA-N Val-Asn-Gly Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)NCC(=O)O)N UDNYEPLJTRDMEJ-RCOVLWMOSA-N 0.000 description 2
- QGFPYRPIUXBYGR-YDHLFZDLSA-N Val-Asn-Phe Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)N QGFPYRPIUXBYGR-YDHLFZDLSA-N 0.000 description 2
- 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 2
- NSUUANXHLKKHQB-BZSNNMDCSA-N Val-Pro-Trp Chemical compound CC(C)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@H](C(O)=O)CC1=CNC2=CC=CC=C12 NSUUANXHLKKHQB-BZSNNMDCSA-N 0.000 description 2
- 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 2
- 240000008042 Zea mays Species 0.000 description 2
- 235000016383 Zea mays subsp huehuetenangensis Nutrition 0.000 description 2
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 2
- 241000222126 [Candida] glabrata Species 0.000 description 2
- HGEVZDLYZYVYHD-UHFFFAOYSA-N acetic acid;2-amino-2-(hydroxymethyl)propane-1,3-diol;2-[2-[bis(carboxymethyl)amino]ethyl-(carboxymethyl)amino]acetic acid Chemical compound CC(O)=O.OCC(N)(CO)CO.OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O HGEVZDLYZYVYHD-UHFFFAOYSA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- DZBUGLKDJFMEHC-UHFFFAOYSA-N acridine Chemical compound C1=CC=CC2=CC3=CC=CC=C3N=C21 DZBUGLKDJFMEHC-UHFFFAOYSA-N 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 238000007792 addition Methods 0.000 description 2
- 229960000643 adenine Drugs 0.000 description 2
- 239000008272 agar Substances 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
- 239000012491 analyte Substances 0.000 description 2
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 2
- 108010013835 arginine glutamate Proteins 0.000 description 2
- 229940009098 aspartate Drugs 0.000 description 2
- 108010040443 aspartyl-aspartic acid Proteins 0.000 description 2
- 108010069205 aspartyl-phenylalanine Proteins 0.000 description 2
- 108010093581 aspartyl-proline Proteins 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 238000004166 bioassay Methods 0.000 description 2
- 239000003139 biocide Substances 0.000 description 2
- 150000001647 brassinosteroids Chemical class 0.000 description 2
- 208000032343 candida glabrata infection Diseases 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 238000012512 characterization method Methods 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- ONQRKEUAIJMULO-YBXTVTTCSA-N cycloartenol Chemical compound CC(C)([C@@H](O)CC1)[C@H]2[C@@]31C[C@@]13CC[C@]3(C)[C@@H]([C@@H](CCC=C(C)C)C)CC[C@@]3(C)[C@@H]1CC2 ONQRKEUAIJMULO-YBXTVTTCSA-N 0.000 description 2
- YNBJLDSWFGUFRT-UHFFFAOYSA-N cycloartenol Natural products CC(CCC=C(C)C)C1CCC2(C)C1(C)CCC34CC35CCC(O)C(C)(C)C5CCC24C YNBJLDSWFGUFRT-UHFFFAOYSA-N 0.000 description 2
- FODTZLFLDFKIQH-UHFFFAOYSA-N cycloartenol trans-ferulate Natural products C1=C(O)C(OC)=CC(C=CC(=O)OC2C(C3CCC4C5(C)CCC(C5(C)CCC54CC53CC2)C(C)CCC=C(C)C)(C)C)=C1 FODTZLFLDFKIQH-UHFFFAOYSA-N 0.000 description 2
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 2
- 235000018417 cysteine Nutrition 0.000 description 2
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000007824 enzymatic assay Methods 0.000 description 2
- ZEPNVCGPJXYABB-LOIOQLKMSA-N ergosta-5,7,24(28)-trien-3beta-ol Chemical compound C1[C@@H](O)CC[C@]2(C)[C@@H](CC[C@@]3([C@@H]([C@H](C)CCC(=C)C(C)C)CC[C@H]33)C)C3=CC=C21 ZEPNVCGPJXYABB-LOIOQLKMSA-N 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 230000035558 fertility Effects 0.000 description 2
- 238000004817 gas chromatography Methods 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 102000034356 gene-regulatory proteins Human genes 0.000 description 2
- 108091006104 gene-regulatory proteins Proteins 0.000 description 2
- 238000010353 genetic engineering Methods 0.000 description 2
- 239000003448 gibberellin Substances 0.000 description 2
- 229930195712 glutamate Natural products 0.000 description 2
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 2
- 235000004554 glutamine Nutrition 0.000 description 2
- 108010055341 glutamyl-glutamic acid Proteins 0.000 description 2
- 108010049041 glutamylalanine Proteins 0.000 description 2
- 108010089804 glycyl-threonine Proteins 0.000 description 2
- 108010050848 glycylleucine Proteins 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 108010092114 histidylphenylalanine Proteins 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- 238000003780 insertion Methods 0.000 description 2
- 230000037431 insertion Effects 0.000 description 2
- 229930027917 kanamycin Natural products 0.000 description 2
- 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 2
- 229960000318 kanamycin Drugs 0.000 description 2
- 229930182823 kanamycin A Natural products 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- 230000004777 loss-of-function mutation Effects 0.000 description 2
- 229910001629 magnesium chloride Inorganic materials 0.000 description 2
- 235000009973 maize Nutrition 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 229910021645 metal ion Inorganic materials 0.000 description 2
- 229930182817 methionine Natural products 0.000 description 2
- 108010056582 methionylglutamic acid Proteins 0.000 description 2
- 108010005942 methionylglycine Proteins 0.000 description 2
- 238000000520 microinjection Methods 0.000 description 2
- 238000007479 molecular analysis Methods 0.000 description 2
- 238000007491 morphometric analysis Methods 0.000 description 2
- 238000002703 mutagenesis Methods 0.000 description 2
- 231100000350 mutagenesis Toxicity 0.000 description 2
- 230000037434 nonsense mutation Effects 0.000 description 2
- 230000009871 nonspecific binding Effects 0.000 description 2
- 238000007899 nucleic acid hybridization Methods 0.000 description 2
- 239000002751 oligonucleotide probe Substances 0.000 description 2
- 230000036961 partial effect Effects 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 239000000816 peptidomimetic Substances 0.000 description 2
- 108010051242 phenylalanylserine Proteins 0.000 description 2
- 108010083476 phenylalanyltryptophan Proteins 0.000 description 2
- 230000008121 plant development Effects 0.000 description 2
- 229920003023 plastic Polymers 0.000 description 2
- 239000004033 plastic Substances 0.000 description 2
- 239000001103 potassium chloride Substances 0.000 description 2
- 235000011164 potassium chloride Nutrition 0.000 description 2
- 239000002243 precursor Substances 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 108010053725 prolylvaline Proteins 0.000 description 2
- 230000004853 protein function Effects 0.000 description 2
- ZCCUUQDIBDJBTK-UHFFFAOYSA-N psoralen Chemical compound C1=C2OC(=O)C=CC2=CC2=C1OC=C2 ZCCUUQDIBDJBTK-UHFFFAOYSA-N 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 230000009711 regulatory function Effects 0.000 description 2
- 230000001850 reproductive effect Effects 0.000 description 2
- 235000009566 rice Nutrition 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 238000002098 selective ion monitoring Methods 0.000 description 2
- 108010048818 seryl-histidine Proteins 0.000 description 2
- 108010048397 seryl-lysyl-leucine Proteins 0.000 description 2
- 230000035939 shock Effects 0.000 description 2
- 239000000377 silicon dioxide Substances 0.000 description 2
- 239000002689 soil Substances 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 238000010561 standard procedure Methods 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 229940113082 thymine Drugs 0.000 description 2
- 230000005030 transcription termination Effects 0.000 description 2
- 238000011426 transformation method Methods 0.000 description 2
- 108010084932 tryptophyl-proline Proteins 0.000 description 2
- SBSXXCCMIWEPEE-DWMMZRLUSA-N typhasterol Natural products O=C1[C@H]2[C@@](C)([C@@H]3[C@H]([C@H]4[C@](C)([C@@H]([C@@H]([C@@H](O)[C@H](O)[C@H](C(C)C)C)C)CC4)CC3)C1)CC[C@@H](O)C2 SBSXXCCMIWEPEE-DWMMZRLUSA-N 0.000 description 2
- 235000013311 vegetables Nutrition 0.000 description 2
- 239000003643 water by type Substances 0.000 description 2
- 238000001262 western blot Methods 0.000 description 2
- KZJWDPNRJALLNS-VPUBHVLGSA-N (-)-beta-Sitosterol Natural products O[C@@H]1CC=2[C@@](C)([C@@H]3[C@H]([C@H]4[C@@](C)([C@H]([C@H](CC[C@@H](C(C)C)CC)C)CC4)CC3)CC=2)CC1 KZJWDPNRJALLNS-VPUBHVLGSA-N 0.000 description 1
- OSELKOCHBMDKEJ-UHFFFAOYSA-N (10R)-3c-Hydroxy-10r.13c-dimethyl-17c-((R)-1-methyl-4-isopropyl-hexen-(4c)-yl)-(8cH.9tH.14tH)-Delta5-tetradecahydro-1H-cyclopenta[a]phenanthren Natural products C1C=C2CC(O)CCC2(C)C2C1C1CCC(C(C)CCC(=CC)C(C)C)C1(C)CC2 OSELKOCHBMDKEJ-UHFFFAOYSA-N 0.000 description 1
- BQPPJGMMIYJVBR-UHFFFAOYSA-N (10S)-3c-Acetoxy-4.4.10r.13c.14t-pentamethyl-17c-((R)-1.5-dimethyl-hexen-(4)-yl)-(5tH)-Delta8-tetradecahydro-1H-cyclopenta[a]phenanthren Natural products CC12CCC(OC(C)=O)C(C)(C)C1CCC1=C2CCC2(C)C(C(CCC=C(C)C)C)CCC21C BQPPJGMMIYJVBR-UHFFFAOYSA-N 0.000 description 1
- CSVWWLUMXNHWSU-UHFFFAOYSA-N (22E)-(24xi)-24-ethyl-5alpha-cholest-22-en-3beta-ol Natural products C1CC2CC(O)CCC2(C)C2C1C1CCC(C(C)C=CC(CC)C(C)C)C1(C)CC2 CSVWWLUMXNHWSU-UHFFFAOYSA-N 0.000 description 1
- LSZJAIFORSLKOY-PACUACIMSA-N (22S)-22-hydroxycampesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)[C@@H](O)C[C@@H](C)C(C)C)[C@@]1(C)CC2 LSZJAIFORSLKOY-PACUACIMSA-N 0.000 description 1
- CWFMWBHMIMNZLN-NAKRPEOUSA-N (2s)-1-[(2s)-2-[[(2s,3s)-2-amino-3-methylpentanoyl]amino]propanoyl]pyrrolidine-2-carboxylic acid Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](C)C(=O)N1CCC[C@H]1C(O)=O CWFMWBHMIMNZLN-NAKRPEOUSA-N 0.000 description 1
- ZQVJBRJGDVZANE-MXDMHAPNSA-N (2s)-2-[(3s,6s,9z,12s,15s,18s,21r,24r,27s)-18,21-bis(2-aminoethyl)-12-benzyl-3-[(1s)-2-chloro-1-hydroxyethyl]-15-[3-(diaminomethylideneamino)propyl]-9-ethylidene-27-[[(3s)-3-hydroxydodecanoyl]amino]-24-(hydroxymethyl)-2,5,8,11,14,17,20,23,26-nonaoxo-1-oxa Chemical compound N1C(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CCN)NC(=O)[C@@H](CCN)NC(=O)[C@@H](CO)NC(=O)[C@@H](NC(=O)C[C@@H](O)CCCCCCCCC)COC(=O)[C@H]([C@H](O)CCl)NC(=O)[C@H]([C@H](O)C(O)=O)NC(=O)\C(=C\C)NC(=O)[C@@H]1CC1=CC=CC=C1 ZQVJBRJGDVZANE-MXDMHAPNSA-N 0.000 description 1
- OGILYBDMVOATLU-CQJMVLFOSA-N (2s)-2-[[(2s)-2-amino-3-(4-hydroxyphenyl)propanoyl]amino]-n-[(2s)-1-[[(2s)-1-amino-1-oxo-3-phenylpropan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]-4-methylpentanamide Chemical compound C([C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)C1=CC=C(O)C=C1 OGILYBDMVOATLU-CQJMVLFOSA-N 0.000 description 1
- YUXKOWPNKJSTPQ-AXWWPMSFSA-N (2s,3r)-2-amino-3-hydroxybutanoic acid;(2s)-2-amino-3-hydroxypropanoic acid Chemical compound OC[C@H](N)C(O)=O.C[C@@H](O)[C@H](N)C(O)=O YUXKOWPNKJSTPQ-AXWWPMSFSA-N 0.000 description 1
- CHGIKSSZNBCNDW-UHFFFAOYSA-N (3beta,5alpha)-4,4-Dimethylcholesta-8,24-dien-3-ol Natural products CC12CCC(O)C(C)(C)C1CCC1=C2CCC2(C)C(C(CCC=C(C)C)C)CCC21 CHGIKSSZNBCNDW-UHFFFAOYSA-N 0.000 description 1
- GZCWLCBFPRFLKL-UHFFFAOYSA-N 1-prop-2-ynoxypropan-2-ol Chemical compound CC(O)COCC#C GZCWLCBFPRFLKL-UHFFFAOYSA-N 0.000 description 1
- XYTLYKGXLMKYMV-UHFFFAOYSA-N 14alpha-methylzymosterol Natural products CC12CCC(O)CC1CCC1=C2CCC2(C)C(C(CCC=C(C)C)C)CCC21C XYTLYKGXLMKYMV-UHFFFAOYSA-N 0.000 description 1
- DQVAZKGVGKHQDS-UHFFFAOYSA-N 2-[[1-[2-[(2-amino-4-methylpentanoyl)amino]-4-methylpentanoyl]pyrrolidine-2-carbonyl]amino]-4-methylpentanoic acid Chemical compound CC(C)CC(N)C(=O)NC(CC(C)C)C(=O)N1CCCC1C(=O)NC(CC(C)C)C(O)=O DQVAZKGVGKHQDS-UHFFFAOYSA-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
- KLEXDBGYSOIREE-UHFFFAOYSA-N 24xi-n-propylcholesterol Natural products C1C=C2CC(O)CCC2(C)C2C1C1CCC(C(C)CCC(CCC)C(C)C)C1(C)CC2 KLEXDBGYSOIREE-UHFFFAOYSA-N 0.000 description 1
- LRRBQWHWKJDDAW-OTDRPZMHSA-N 28-Homodolicholide Natural products O=C1OC[C@@H]2[C@@H]3[C@@](C)([C@@H]([C@@H]([C@@H](O)[C@H](O)/C(=C\C)/C(C)C)C)CC3)CC[C@@H]2[C@]2(C)[C@@H]1C[C@H](O)[C@H](O)C2 LRRBQWHWKJDDAW-OTDRPZMHSA-N 0.000 description 1
- 150000000459 28-homodolicholides Chemical class 0.000 description 1
- 108010029908 3-oxo-5-alpha-steroid 4-dehydrogenase Proteins 0.000 description 1
- 102000001779 3-oxo-5-alpha-steroid 4-dehydrogenase Human genes 0.000 description 1
- OSJPPGNTCRNQQC-UWTATZPHSA-N 3-phospho-D-glyceric acid Chemical compound OC(=O)[C@H](O)COP(O)(O)=O OSJPPGNTCRNQQC-UWTATZPHSA-N 0.000 description 1
- FPTJELQXIUUCEY-UHFFFAOYSA-N 3beta-Hydroxy-lanostan Natural products C1CC2C(C)(C)C(O)CCC2(C)C2C1C1(C)CCC(C(C)CCCC(C)C)C1(C)CC2 FPTJELQXIUUCEY-UHFFFAOYSA-N 0.000 description 1
- VXGRJERITKFWPL-UHFFFAOYSA-N 4',5'-Dihydropsoralen Natural products C1=C2OC(=O)C=CC2=CC2=C1OCC2 VXGRJERITKFWPL-UHFFFAOYSA-N 0.000 description 1
- IJJWOSAXNHWBPR-HUBLWGQQSA-N 5-[(3as,4s,6ar)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]-n-(6-hydrazinyl-6-oxohexyl)pentanamide Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)NCCCCCC(=O)NN)SC[C@@H]21 IJJWOSAXNHWBPR-HUBLWGQQSA-N 0.000 description 1
- VXBLCLVRWCLEOX-TVMSINTGSA-N 6-Deoxocastasterone Natural products O[C@@H]([C@H](O)[C@@H](C)[C@@H]1[C@@]2(C)[C@H]([C@H]3[C@@H]([C@]4(C)[C@@H](C[C@H](O)[C@H](O)C4)CC3)CC2)CC1)[C@H](C(C)C)C VXBLCLVRWCLEOX-TVMSINTGSA-N 0.000 description 1
- WPHVOXMMNSLJSF-MSEICUJVSA-N 6-Deoxoteasterone Natural products O[C@@H]([C@H](O)[C@@H](C)[C@@H]1[C@@]2(C)[C@H]([C@@H]3[C@@H]([C@@]4(C)[C@H](C[C@@H](O)CC4)CC3)CC2)CC1)[C@H](C(C)C)C WPHVOXMMNSLJSF-MSEICUJVSA-N 0.000 description 1
- VXBLCLVRWCLEOX-BFYSZXNBSA-N 6-deoxocastasterone Chemical compound C([C@@H]1CC2)[C@H](O)[C@H](O)C[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@H](C)[C@@H](O)[C@H](O)[C@@H](C)C(C)C)[C@@]2(C)CC1 VXBLCLVRWCLEOX-BFYSZXNBSA-N 0.000 description 1
- WPHVOXMMNSLJSF-GUOPQYDVSA-N 6-deoxoteasterone Chemical compound C([C@@H]1CC2)[C@@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@H](C)[C@@H](O)[C@H](O)[C@@H](C)C(C)C)[C@@]2(C)CC1 WPHVOXMMNSLJSF-GUOPQYDVSA-N 0.000 description 1
- WPHVOXMMNSLJSF-DAWJDVIISA-N 6-deoxotyphasterol Chemical compound C([C@@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@H](C)[C@@H](O)[C@H](O)[C@@H](C)C(C)C)[C@@]2(C)CC1 WPHVOXMMNSLJSF-DAWJDVIISA-N 0.000 description 1
- NBJZGNFIZZWBOJ-JSHJXQBASA-N 6-oxocampestanol Chemical compound C([C@@H]1C(=O)C2)[C@@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@H](C)CC[C@@H](C)C(C)C)[C@@]2(C)CC1 NBJZGNFIZZWBOJ-JSHJXQBASA-N 0.000 description 1
- NBJZGNFIZZWBOJ-JUFNBKTGSA-N 6-oxocampestanol Natural products O=C1[C@@H]2[C@@](C)([C@H]3[C@@H]([C@@H]4[C@@](C)([C@H]([C@@H](CC[C@H](C(C)C)C)C)CC4)CC3)C1)CC[C@H](O)C2 NBJZGNFIZZWBOJ-JUFNBKTGSA-N 0.000 description 1
- 102000013563 Acid Phosphatase Human genes 0.000 description 1
- 108010051457 Acid Phosphatase Proteins 0.000 description 1
- AAQGRPOPTAUUBM-ZLUOBGJFSA-N Ala-Ala-Asn Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(N)=O)C(O)=O AAQGRPOPTAUUBM-ZLUOBGJFSA-N 0.000 description 1
- SVBXIUDNTRTKHE-CIUDSAMLSA-N Ala-Arg-Glu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(O)=O SVBXIUDNTRTKHE-CIUDSAMLSA-N 0.000 description 1
- 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 1
- NHLAEBFGWPXFGI-WHFBIAKZSA-N Ala-Gly-Asn Chemical compound C[C@@H](C(=O)NCC(=O)N[C@@H](CC(=O)N)C(=O)O)N NHLAEBFGWPXFGI-WHFBIAKZSA-N 0.000 description 1
- 108010076441 Ala-His-His Proteins 0.000 description 1
- ATAKEVCGTRZKLI-UWJYBYFXSA-N Ala-His-His Chemical compound C([C@H](NC(=O)[C@@H](N)C)C(=O)N[C@@H](CC=1NC=NC=1)C(O)=O)C1=CN=CN1 ATAKEVCGTRZKLI-UWJYBYFXSA-N 0.000 description 1
- LXAARTARZJJCMB-CIQUZCHMSA-N Ala-Ile-Thr Chemical compound [H]N[C@@H](C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(O)=O LXAARTARZJJCMB-CIQUZCHMSA-N 0.000 description 1
- AWZKCUCQJNTBAD-SRVKXCTJSA-N Ala-Leu-Lys Chemical compound C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CCCCN AWZKCUCQJNTBAD-SRVKXCTJSA-N 0.000 description 1
- SOBIAADAMRHGKH-CIUDSAMLSA-N Ala-Leu-Ser Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O SOBIAADAMRHGKH-CIUDSAMLSA-N 0.000 description 1
- MDNAVFBZPROEHO-UHFFFAOYSA-N Ala-Lys-Val Natural products CC(C)C(C(O)=O)NC(=O)C(NC(=O)C(C)N)CCCCN MDNAVFBZPROEHO-UHFFFAOYSA-N 0.000 description 1
- XSTZMVAYYCJTNR-DCAQKATOSA-N Ala-Met-Leu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(C)C)C(O)=O XSTZMVAYYCJTNR-DCAQKATOSA-N 0.000 description 1
- ADSGHMXEAZJJNF-DCAQKATOSA-N Ala-Pro-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@H](C)N ADSGHMXEAZJJNF-DCAQKATOSA-N 0.000 description 1
- JNLDTVRGXMSYJC-UVBJJODRSA-N Ala-Pro-Trp Chemical compound C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](Cc1c[nH]c2ccccc12)C(O)=O JNLDTVRGXMSYJC-UVBJJODRSA-N 0.000 description 1
- MMLHRUJLOUSRJX-CIUDSAMLSA-N Ala-Ser-Lys Chemical compound C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCCCN MMLHRUJLOUSRJX-CIUDSAMLSA-N 0.000 description 1
- JJHBEVZAZXZREW-LFSVMHDDSA-N Ala-Thr-Phe Chemical compound C[C@@H](O)[C@H](NC(=O)[C@H](C)N)C(=O)N[C@@H](Cc1ccccc1)C(O)=O JJHBEVZAZXZREW-LFSVMHDDSA-N 0.000 description 1
- XCIGOVDXZULBBV-DCAQKATOSA-N Ala-Val-Lys Chemical compound CC(C)[C@H](NC(=O)[C@H](C)N)C(=O)N[C@@H](CCCCN)C(O)=O XCIGOVDXZULBBV-DCAQKATOSA-N 0.000 description 1
- REWSWYIDQIELBE-FXQIFTODSA-N Ala-Val-Ser Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(O)=O REWSWYIDQIELBE-FXQIFTODSA-N 0.000 description 1
- 235000003840 Amygdalus nana Nutrition 0.000 description 1
- 244000296825 Amygdalus nana Species 0.000 description 1
- 241000219195 Arabidopsis thaliana Species 0.000 description 1
- 101001037270 Arabidopsis thaliana Auxin-responsive protein IAA7 Proteins 0.000 description 1
- 101000946587 Arabidopsis thaliana Cycloeucalenol cycloisomerase Proteins 0.000 description 1
- JGDGLDNAQJJGJI-AVGNSLFASA-N Arg-Arg-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CCCN=C(N)N)N JGDGLDNAQJJGJI-AVGNSLFASA-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
- PNQWAUXQDBIJDY-GUBZILKMSA-N Arg-Glu-Glu Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O PNQWAUXQDBIJDY-GUBZILKMSA-N 0.000 description 1
- FNXCAFKDGBROCU-STECZYCISA-N Arg-Ile-Tyr Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 FNXCAFKDGBROCU-STECZYCISA-N 0.000 description 1
- YBZMTKUDWXZLIX-UWVGGRQHSA-N Arg-Leu-Gly Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)NCC(O)=O YBZMTKUDWXZLIX-UWVGGRQHSA-N 0.000 description 1
- OGZBJJLRKQZRHL-KJEVXHAQSA-N Arg-Thr-Tyr Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H]([C@H](O)C)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 OGZBJJLRKQZRHL-KJEVXHAQSA-N 0.000 description 1
- YHZQOSXDTFRZKU-WDSOQIARSA-N Arg-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)CCCN=C(N)N)=CNC2=C1 YHZQOSXDTFRZKU-WDSOQIARSA-N 0.000 description 1
- CGWVCWFQGXOUSJ-ULQDDVLXSA-N Arg-Tyr-Leu Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(C)C)C(O)=O CGWVCWFQGXOUSJ-ULQDDVLXSA-N 0.000 description 1
- PSUXEQYPYZLNER-QXEWZRGKSA-N Arg-Val-Asn Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O PSUXEQYPYZLNER-QXEWZRGKSA-N 0.000 description 1
- 108010082340 Arginine deiminase Proteins 0.000 description 1
- PSZDKHGMNWAHGO-UHFFFAOYSA-N Asn Asn Pro Val Chemical compound CC(C)C(C(O)=O)NC(=O)C1CCCN1C(=O)C(CC(N)=O)NC(=O)C(N)CC(N)=O PSZDKHGMNWAHGO-UHFFFAOYSA-N 0.000 description 1
- PTNFNTOBUDWHNZ-GUBZILKMSA-N Asn-Arg-Met Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCSC)C(O)=O PTNFNTOBUDWHNZ-GUBZILKMSA-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
- JLNFZLNDHONLND-GARJFASQSA-N Asn-Leu-Pro Chemical compound CC(C)C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CC(=O)N)N JLNFZLNDHONLND-GARJFASQSA-N 0.000 description 1
- PLTGTJAZQRGMPP-FXQIFTODSA-N Asn-Pro-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)CC(N)=O PLTGTJAZQRGMPP-FXQIFTODSA-N 0.000 description 1
- CPYHLXSGDBDULY-IHPCNDPISA-N Asn-Trp-Phe Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O CPYHLXSGDBDULY-IHPCNDPISA-N 0.000 description 1
- RTFXPCYMDYBZNQ-SRVKXCTJSA-N Asn-Tyr-Asn Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(N)=O)C(O)=O RTFXPCYMDYBZNQ-SRVKXCTJSA-N 0.000 description 1
- HBUJSDCLZCXXCW-YDHLFZDLSA-N Asn-Val-Tyr Chemical compound NC(=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 HBUJSDCLZCXXCW-YDHLFZDLSA-N 0.000 description 1
- VTYQAQFKMQTKQD-ACZMJKKPSA-N Asp-Ala-Gln Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(N)=O)C(O)=O VTYQAQFKMQTKQD-ACZMJKKPSA-N 0.000 description 1
- XEDQMTWEYFBOIK-ACZMJKKPSA-N Asp-Ala-Glu Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(O)=O XEDQMTWEYFBOIK-ACZMJKKPSA-N 0.000 description 1
- VPPXTHJNTYDNFJ-CIUDSAMLSA-N Asp-Ala-Lys Chemical compound C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC(=O)O)N VPPXTHJNTYDNFJ-CIUDSAMLSA-N 0.000 description 1
- NYLBGYLHBDFRHL-VEVYYDQMSA-N Asp-Arg-Thr Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(O)=O NYLBGYLHBDFRHL-VEVYYDQMSA-N 0.000 description 1
- ILJQISGMGXRZQQ-IHRRRGAJSA-N Asp-Arg-Tyr Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O ILJQISGMGXRZQQ-IHRRRGAJSA-N 0.000 description 1
- XYBJLTKSGFBLCS-QXEWZRGKSA-N Asp-Arg-Val Chemical compound NC(N)=NCCC[C@@H](C(=O)N[C@@H](C(C)C)C(O)=O)NC(=O)[C@@H](N)CC(O)=O XYBJLTKSGFBLCS-QXEWZRGKSA-N 0.000 description 1
- QOVWVLLHMMCFFY-ZLUOBGJFSA-N Asp-Asp-Asn Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O QOVWVLLHMMCFFY-ZLUOBGJFSA-N 0.000 description 1
- QXHVOUSPVAWEMX-ZLUOBGJFSA-N Asp-Asp-Ser Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(O)=O QXHVOUSPVAWEMX-ZLUOBGJFSA-N 0.000 description 1
- VZNOVQKGJQJOCS-SRVKXCTJSA-N Asp-Asp-Tyr Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O VZNOVQKGJQJOCS-SRVKXCTJSA-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
- PMEHKVHZQKJACS-PEFMBERDSA-N Asp-Gln-Ile 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)CC)C(O)=O PMEHKVHZQKJACS-PEFMBERDSA-N 0.000 description 1
- KHGPWGKPYHPOIK-QWRGUYRKSA-N Asp-Gly-Phe Chemical compound [H]N[C@@H](CC(O)=O)C(=O)NCC(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O KHGPWGKPYHPOIK-QWRGUYRKSA-N 0.000 description 1
- RWHHSFSWKFBTCF-KKUMJFAQSA-N Asp-His-Phe Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)O)NC(=O)[C@H](CC2=CN=CN2)NC(=O)[C@H](CC(=O)O)N RWHHSFSWKFBTCF-KKUMJFAQSA-N 0.000 description 1
- GKWFMNNNYZHJHV-SRVKXCTJSA-N Asp-Lys-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CC(O)=O GKWFMNNNYZHJHV-SRVKXCTJSA-N 0.000 description 1
- NVFSJIXJZCDICF-SRVKXCTJSA-N Asp-Lys-Lys Chemical compound C(CCN)C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC(=O)O)N NVFSJIXJZCDICF-SRVKXCTJSA-N 0.000 description 1
- IOXWDLNHXZOXQP-FXQIFTODSA-N Asp-Met-Ser Chemical compound CSCC[C@@H](C(=O)N[C@@H](CO)C(=O)O)NC(=O)[C@H](CC(=O)O)N IOXWDLNHXZOXQP-FXQIFTODSA-N 0.000 description 1
- UAXIKORUDGGIGA-DCAQKATOSA-N Asp-Pro-Lys Chemical compound C1C[C@H](N(C1)C(=O)[C@H](CC(=O)O)N)C(=O)N[C@@H](CCCCN)C(=O)O UAXIKORUDGGIGA-DCAQKATOSA-N 0.000 description 1
- QSFHZPQUAAQHAQ-CIUDSAMLSA-N Asp-Ser-Leu Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(O)=O QSFHZPQUAAQHAQ-CIUDSAMLSA-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
- NJLLRXWFPQQPHV-SRVKXCTJSA-N Asp-Tyr-Asn 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](CC(N)=O)C(O)=O NJLLRXWFPQQPHV-SRVKXCTJSA-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
- 235000005340 Asparagus officinalis Nutrition 0.000 description 1
- 235000005781 Avena Nutrition 0.000 description 1
- 244000075850 Avena orientalis Species 0.000 description 1
- 241000726301 Avocado sunblotch viroid Species 0.000 description 1
- 241000219310 Beta vulgaris subsp. vulgaris Species 0.000 description 1
- 108010006654 Bleomycin Proteins 0.000 description 1
- ZOXJGFHDIHLPTG-UHFFFAOYSA-N Boron Chemical compound [B] ZOXJGFHDIHLPTG-UHFFFAOYSA-N 0.000 description 1
- 235000011331 Brassica Nutrition 0.000 description 1
- 241000219198 Brassica Species 0.000 description 1
- 240000002791 Brassica napus Species 0.000 description 1
- 235000004977 Brassica sinapistrum Nutrition 0.000 description 1
- 101710082260 C-4 methylsterol oxidase Proteins 0.000 description 1
- QCMYYKRYFNMIEC-UHFFFAOYSA-N COP(O)=O Chemical class COP(O)=O QCMYYKRYFNMIEC-UHFFFAOYSA-N 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 235000002566 Capsicum Nutrition 0.000 description 1
- 240000008574 Capsicum frutescens Species 0.000 description 1
- UGFAIRIUMAVXCW-UHFFFAOYSA-N Carbon monoxide Chemical compound [O+]#[C-] UGFAIRIUMAVXCW-UHFFFAOYSA-N 0.000 description 1
- 102000014914 Carrier Proteins Human genes 0.000 description 1
- JSVPGVHCEQDJCZ-PHQFZZKCSA-N Cathasterone Natural products O=C1[C@@H]2[C@@](C)([C@@H]3[C@H]([C@@H]4[C@](C)([C@H]([C@@H]([C@@H](O)C[C@H](C(C)C)C)C)CC4)CC3)C1)CC[C@H](O)C2 JSVPGVHCEQDJCZ-PHQFZZKCSA-N 0.000 description 1
- 108010066551 Cholestenone 5 alpha-Reductase Proteins 0.000 description 1
- 235000007516 Chrysanthemum Nutrition 0.000 description 1
- 244000189548 Chrysanthemum x morifolium Species 0.000 description 1
- 108091028075 Circular RNA Proteins 0.000 description 1
- LPZCCMIISIBREI-MTFRKTCUSA-N Citrostadienol Natural products CC=C(CC[C@@H](C)[C@H]1CC[C@H]2C3=CC[C@H]4[C@H](C)[C@@H](O)CC[C@]4(C)[C@H]3CC[C@]12C)C(C)C LPZCCMIISIBREI-MTFRKTCUSA-N 0.000 description 1
- 241000219109 Citrullus Species 0.000 description 1
- 241000207199 Citrus Species 0.000 description 1
- 240000004270 Colocasia esculenta var. antiquorum Species 0.000 description 1
- 108091028732 Concatemer Proteins 0.000 description 1
- 241000218631 Coniferophyta Species 0.000 description 1
- 241000219122 Cucurbita Species 0.000 description 1
- XFXPMWWXUTWYJX-UHFFFAOYSA-N Cyanide Chemical compound N#[C-] XFXPMWWXUTWYJX-UHFFFAOYSA-N 0.000 description 1
- 241000192700 Cyanobacteria Species 0.000 description 1
- PRHGYQOSEHLDRW-VGDYDELISA-N Cys-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](CS)N PRHGYQOSEHLDRW-VGDYDELISA-N 0.000 description 1
- RESAHOSBQHMOKH-KKUMJFAQSA-N Cys-Phe-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)NC(=O)[C@H](CS)N RESAHOSBQHMOKH-KKUMJFAQSA-N 0.000 description 1
- 102000005297 Cytochrome P-450 CYP4A Human genes 0.000 description 1
- 108010081498 Cytochrome P-450 CYP4A Proteins 0.000 description 1
- 241000701022 Cytomegalovirus Species 0.000 description 1
- DCXYFEDJOCDNAF-UWTATZPHSA-N D-Asparagine Chemical compound OC(=O)[C@H](N)CC(N)=O DCXYFEDJOCDNAF-UWTATZPHSA-N 0.000 description 1
- 229930182846 D-asparagine Natural products 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 1
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 1
- 241000208175 Daucus Species 0.000 description 1
- 244000000626 Daucus carota Species 0.000 description 1
- 235000002767 Daucus carota Nutrition 0.000 description 1
- ARVGMISWLZPBCH-UHFFFAOYSA-N Dehydro-beta-sitosterol Natural products C1C(O)CCC2(C)C(CCC3(C(C(C)CCC(CC)C(C)C)CCC33)C)C3=CC=C21 ARVGMISWLZPBCH-UHFFFAOYSA-N 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
- 101100136092 Drosophila melanogaster peng gene Proteins 0.000 description 1
- 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 1
- 101150106008 ERG11 gene Proteins 0.000 description 1
- YQYJSBFKSSDGFO-UHFFFAOYSA-N Epihygromycin Natural products OC1C(O)C(C(=O)C)OC1OC(C(=C1)O)=CC=C1C=C(C)C(=O)NC1C(O)C(O)C2OCOC2C1O YQYJSBFKSSDGFO-UHFFFAOYSA-N 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 101000933461 Escherichia coli (strain K12) Beta-glucuronidase Proteins 0.000 description 1
- 102000009114 Fatty acid desaturases Human genes 0.000 description 1
- 108010087894 Fatty acid desaturases Proteins 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 235000016623 Fragaria vesca Nutrition 0.000 description 1
- 240000009088 Fragaria x ananassa Species 0.000 description 1
- 235000011363 Fragaria x ananassa Nutrition 0.000 description 1
- 108010058643 Fungal Proteins Proteins 0.000 description 1
- 108010010803 Gelatin 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
- LZRMPXRYLLTAJX-GUBZILKMSA-N Gln-Arg-Glu Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(O)=O LZRMPXRYLLTAJX-GUBZILKMSA-N 0.000 description 1
- PGPJSRSLQNXBDT-YUMQZZPRSA-N Gln-Arg-Gly Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(O)=O PGPJSRSLQNXBDT-YUMQZZPRSA-N 0.000 description 1
- ZQPOVSJFBBETHQ-CIUDSAMLSA-N Gln-Glu-Gln Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O ZQPOVSJFBBETHQ-CIUDSAMLSA-N 0.000 description 1
- LLRJEFPKIIBGJP-DCAQKATOSA-N Gln-Glu-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CCC(=O)N)N LLRJEFPKIIBGJP-DCAQKATOSA-N 0.000 description 1
- GQTNWYFWSUFFRA-KKUMJFAQSA-N Gln-Met-Tyr Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O GQTNWYFWSUFFRA-KKUMJFAQSA-N 0.000 description 1
- DOQUICBEISTQHE-CIUDSAMLSA-N Gln-Pro-Asp Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(O)=O)C(O)=O DOQUICBEISTQHE-CIUDSAMLSA-N 0.000 description 1
- VYOILACOFPPNQH-UMNHJUIQSA-N Gln-Val-Pro Chemical compound CC(C)[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CCC(=O)N)N VYOILACOFPPNQH-UMNHJUIQSA-N 0.000 description 1
- YKLNMGJYMNPBCP-ACZMJKKPSA-N Glu-Asn-Asp Chemical compound C(CC(=O)O)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CC(=O)O)C(=O)O)N YKLNMGJYMNPBCP-ACZMJKKPSA-N 0.000 description 1
- JRCUFCXYZLPSDZ-ACZMJKKPSA-N Glu-Asp-Ser Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(O)=O JRCUFCXYZLPSDZ-ACZMJKKPSA-N 0.000 description 1
- FLQAKQOBSPFGKG-CIUDSAMLSA-N Glu-Cys-Arg Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CS)C(=O)N[C@H](C(O)=O)CCCN=C(N)N FLQAKQOBSPFGKG-CIUDSAMLSA-N 0.000 description 1
- CUXJIASLBRJOFV-LAEOZQHASA-N Glu-Gly-Ile Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H]([C@@H](C)CC)C(O)=O CUXJIASLBRJOFV-LAEOZQHASA-N 0.000 description 1
- XIKYNVKEUINBGL-IUCAKERBSA-N Glu-His-Gly Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC1=CNC=N1)C(=O)NCC(O)=O XIKYNVKEUINBGL-IUCAKERBSA-N 0.000 description 1
- BIHMNDPWRUROFZ-JYJNAYRXSA-N Glu-His-Phe Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O BIHMNDPWRUROFZ-JYJNAYRXSA-N 0.000 description 1
- QIQABBIDHGQXGA-ZPFDUUQYSA-N Glu-Ile-Arg Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O QIQABBIDHGQXGA-ZPFDUUQYSA-N 0.000 description 1
- XTZDZAXYPDISRR-MNXVOIDGSA-N Glu-Ile-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CCC(=O)O)N XTZDZAXYPDISRR-MNXVOIDGSA-N 0.000 description 1
- VMKCPNBBPGGQBJ-GUBZILKMSA-N Glu-Leu-Asn Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](CCC(=O)O)N VMKCPNBBPGGQBJ-GUBZILKMSA-N 0.000 description 1
- WNRZUESNGGDCJX-JYJNAYRXSA-N Glu-Leu-Phe Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O WNRZUESNGGDCJX-JYJNAYRXSA-N 0.000 description 1
- BCYGDJXHAGZNPQ-DCAQKATOSA-N Glu-Lys-Glu Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(O)=O BCYGDJXHAGZNPQ-DCAQKATOSA-N 0.000 description 1
- ILWHFUZZCFYSKT-AVGNSLFASA-N Glu-Lys-Leu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(O)=O ILWHFUZZCFYSKT-AVGNSLFASA-N 0.000 description 1
- YTRBQAQSUDSIQE-FHWLQOOXSA-N Glu-Phe-Phe Chemical compound C([C@H](NC(=O)[C@H](CCC(O)=O)N)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 YTRBQAQSUDSIQE-FHWLQOOXSA-N 0.000 description 1
- SYAYROHMAIHWFB-KBIXCLLPSA-N Glu-Ser-Ile Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O SYAYROHMAIHWFB-KBIXCLLPSA-N 0.000 description 1
- QVXWAFZDWRLXTI-NWLDYVSISA-N Glu-Thr-Trp Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)O)NC(=O)[C@H](CCC(=O)O)N)O QVXWAFZDWRLXTI-NWLDYVSISA-N 0.000 description 1
- HAGKYCXGTRUUFI-RYUDHWBXSA-N Glu-Tyr-Gly Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)NCC(=O)O)NC(=O)[C@H](CCC(=O)O)N)O HAGKYCXGTRUUFI-RYUDHWBXSA-N 0.000 description 1
- BKLIAINBCQPSOV-UHFFFAOYSA-N Gluanol Natural products CC(C)CC=CC(C)C1CCC2(C)C3=C(CCC12C)C4(C)CCC(O)C(C)(C)C4CC3 BKLIAINBCQPSOV-UHFFFAOYSA-N 0.000 description 1
- 108010060309 Glucuronidase Proteins 0.000 description 1
- 102000053187 Glucuronidase Human genes 0.000 description 1
- PYTZFYUXZZHOAD-WHFBIAKZSA-N Gly-Ala-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)CN PYTZFYUXZZHOAD-WHFBIAKZSA-N 0.000 description 1
- GQGAFTPXAPKSCF-WHFBIAKZSA-N Gly-Ala-Cys Chemical compound NCC(=O)N[C@@H](C)C(=O)N[C@@H](CS)C(=O)O GQGAFTPXAPKSCF-WHFBIAKZSA-N 0.000 description 1
- BGVYNAQWHSTTSP-BYULHYEWSA-N Gly-Asn-Ile Chemical compound [H]NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O BGVYNAQWHSTTSP-BYULHYEWSA-N 0.000 description 1
- JUGQPPOVWXSPKJ-RYUDHWBXSA-N Gly-Gln-Phe Chemical compound [H]NCC(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O JUGQPPOVWXSPKJ-RYUDHWBXSA-N 0.000 description 1
- JLJLBWDKDRYOPA-RYUDHWBXSA-N Gly-Gln-Tyr Chemical compound NCC(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 JLJLBWDKDRYOPA-RYUDHWBXSA-N 0.000 description 1
- 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 1
- 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 1
- DBJYVKDPGIFXFO-BQBZGAKWSA-N Gly-Met-Ala Chemical compound [H]NCC(=O)N[C@@H](CCSC)C(=O)N[C@@H](C)C(O)=O DBJYVKDPGIFXFO-BQBZGAKWSA-N 0.000 description 1
- JPVGHHQGKPQYIL-KBPBESRZSA-N Gly-Phe-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CC1=CC=CC=C1 JPVGHHQGKPQYIL-KBPBESRZSA-N 0.000 description 1
- 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 1
- IXHQLZIWBCQBLQ-STQMWFEESA-N Gly-Pro-Phe Chemical compound NCC(=O)N1CCC[C@H]1C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 IXHQLZIWBCQBLQ-STQMWFEESA-N 0.000 description 1
- FKYQEVBRZSFAMJ-QWRGUYRKSA-N Gly-Ser-Tyr Chemical compound NCC(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 FKYQEVBRZSFAMJ-QWRGUYRKSA-N 0.000 description 1
- UMBDRSMLCUYIRI-DVJZZOLTSA-N Gly-Trp-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)NC(=O)CN)O UMBDRSMLCUYIRI-DVJZZOLTSA-N 0.000 description 1
- HQSKKSLNLSTONK-JTQLQIEISA-N Gly-Tyr-Gly Chemical compound OC(=O)CNC(=O)[C@@H](NC(=O)CN)CC1=CC=C(O)C=C1 HQSKKSLNLSTONK-JTQLQIEISA-N 0.000 description 1
- KBBFOULZCHWGJX-KBPBESRZSA-N Gly-Tyr-His Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)NC(=O)CN)O KBBFOULZCHWGJX-KBPBESRZSA-N 0.000 description 1
- GJHWILMUOANXTG-WPRPVWTQSA-N Gly-Val-Arg Chemical compound [H]NCC(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O GJHWILMUOANXTG-WPRPVWTQSA-N 0.000 description 1
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 1
- 102000004144 Green Fluorescent Proteins Human genes 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
- 101150107195 HDF7 gene Proteins 0.000 description 1
- 244000020551 Helianthus annuus Species 0.000 description 1
- 235000003222 Helianthus annuus Nutrition 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- TVQGUFGDVODUIF-LSJOCFKGSA-N His-Arg-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC1=CN=CN1)N TVQGUFGDVODUIF-LSJOCFKGSA-N 0.000 description 1
- LSQHWKPPOFDHHZ-YUMQZZPRSA-N His-Asp-Gly Chemical compound C1=C(NC=N1)C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)NCC(=O)O)N LSQHWKPPOFDHHZ-YUMQZZPRSA-N 0.000 description 1
- RGPWUJOMKFYFSR-QWRGUYRKSA-N His-Gly-Leu Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)NCC(=O)N[C@@H](CC(C)C)C(O)=O RGPWUJOMKFYFSR-QWRGUYRKSA-N 0.000 description 1
- KWBISLAEQZUYIC-UWJYBYFXSA-N His-His-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CC1=CN=CN1)NC(=O)[C@H](CC2=CN=CN2)N KWBISLAEQZUYIC-UWJYBYFXSA-N 0.000 description 1
- AKAPKBNIVNPIPO-KKUMJFAQSA-N His-His-Lys Chemical compound C([C@@H](C(=O)N[C@@H](CCCCN)C(O)=O)NC(=O)[C@@H](N)CC=1NC=NC=1)C1=CN=CN1 AKAPKBNIVNPIPO-KKUMJFAQSA-N 0.000 description 1
- YAALVYQFVJNXIV-KKUMJFAQSA-N His-Leu-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CC1=CN=CN1 YAALVYQFVJNXIV-KKUMJFAQSA-N 0.000 description 1
- KHUFDBQXGLEIHC-BZSNNMDCSA-N His-Leu-Tyr Chemical compound C([C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)C1=CN=CN1 KHUFDBQXGLEIHC-BZSNNMDCSA-N 0.000 description 1
- JUIOPCXACJLRJK-AVGNSLFASA-N His-Lys-Glu Chemical compound C1=C(NC=N1)C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N JUIOPCXACJLRJK-AVGNSLFASA-N 0.000 description 1
- UXSATKFPUVZVDK-KKUMJFAQSA-N His-Lys-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC1=CN=CN1)N UXSATKFPUVZVDK-KKUMJFAQSA-N 0.000 description 1
- CKRJBQJIGOEKMC-SRVKXCTJSA-N His-Lys-Ser Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(O)=O CKRJBQJIGOEKMC-SRVKXCTJSA-N 0.000 description 1
- FJCGVRRVBKYYOU-DCAQKATOSA-N His-Met-Ser Chemical compound CSCC[C@@H](C(=O)N[C@@H](CO)C(=O)O)NC(=O)[C@H](CC1=CN=CN1)N FJCGVRRVBKYYOU-DCAQKATOSA-N 0.000 description 1
- FLXCRBXJRJSDHX-AVGNSLFASA-N His-Pro-Val Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C(C)C)C(O)=O FLXCRBXJRJSDHX-AVGNSLFASA-N 0.000 description 1
- UOYGZBIPZYKGSH-SRVKXCTJSA-N His-Ser-Lys Chemical compound C1=C(NC=N1)C[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)O)N UOYGZBIPZYKGSH-SRVKXCTJSA-N 0.000 description 1
- IAYPZSHNZQHQNO-KKUMJFAQSA-N His-Ser-Phe Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC2=CN=CN2)N IAYPZSHNZQHQNO-KKUMJFAQSA-N 0.000 description 1
- PBJOQLUVSGXRSW-YTQUADARSA-N His-Trp-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC2=CNC3=CC=CC=C32)NC(=O)[C@H](CC4=CN=CN4)N)C(=O)O PBJOQLUVSGXRSW-YTQUADARSA-N 0.000 description 1
- UWNUQPZUSRFIIN-JUKXBJQTSA-N His-Tyr-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)NC(=O)[C@H](CC2=CN=CN2)N UWNUQPZUSRFIIN-JUKXBJQTSA-N 0.000 description 1
- 101000615488 Homo sapiens Methyl-CpG-binding domain protein 2 Proteins 0.000 description 1
- 241000209219 Hordeum Species 0.000 description 1
- 240000005979 Hordeum vulgare Species 0.000 description 1
- 235000007340 Hordeum vulgare Nutrition 0.000 description 1
- AVXURJPOCDRRFD-UHFFFAOYSA-N Hydroxylamine Chemical compound ON AVXURJPOCDRRFD-UHFFFAOYSA-N 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- JRHFQUPIZOYKQP-KBIXCLLPSA-N Ile-Ala-Glu Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCC(O)=O JRHFQUPIZOYKQP-KBIXCLLPSA-N 0.000 description 1
- VZIFYHYNQDIPLI-HJWJTTGWSA-N Ile-Arg-Phe Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)N VZIFYHYNQDIPLI-HJWJTTGWSA-N 0.000 description 1
- HVWXAQVMRBKKFE-UGYAYLCHSA-N Ile-Asp-Asp Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CC(=O)O)C(=O)O)N HVWXAQVMRBKKFE-UGYAYLCHSA-N 0.000 description 1
- JXMSHKFPDIUYGS-SIUGBPQLSA-N Ile-Glu-Tyr Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)O)N JXMSHKFPDIUYGS-SIUGBPQLSA-N 0.000 description 1
- AMSYMDIIIRJRKZ-HJPIBITLSA-N Ile-His-His Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)N AMSYMDIIIRJRKZ-HJPIBITLSA-N 0.000 description 1
- CMNMPCTVCWWYHY-MXAVVETBSA-N Ile-His-Leu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)N[C@@H](CC(C)C)C(=O)O)N CMNMPCTVCWWYHY-MXAVVETBSA-N 0.000 description 1
- PHRWFSFCNJPWRO-PPCPHDFISA-N Ile-Leu-Thr Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(=O)O)N PHRWFSFCNJPWRO-PPCPHDFISA-N 0.000 description 1
- PWUMCBLVWPCKNO-MGHWNKPDSA-N Ile-Leu-Tyr Chemical compound CC[C@H](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 PWUMCBLVWPCKNO-MGHWNKPDSA-N 0.000 description 1
- ADDYYRVQQZFIMW-MNXVOIDGSA-N Ile-Lys-Glu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N ADDYYRVQQZFIMW-MNXVOIDGSA-N 0.000 description 1
- IDMNOFVUXYYZPF-DKIMLUQUSA-N Ile-Lys-Phe Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)N IDMNOFVUXYYZPF-DKIMLUQUSA-N 0.000 description 1
- OTSVBELRDMSPKY-PCBIJLKTSA-N Ile-Phe-Asn Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(=O)N)C(=O)O)N OTSVBELRDMSPKY-PCBIJLKTSA-N 0.000 description 1
- UAELWXJFLZBKQS-WHOFXGATSA-N Ile-Phe-Gly Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](Cc1ccccc1)C(=O)NCC(O)=O UAELWXJFLZBKQS-WHOFXGATSA-N 0.000 description 1
- CIDLJWVDMNDKPT-FIRPJDEBSA-N Ile-Phe-Phe Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC2=CC=CC=C2)C(=O)O)N CIDLJWVDMNDKPT-FIRPJDEBSA-N 0.000 description 1
- MLSUZXHSNRBDCI-CYDGBPFRSA-N Ile-Pro-Val Chemical compound CC[C@H](C)[C@@H](C(=O)N1CCC[C@H]1C(=O)N[C@@H](C(C)C)C(=O)O)N MLSUZXHSNRBDCI-CYDGBPFRSA-N 0.000 description 1
- 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 1
- GMUYXHHJAGQHGB-TUBUOCAGSA-N Ile-Thr-His Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N GMUYXHHJAGQHGB-TUBUOCAGSA-N 0.000 description 1
- NXRNRBOKDBIVKQ-CXTHYWKRSA-N Ile-Tyr-Tyr Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CC2=CC=C(C=C2)O)C(=O)O)N NXRNRBOKDBIVKQ-CXTHYWKRSA-N 0.000 description 1
- JZBVBOKASHNXAD-NAKRPEOUSA-N Ile-Val-Ser Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(=O)O)N JZBVBOKASHNXAD-NAKRPEOUSA-N 0.000 description 1
- APQYGMBHIVXFML-OSUNSFLBSA-N Ile-Val-Thr Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)O)C(=O)O)N APQYGMBHIVXFML-OSUNSFLBSA-N 0.000 description 1
- 108700002232 Immediate-Early Genes Proteins 0.000 description 1
- 108010065920 Insulin Lispro Proteins 0.000 description 1
- 235000006350 Ipomoea batatas var. batatas Nutrition 0.000 description 1
- 102000008133 Iron-Binding Proteins Human genes 0.000 description 1
- 108010035210 Iron-Binding Proteins Proteins 0.000 description 1
- OSELKOCHBMDKEJ-VRUYXKNBSA-N Isofucosterol Natural products CC=C(CC[C@@H](C)[C@H]1CC[C@@H]2[C@H]3CC=C4C[C@@H](O)CC[C@]4(C)[C@@H]3CC[C@]12C)C(C)C OSELKOCHBMDKEJ-VRUYXKNBSA-N 0.000 description 1
- 108090000769 Isomerases Proteins 0.000 description 1
- 102000004195 Isomerases Human genes 0.000 description 1
- 101100288095 Klebsiella pneumoniae neo gene Proteins 0.000 description 1
- 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 1
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 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
- 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 1
- 241000208822 Lactuca Species 0.000 description 1
- 235000003228 Lactuca sativa Nutrition 0.000 description 1
- 240000008415 Lactuca sativa Species 0.000 description 1
- LOPKHWOTGJIQLC-UHFFFAOYSA-N Lanosterol Natural products CC(CCC=C(C)C)C1CCC2(C)C3=C(CCC12C)C4(C)CCC(C)(O)C(C)(C)C4CC3 LOPKHWOTGJIQLC-UHFFFAOYSA-N 0.000 description 1
- 108010059597 Lanosterol synthase Proteins 0.000 description 1
- 102100032011 Lanosterol synthase Human genes 0.000 description 1
- 108091026898 Leader sequence (mRNA) Proteins 0.000 description 1
- 241000880493 Leptailurus serval Species 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
- PBCHMHROGNUXMK-DLOVCJGASA-N Leu-Ala-His Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CC1=CN=CN1 PBCHMHROGNUXMK-DLOVCJGASA-N 0.000 description 1
- QPRQGENIBFLVEB-BJDJZHNGSA-N Leu-Ala-Ile Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O QPRQGENIBFLVEB-BJDJZHNGSA-N 0.000 description 1
- HBJZFCIVFIBNSV-DCAQKATOSA-N Leu-Arg-Asn Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CC(N)=O)C(O)=O HBJZFCIVFIBNSV-DCAQKATOSA-N 0.000 description 1
- 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 1
- OIARJGNVARWKFP-YUMQZZPRSA-N Leu-Asn-Gly Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(O)=O OIARJGNVARWKFP-YUMQZZPRSA-N 0.000 description 1
- OGCQGUIWMSBHRZ-CIUDSAMLSA-N Leu-Asn-Ser Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(O)=O OGCQGUIWMSBHRZ-CIUDSAMLSA-N 0.000 description 1
- TWQIYNGNYNJUFM-NHCYSSNCSA-N Leu-Asn-Val Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C(C)C)C(O)=O TWQIYNGNYNJUFM-NHCYSSNCSA-N 0.000 description 1
- YKNBJXOJTURHCU-DCAQKATOSA-N Leu-Asp-Arg Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CCCN=C(N)N YKNBJXOJTURHCU-DCAQKATOSA-N 0.000 description 1
- PNUCWVAGVNLUMW-CIUDSAMLSA-N Leu-Cys-Ser Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CS)C(=O)N[C@@H](CO)C(O)=O PNUCWVAGVNLUMW-CIUDSAMLSA-N 0.000 description 1
- ZTLGVASZOIKNIX-DCAQKATOSA-N Leu-Gln-Glu Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N ZTLGVASZOIKNIX-DCAQKATOSA-N 0.000 description 1
- RSFGIMMPWAXNML-MNXVOIDGSA-N Leu-Gln-Ile Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O RSFGIMMPWAXNML-MNXVOIDGSA-N 0.000 description 1
- GPICTNQYKHHHTH-GUBZILKMSA-N Leu-Gln-Ser Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(O)=O GPICTNQYKHHHTH-GUBZILKMSA-N 0.000 description 1
- HPBCTWSUJOGJSH-MNXVOIDGSA-N Leu-Glu-Ile Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O HPBCTWSUJOGJSH-MNXVOIDGSA-N 0.000 description 1
- QVFGXCVIXXBFHO-AVGNSLFASA-N Leu-Glu-Leu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O QVFGXCVIXXBFHO-AVGNSLFASA-N 0.000 description 1
- HQUXQAMSWFIRET-AVGNSLFASA-N Leu-Glu-Lys Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@H](C(O)=O)CCCCN HQUXQAMSWFIRET-AVGNSLFASA-N 0.000 description 1
- ZFNLIDNJUWNIJL-WDCWCFNPSA-N Leu-Glu-Thr Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O ZFNLIDNJUWNIJL-WDCWCFNPSA-N 0.000 description 1
- UCDHVOALNXENLC-KBPBESRZSA-N Leu-Gly-Tyr Chemical compound CC(C)C[C@H]([NH3+])C(=O)NCC(=O)N[C@H](C([O-])=O)CC1=CC=C(O)C=C1 UCDHVOALNXENLC-KBPBESRZSA-N 0.000 description 1
- PBGDOSARRIJMEV-DLOVCJGASA-N Leu-His-Ala Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](C)C(O)=O PBGDOSARRIJMEV-DLOVCJGASA-N 0.000 description 1
- YWYQSLOTVIRCFE-SRVKXCTJSA-N Leu-His-Asp Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC(O)=O)C(O)=O YWYQSLOTVIRCFE-SRVKXCTJSA-N 0.000 description 1
- KXODZBLFVFSLAI-AVGNSLFASA-N Leu-His-Glu Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)CC(C)C)CC1=CN=CN1 KXODZBLFVFSLAI-AVGNSLFASA-N 0.000 description 1
- AOFYPTOHESIBFZ-KKUMJFAQSA-N Leu-His-His Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](Cc1cnc[nH]1)C(=O)N[C@@H](Cc1cnc[nH]1)C(O)=O AOFYPTOHESIBFZ-KKUMJFAQSA-N 0.000 description 1
- KVOFSTUWVSQMDK-KKUMJFAQSA-N Leu-His-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)CC(C)C)CC1=CN=CN1 KVOFSTUWVSQMDK-KKUMJFAQSA-N 0.000 description 1
- AVEGDIAXTDVBJS-XUXIUFHCSA-N Leu-Ile-Arg Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O AVEGDIAXTDVBJS-XUXIUFHCSA-N 0.000 description 1
- HGFGEMSVBMCFKK-MNXVOIDGSA-N Leu-Ile-Glu Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(O)=O)C(O)=O HGFGEMSVBMCFKK-MNXVOIDGSA-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
- 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
- IEWBEPKLKUXQBU-VOAKCMCISA-N Leu-Leu-Thr Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O IEWBEPKLKUXQBU-VOAKCMCISA-N 0.000 description 1
- BGZCJDGBBUUBHA-KKUMJFAQSA-N Leu-Lys-Leu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(O)=O BGZCJDGBBUUBHA-KKUMJFAQSA-N 0.000 description 1
- INCJJHQRZGQLFC-KBPBESRZSA-N Leu-Phe-Gly Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)NCC(O)=O INCJJHQRZGQLFC-KBPBESRZSA-N 0.000 description 1
- 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 1
- QMKFDEUJGYNFMC-AVGNSLFASA-N Leu-Pro-Arg Chemical compound CC(C)C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCN=C(N)N)C(O)=O QMKFDEUJGYNFMC-AVGNSLFASA-N 0.000 description 1
- MUCIDQMDOYQYBR-IHRRRGAJSA-N Leu-Pro-His Chemical compound CC(C)C[C@@H](C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)N MUCIDQMDOYQYBR-IHRRRGAJSA-N 0.000 description 1
- IRMLZWSRWSGTOP-CIUDSAMLSA-N Leu-Ser-Ala Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(O)=O IRMLZWSRWSGTOP-CIUDSAMLSA-N 0.000 description 1
- KZZCOWMDDXDKSS-CIUDSAMLSA-N Leu-Ser-Asn Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(O)=O KZZCOWMDDXDKSS-CIUDSAMLSA-N 0.000 description 1
- XOWMDXHFSBCAKQ-SRVKXCTJSA-N Leu-Ser-Leu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CC(C)C XOWMDXHFSBCAKQ-SRVKXCTJSA-N 0.000 description 1
- SQUFDMCWMFOEBA-KKUMJFAQSA-N Leu-Ser-Tyr Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 SQUFDMCWMFOEBA-KKUMJFAQSA-N 0.000 description 1
- CNWDWAMPKVYJJB-NUTKFTJISA-N Leu-Trp-Ala Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@@H](N)CC(C)C)C(=O)N[C@@H](C)C(O)=O)=CNC2=C1 CNWDWAMPKVYJJB-NUTKFTJISA-N 0.000 description 1
- IDGRADDMTTWOQC-WDSOQIARSA-N Leu-Trp-Arg Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O IDGRADDMTTWOQC-WDSOQIARSA-N 0.000 description 1
- LSLUTXRANSUGFY-XIRDDKMYSA-N Leu-Trp-Asp Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CC(O)=O)C(O)=O LSLUTXRANSUGFY-XIRDDKMYSA-N 0.000 description 1
- FPFOYSCDUWTZBF-IHPCNDPISA-N Leu-Trp-Leu Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@@H]([NH3+])CC(C)C)C(=O)N[C@@H](CC(C)C)C([O-])=O)=CNC2=C1 FPFOYSCDUWTZBF-IHPCNDPISA-N 0.000 description 1
- JGKHAFUAPZCCDU-BZSNNMDCSA-N Leu-Tyr-Leu Chemical compound CC(C)C[C@H]([NH3+])C(=O)N[C@H](C(=O)N[C@@H](CC(C)C)C([O-])=O)CC1=CC=C(O)C=C1 JGKHAFUAPZCCDU-BZSNNMDCSA-N 0.000 description 1
- BTEMNFBEAAOGBR-BZSNNMDCSA-N Leu-Tyr-Lys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CCCCN)C(=O)O)N BTEMNFBEAAOGBR-BZSNNMDCSA-N 0.000 description 1
- UFPLDOKWDNTTRP-ULQDDVLXSA-N Leu-Tyr-Met Chemical compound CSCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)CC(C)C)CC1=CC=C(O)C=C1 UFPLDOKWDNTTRP-ULQDDVLXSA-N 0.000 description 1
- XZNJZXJZBMBGGS-NHCYSSNCSA-N Leu-Val-Asn Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O XZNJZXJZBMBGGS-NHCYSSNCSA-N 0.000 description 1
- AIMGJYMCTAABEN-GVXVVHGQSA-N Leu-Val-Glu Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O AIMGJYMCTAABEN-GVXVVHGQSA-N 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 241000724705 Lucerne transient streak virus Species 0.000 description 1
- 235000002262 Lycopersicon Nutrition 0.000 description 1
- KPJJOZUXFOLGMQ-CIUDSAMLSA-N Lys-Asp-Asn Chemical compound C(CCN)C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CC(=O)N)C(=O)O)N KPJJOZUXFOLGMQ-CIUDSAMLSA-N 0.000 description 1
- DZQYZKPINJLLEN-KKUMJFAQSA-N Lys-Cys-Tyr Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H](CCCCN)N)O DZQYZKPINJLLEN-KKUMJFAQSA-N 0.000 description 1
- YFGWNAROEYWGNL-GUBZILKMSA-N Lys-Gln-Asn Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O YFGWNAROEYWGNL-GUBZILKMSA-N 0.000 description 1
- ULUQBUKAPDUKOC-GVXVVHGQSA-N Lys-Glu-Val Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O ULUQBUKAPDUKOC-GVXVVHGQSA-N 0.000 description 1
- SQJSXOQXJYAVRV-SRVKXCTJSA-N Lys-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](CCCCN)N SQJSXOQXJYAVRV-SRVKXCTJSA-N 0.000 description 1
- IZJGPPIGYTVXLB-FQUUOJAGSA-N Lys-Ile-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CCCCN)N IZJGPPIGYTVXLB-FQUUOJAGSA-N 0.000 description 1
- 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 1
- MYZMQWHPDAYKIE-SRVKXCTJSA-N Lys-Leu-Ala Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(O)=O MYZMQWHPDAYKIE-SRVKXCTJSA-N 0.000 description 1
- OVAOHZIOUBEQCJ-IHRRRGAJSA-N Lys-Leu-Arg Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O OVAOHZIOUBEQCJ-IHRRRGAJSA-N 0.000 description 1
- WVJNGSFKBKOKRV-AJNGGQMLSA-N Lys-Leu-Ile Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O WVJNGSFKBKOKRV-AJNGGQMLSA-N 0.000 description 1
- VUTWYNQUSJWBHO-BZSNNMDCSA-N Lys-Leu-Tyr Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O VUTWYNQUSJWBHO-BZSNNMDCSA-N 0.000 description 1
- QQPSCXKFDSORFT-IHRRRGAJSA-N Lys-Lys-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CCCCN QQPSCXKFDSORFT-IHRRRGAJSA-N 0.000 description 1
- GZGWILAQHOVXTD-DCAQKATOSA-N Lys-Met-Asp Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(O)=O GZGWILAQHOVXTD-DCAQKATOSA-N 0.000 description 1
- JOSAKOKSPXROGQ-BJDJZHNGSA-N Lys-Ser-Ile Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O JOSAKOKSPXROGQ-BJDJZHNGSA-N 0.000 description 1
- CUHGAUZONORRIC-HJGDQZAQSA-N Lys-Thr-Asn Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](CCCCN)N)O CUHGAUZONORRIC-HJGDQZAQSA-N 0.000 description 1
- KXYLFJIQDIMURW-IHPCNDPISA-N Lys-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)CCCCN)=CNC2=C1 KXYLFJIQDIMURW-IHPCNDPISA-N 0.000 description 1
- MIMXMVDLMDMOJD-BZSNNMDCSA-N Lys-Tyr-Leu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(C)C)C(O)=O MIMXMVDLMDMOJD-BZSNNMDCSA-N 0.000 description 1
- LMMBAXJRYSXCOQ-ACRUOGEOSA-N Lys-Tyr-Phe Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H](Cc1ccccc1)C(O)=O LMMBAXJRYSXCOQ-ACRUOGEOSA-N 0.000 description 1
- VVURYEVJJTXWNE-ULQDDVLXSA-N Lys-Tyr-Val Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](C(C)C)C(O)=O VVURYEVJJTXWNE-ULQDDVLXSA-N 0.000 description 1
- MDDUIRLQCYVRDO-NHCYSSNCSA-N Lys-Val-Asn Chemical compound NC(=O)C[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CCCCN MDDUIRLQCYVRDO-NHCYSSNCSA-N 0.000 description 1
- GILLQRYAWOMHED-DCAQKATOSA-N Lys-Val-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CCCCN GILLQRYAWOMHED-DCAQKATOSA-N 0.000 description 1
- 241000218922 Magnoliophyta Species 0.000 description 1
- 235000011430 Malus pumila Nutrition 0.000 description 1
- 235000015103 Malus silvestris Nutrition 0.000 description 1
- 240000003183 Manihot esculenta Species 0.000 description 1
- VPRLICVDSGMIKO-UHFFFAOYSA-N Mannopine Natural products NC(=O)CCC(C(O)=O)NCC(O)C(O)C(O)C(O)CO VPRLICVDSGMIKO-UHFFFAOYSA-N 0.000 description 1
- 240000004658 Medicago sativa Species 0.000 description 1
- 235000017587 Medicago sativa ssp. sativa Nutrition 0.000 description 1
- VTKPSXWRUGCOAC-GUBZILKMSA-N Met-Ala-Met Chemical compound CSCC[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCSC VTKPSXWRUGCOAC-GUBZILKMSA-N 0.000 description 1
- MNNKPHGAPRUKMW-BPUTZDHNSA-N Met-Asp-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](N)CCSC)C(O)=O)=CNC2=C1 MNNKPHGAPRUKMW-BPUTZDHNSA-N 0.000 description 1
- FVKRQMQQFGBXHV-QXEWZRGKSA-N Met-Asp-Val Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O FVKRQMQQFGBXHV-QXEWZRGKSA-N 0.000 description 1
- JPCHYAUKOUGOIB-HJGDQZAQSA-N Met-Glu-Thr Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O JPCHYAUKOUGOIB-HJGDQZAQSA-N 0.000 description 1
- IUYCGMNKIZDRQI-BQBZGAKWSA-N Met-Gly-Ala Chemical compound CSCC[C@H](N)C(=O)NCC(=O)N[C@@H](C)C(O)=O IUYCGMNKIZDRQI-BQBZGAKWSA-N 0.000 description 1
- SXWQMBGNFXAGAT-FJXKBIBVSA-N Met-Gly-Thr Chemical compound CSCC[C@H](N)C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(O)=O SXWQMBGNFXAGAT-FJXKBIBVSA-N 0.000 description 1
- QGRJTULYDZUBAY-ZPFDUUQYSA-N Met-Ile-Glu Chemical compound [H]N[C@@H](CCSC)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(O)=O)C(O)=O QGRJTULYDZUBAY-ZPFDUUQYSA-N 0.000 description 1
- WPTDJKDGICUFCP-XUXIUFHCSA-N Met-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](CCSC)N WPTDJKDGICUFCP-XUXIUFHCSA-N 0.000 description 1
- HGAJNEWOUHDUMZ-SRVKXCTJSA-N Met-Leu-Glu Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CCC(O)=O HGAJNEWOUHDUMZ-SRVKXCTJSA-N 0.000 description 1
- XDGFFEZAZHRZFR-RHYQMDGZSA-N Met-Leu-Thr Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O XDGFFEZAZHRZFR-RHYQMDGZSA-N 0.000 description 1
- UNPGTBHYKJOCCZ-DCAQKATOSA-N Met-Lys-Ala Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(O)=O UNPGTBHYKJOCCZ-DCAQKATOSA-N 0.000 description 1
- KRLKICLNEICJGV-STQMWFEESA-N Met-Phe-Gly Chemical compound CSCC[C@H](N)C(=O)N[C@H](C(=O)NCC(O)=O)CC1=CC=CC=C1 KRLKICLNEICJGV-STQMWFEESA-N 0.000 description 1
- RMLWDZINJUDMEB-IHRRRGAJSA-N Met-Tyr-Asn Chemical compound CSCC[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 RMLWDZINJUDMEB-IHRRRGAJSA-N 0.000 description 1
- ATBJCCFCJXCNGZ-UFYCRDLUSA-N Met-Tyr-Phe Chemical compound C([C@H](NC(=O)[C@@H](N)CCSC)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=C(O)C=C1 ATBJCCFCJXCNGZ-UFYCRDLUSA-N 0.000 description 1
- 102100021299 Methyl-CpG-binding domain protein 2 Human genes 0.000 description 1
- 101710142850 Methylsterol monooxygenase 1 Proteins 0.000 description 1
- 102100021091 Methylsterol monooxygenase 1 Human genes 0.000 description 1
- AUEJLPRZGVVDNU-UHFFFAOYSA-N N-L-tyrosyl-L-leucine Natural products CC(C)CC(C(O)=O)NC(=O)C(N)CC1=CC=C(O)C=C1 AUEJLPRZGVVDNU-UHFFFAOYSA-N 0.000 description 1
- 108010066427 N-valyltryptophan Proteins 0.000 description 1
- 108010087066 N2-tryptophyllysine Proteins 0.000 description 1
- 108091061960 Naked DNA Proteins 0.000 description 1
- CAHGCLMLTWQZNJ-UHFFFAOYSA-N Nerifoliol Natural products CC12CCC(O)C(C)(C)C1CCC1=C2CCC2(C)C(C(CCC=C(C)C)C)CCC21C CAHGCLMLTWQZNJ-UHFFFAOYSA-N 0.000 description 1
- 101100342977 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) leu-1 gene Proteins 0.000 description 1
- 241000208125 Nicotiana Species 0.000 description 1
- 108010038807 Oligopeptides Proteins 0.000 description 1
- 102000015636 Oligopeptides Human genes 0.000 description 1
- 102000043276 Oncogene Human genes 0.000 description 1
- 108700020796 Oncogene Proteins 0.000 description 1
- 108700005081 Overlapping Genes Proteins 0.000 description 1
- 239000012807 PCR reagent Substances 0.000 description 1
- 101710091688 Patatin Proteins 0.000 description 1
- 241000218196 Persea Species 0.000 description 1
- 240000007377 Petunia x hybrida Species 0.000 description 1
- 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 1
- FPTXMUIBLMGTQH-ONGXEEELSA-N Phe-Ala-Gly Chemical compound OC(=O)CNC(=O)[C@H](C)NC(=O)[C@@H](N)CC1=CC=CC=C1 FPTXMUIBLMGTQH-ONGXEEELSA-N 0.000 description 1
- UHRNIXJAGGLKHP-DLOVCJGASA-N Phe-Ala-Ser Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O UHRNIXJAGGLKHP-DLOVCJGASA-N 0.000 description 1
- LXVFHIBXOWJTKZ-BZSNNMDCSA-N Phe-Asn-Tyr Chemical compound N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)O LXVFHIBXOWJTKZ-BZSNNMDCSA-N 0.000 description 1
- XMPUYNHKEPFERE-IHRRRGAJSA-N Phe-Asp-Arg Chemical compound NC(N)=NCCC[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](N)CC1=CC=CC=C1 XMPUYNHKEPFERE-IHRRRGAJSA-N 0.000 description 1
- 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 1
- IUVYJBMTHARMIP-PCBIJLKTSA-N Phe-Asp-Ile 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)CC)C(O)=O IUVYJBMTHARMIP-PCBIJLKTSA-N 0.000 description 1
- DJPXNKUDJKGQEE-BZSNNMDCSA-N Phe-Asp-Phe Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O DJPXNKUDJKGQEE-BZSNNMDCSA-N 0.000 description 1
- MQVFHOPCKNTHGT-MELADBBJSA-N Phe-Asp-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC(=O)O)NC(=O)[C@H](CC2=CC=CC=C2)N)C(=O)O MQVFHOPCKNTHGT-MELADBBJSA-N 0.000 description 1
- 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 1
- APJPXSFJBMMOLW-KBPBESRZSA-N Phe-Gly-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CC1=CC=CC=C1 APJPXSFJBMMOLW-KBPBESRZSA-N 0.000 description 1
- KRYSMKKRRRWOCZ-QEWYBTABSA-N Phe-Ile-Glu Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(O)=O)C(O)=O KRYSMKKRRRWOCZ-QEWYBTABSA-N 0.000 description 1
- BWTKUQPNOMMKMA-FIRPJDEBSA-N Phe-Ile-Phe Chemical compound C([C@H](N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 BWTKUQPNOMMKMA-FIRPJDEBSA-N 0.000 description 1
- CWFGECHCRMGPPT-MXAVVETBSA-N Phe-Ile-Ser Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(O)=O CWFGECHCRMGPPT-MXAVVETBSA-N 0.000 description 1
- KXUZHWXENMYOHC-QEJZJMRPSA-N Phe-Leu-Ala Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(O)=O KXUZHWXENMYOHC-QEJZJMRPSA-N 0.000 description 1
- RSPUIENXSJYZQO-JYJNAYRXSA-N Phe-Leu-Gln Chemical compound NC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CC1=CC=CC=C1 RSPUIENXSJYZQO-JYJNAYRXSA-N 0.000 description 1
- MSHZERMPZKCODG-ACRUOGEOSA-N Phe-Leu-Phe Chemical compound C([C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 MSHZERMPZKCODG-ACRUOGEOSA-N 0.000 description 1
- QRUOLOPKCOEZKU-HJWJTTGWSA-N Phe-Met-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CCSC)NC(=O)[C@H](CC1=CC=CC=C1)N QRUOLOPKCOEZKU-HJWJTTGWSA-N 0.000 description 1
- WKLMCMXFMQEKCX-SLFFLAALSA-N Phe-Phe-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC2=CC=CC=C2)NC(=O)[C@H](CC3=CC=CC=C3)N)C(=O)O WKLMCMXFMQEKCX-SLFFLAALSA-N 0.000 description 1
- CKJACGQPCPMWIT-UFYCRDLUSA-N Phe-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=CC=C1 CKJACGQPCPMWIT-UFYCRDLUSA-N 0.000 description 1
- XDMMOISUAHXXFD-SRVKXCTJSA-N Phe-Ser-Asp Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(O)=O XDMMOISUAHXXFD-SRVKXCTJSA-N 0.000 description 1
- RAGOJJCBGXARPO-XVSYOHENSA-N Phe-Thr-Asp Chemical compound OC(=O)C[C@@H](C(O)=O)NC(=O)[C@H]([C@H](O)C)NC(=O)[C@@H](N)CC1=CC=CC=C1 RAGOJJCBGXARPO-XVSYOHENSA-N 0.000 description 1
- KLYYKKGCPOGDPE-OEAJRASXSA-N Phe-Thr-Leu Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(O)=O KLYYKKGCPOGDPE-OEAJRASXSA-N 0.000 description 1
- WDOCBGZHAQQIBL-IHPCNDPISA-N Phe-Trp-Ser Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CO)C(O)=O)C1=CC=CC=C1 WDOCBGZHAQQIBL-IHPCNDPISA-N 0.000 description 1
- GCFNFKNPCMBHNT-IRXDYDNUSA-N Phe-Tyr-Gly Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CC2=CC=C(C=C2)O)C(=O)NCC(=O)O)N GCFNFKNPCMBHNT-IRXDYDNUSA-N 0.000 description 1
- GOUWCZRDTWTODO-YDHLFZDLSA-N Phe-Val-Asn Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O GOUWCZRDTWTODO-YDHLFZDLSA-N 0.000 description 1
- GNZCMRRSXOBHLC-JYJNAYRXSA-N Phe-Val-Met Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCSC)C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)N GNZCMRRSXOBHLC-JYJNAYRXSA-N 0.000 description 1
- VDTYRPWRWRCROL-UFYCRDLUSA-N Phe-Val-Phe Chemical compound C([C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 VDTYRPWRWRCROL-UFYCRDLUSA-N 0.000 description 1
- 241000218657 Picea Species 0.000 description 1
- 241000219843 Pisum Species 0.000 description 1
- 108020005120 Plant DNA Proteins 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- OOLOTUZJUBOMAX-GUBZILKMSA-N Pro-Ala-Val Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](C)C(=O)N[C@@H](C(C)C)C(O)=O OOLOTUZJUBOMAX-GUBZILKMSA-N 0.000 description 1
- SWXSLPHTJVAWDF-VEVYYDQMSA-N Pro-Asn-Thr Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O SWXSLPHTJVAWDF-VEVYYDQMSA-N 0.000 description 1
- JUJGNDZIKKQMDJ-IHRRRGAJSA-N Pro-His-His Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC1=CNC=N1)C(O)=O JUJGNDZIKKQMDJ-IHRRRGAJSA-N 0.000 description 1
- LPGSNRSLPHRNBW-AVGNSLFASA-N Pro-His-Val Chemical compound C([C@@H](C(=O)N[C@@H](C(C)C)C([O-])=O)NC(=O)[C@H]1[NH2+]CCC1)C1=CN=CN1 LPGSNRSLPHRNBW-AVGNSLFASA-N 0.000 description 1
- CLJLVCYFABNTHP-DCAQKATOSA-N Pro-Leu-Asp Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O CLJLVCYFABNTHP-DCAQKATOSA-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
- 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 1
- SMFQZMGHCODUPQ-ULQDDVLXSA-N Pro-Lys-Phe Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O SMFQZMGHCODUPQ-ULQDDVLXSA-N 0.000 description 1
- ZUZINZIJHJFJRN-UBHSHLNASA-N Pro-Phe-Ala Chemical compound C([C@@H](C(=O)N[C@@H](C)C(O)=O)NC(=O)[C@H]1NCCC1)C1=CC=CC=C1 ZUZINZIJHJFJRN-UBHSHLNASA-N 0.000 description 1
- WHNJMTHJGCEKGA-ULQDDVLXSA-N Pro-Phe-Leu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(C)C)C(O)=O WHNJMTHJGCEKGA-ULQDDVLXSA-N 0.000 description 1
- GXWRTSIVLSQACD-RCWTZXSCSA-N Pro-Thr-Met Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCSC)C(=O)O)NC(=O)[C@@H]1CCCN1)O GXWRTSIVLSQACD-RCWTZXSCSA-N 0.000 description 1
- DIDLUFMLRUJLFB-FKBYEOEOSA-N Pro-Trp-Tyr Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CC2=CNC3=CC=CC=C32)C(=O)N[C@@H](CC4=CC=C(C=C4)O)C(=O)O DIDLUFMLRUJLFB-FKBYEOEOSA-N 0.000 description 1
- ZAUHSLVPDLNTRZ-QXEWZRGKSA-N Pro-Val-Asn Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O ZAUHSLVPDLNTRZ-QXEWZRGKSA-N 0.000 description 1
- 102000001253 Protein Kinase Human genes 0.000 description 1
- 235000011432 Prunus Nutrition 0.000 description 1
- 235000014443 Pyrus communis Nutrition 0.000 description 1
- 241000220259 Raphanus Species 0.000 description 1
- 101001115837 Rattus norvegicus Methylsterol monooxygenase 1 Proteins 0.000 description 1
- 102000006382 Ribonucleases Human genes 0.000 description 1
- 108010083644 Ribonucleases Proteins 0.000 description 1
- 108091028664 Ribonucleotide Proteins 0.000 description 1
- 108010003581 Ribulose-bisphosphate carboxylase Proteins 0.000 description 1
- 241000220317 Rosa Species 0.000 description 1
- 241000209056 Secale Species 0.000 description 1
- WTWGOQRNRFHFQD-JBDRJPRFSA-N Ser-Ala-Ile Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O WTWGOQRNRFHFQD-JBDRJPRFSA-N 0.000 description 1
- RDFQNDHEHVSONI-ZLUOBGJFSA-N Ser-Asn-Ser Chemical compound OC[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(O)=O RDFQNDHEHVSONI-ZLUOBGJFSA-N 0.000 description 1
- WBINSDOPZHQPPM-AVGNSLFASA-N Ser-Glu-Tyr Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CO)N)O WBINSDOPZHQPPM-AVGNSLFASA-N 0.000 description 1
- GZFAWAQTEYDKII-YUMQZZPRSA-N Ser-Gly-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CO GZFAWAQTEYDKII-YUMQZZPRSA-N 0.000 description 1
- SFTZTYBXIXLRGQ-JBDRJPRFSA-N Ser-Ile-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(O)=O SFTZTYBXIXLRGQ-JBDRJPRFSA-N 0.000 description 1
- BEAFYHFQTOTVFS-VGDYDELISA-N Ser-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](CO)N BEAFYHFQTOTVFS-VGDYDELISA-N 0.000 description 1
- VMLONWHIORGALA-SRVKXCTJSA-N Ser-Leu-Leu Chemical compound CC(C)C[C@@H](C([O-])=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H]([NH3+])CO VMLONWHIORGALA-SRVKXCTJSA-N 0.000 description 1
- 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 1
- XXNYYSXNXCJYKX-DCAQKATOSA-N Ser-Leu-Met Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(O)=O XXNYYSXNXCJYKX-DCAQKATOSA-N 0.000 description 1
- VZQRNAYURWAEFE-KKUMJFAQSA-N Ser-Leu-Phe Chemical compound OC[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 VZQRNAYURWAEFE-KKUMJFAQSA-N 0.000 description 1
- KCGIREHVWRXNDH-GARJFASQSA-N Ser-Leu-Pro Chemical compound CC(C)C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CO)N KCGIREHVWRXNDH-GARJFASQSA-N 0.000 description 1
- 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 1
- JWOBLHJRDADHLN-KKUMJFAQSA-N Ser-Leu-Tyr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O JWOBLHJRDADHLN-KKUMJFAQSA-N 0.000 description 1
- HDBOEVPDIDDEPC-CIUDSAMLSA-N Ser-Lys-Asn Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(O)=O HDBOEVPDIDDEPC-CIUDSAMLSA-N 0.000 description 1
- 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 1
- NQZFFLBPNDLTPO-DLOVCJGASA-N Ser-Phe-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)NC(=O)[C@H](CO)N NQZFFLBPNDLTPO-DLOVCJGASA-N 0.000 description 1
- UGTZYIPOBYXWRW-SRVKXCTJSA-N Ser-Phe-Asp Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(O)=O)C(O)=O UGTZYIPOBYXWRW-SRVKXCTJSA-N 0.000 description 1
- MQUZANJDFOQOBX-SRVKXCTJSA-N Ser-Phe-Ser Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(O)=O MQUZANJDFOQOBX-SRVKXCTJSA-N 0.000 description 1
- ZKBKUWQVDWWSRI-BZSNNMDCSA-N Ser-Phe-Tyr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O ZKBKUWQVDWWSRI-BZSNNMDCSA-N 0.000 description 1
- NVNPWELENFJOHH-CIUDSAMLSA-N Ser-Ser-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](CO)N NVNPWELENFJOHH-CIUDSAMLSA-N 0.000 description 1
- JCLAFVNDBJMLBC-JBDRJPRFSA-N Ser-Ser-Ile Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O JCLAFVNDBJMLBC-JBDRJPRFSA-N 0.000 description 1
- JURQXQBJKUHGJS-UHFFFAOYSA-N Ser-Ser-Ser-Ser Chemical compound OCC(N)C(=O)NC(CO)C(=O)NC(CO)C(=O)NC(CO)C(O)=O JURQXQBJKUHGJS-UHFFFAOYSA-N 0.000 description 1
- NADLKBTYNKUJEP-KATARQTJSA-N Ser-Thr-Leu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(O)=O NADLKBTYNKUJEP-KATARQTJSA-N 0.000 description 1
- FGBLCMLXHRPVOF-IHRRRGAJSA-N Ser-Tyr-Arg Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O FGBLCMLXHRPVOF-IHRRRGAJSA-N 0.000 description 1
- ZVBCMFDJIMUELU-BZSNNMDCSA-N Ser-Tyr-Phe Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)O)NC(=O)[C@H](CC2=CC=C(C=C2)O)NC(=O)[C@H](CO)N ZVBCMFDJIMUELU-BZSNNMDCSA-N 0.000 description 1
- HAYADTTXNZFUDM-IHRRRGAJSA-N Ser-Tyr-Val Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](C(C)C)C(O)=O HAYADTTXNZFUDM-IHRRRGAJSA-N 0.000 description 1
- PCMZJFMUYWIERL-ZKWXMUAHSA-N Ser-Val-Asn Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O PCMZJFMUYWIERL-ZKWXMUAHSA-N 0.000 description 1
- 241000207763 Solanum Species 0.000 description 1
- 235000002634 Solanum Nutrition 0.000 description 1
- 241000724704 Solanum nodiflorum mottle virus Species 0.000 description 1
- 235000002595 Solanum tuberosum Nutrition 0.000 description 1
- 244000061456 Solanum tuberosum Species 0.000 description 1
- 235000007230 Sorghum bicolor Nutrition 0.000 description 1
- 235000011684 Sorghum saccharatum Nutrition 0.000 description 1
- 235000009337 Spinacia oleracea Nutrition 0.000 description 1
- 244000300264 Spinacia oleracea Species 0.000 description 1
- 108091081024 Start codon Proteins 0.000 description 1
- 241000724703 Subterranean clover mottle virus Species 0.000 description 1
- 235000021536 Sugar beet Nutrition 0.000 description 1
- ZQVJBRJGDVZANE-UHFFFAOYSA-N Syringomycin Natural products N1C(=O)C(CCCN=C(N)N)NC(=O)C(CCN)NC(=O)C(CCN)NC(=O)C(CO)NC(=O)C(NC(=O)CC(O)CCCCCCCCC)COC(=O)C(C(O)CCl)NC(=O)C(C(O)C(O)=O)NC(=O)C(=CC)NC(=O)C1CC1=CC=CC=C1 ZQVJBRJGDVZANE-UHFFFAOYSA-N 0.000 description 1
- SBSXXCCMIWEPEE-UDNJSVTLSA-N Teasterone Natural products O=C1[C@H]2[C@@](C)([C@@H]3[C@H]([C@H]4[C@](C)([C@@H]([C@@H]([C@@H](O)[C@H](O)[C@H](C(C)C)C)C)CC4)CC3)C1)CC[C@H](O)C2 SBSXXCCMIWEPEE-UDNJSVTLSA-N 0.000 description 1
- RYYWUUFWQRZTIU-UHFFFAOYSA-N Thiophosphoric acid Chemical class OP(O)(S)=O RYYWUUFWQRZTIU-UHFFFAOYSA-N 0.000 description 1
- IGROJMCBGRFRGI-YTLHQDLWSA-N Thr-Ala-Ala Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(O)=O IGROJMCBGRFRGI-YTLHQDLWSA-N 0.000 description 1
- XSLXHSYIVPGEER-KZVJFYERSA-N Thr-Ala-Val Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(=O)N[C@@H](C(C)C)C(O)=O XSLXHSYIVPGEER-KZVJFYERSA-N 0.000 description 1
- NAXBBCLCEOTAIG-RHYQMDGZSA-N Thr-Arg-Lys Chemical compound NC(N)=NCCC[C@H](NC(=O)[C@@H](N)[C@H](O)C)C(=O)N[C@@H](CCCCN)C(O)=O NAXBBCLCEOTAIG-RHYQMDGZSA-N 0.000 description 1
- NLJKZUGAIIRWJN-LKXGYXEUSA-N Thr-Asp-Cys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CS)C(=O)O)N)O NLJKZUGAIIRWJN-LKXGYXEUSA-N 0.000 description 1
- HJOSVGCWOTYJFG-WDCWCFNPSA-N Thr-Glu-Lys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CCCCN)C(=O)O)N)O HJOSVGCWOTYJFG-WDCWCFNPSA-N 0.000 description 1
- XOTBWOCSLMBGMF-SUSMZKCASA-N Thr-Glu-Thr Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O XOTBWOCSLMBGMF-SUSMZKCASA-N 0.000 description 1
- XSTGOZBBXFKGHA-YJRXYDGGSA-N Thr-His-His Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)N)O XSTGOZBBXFKGHA-YJRXYDGGSA-N 0.000 description 1
- IMDMLDSVUSMAEJ-HJGDQZAQSA-N Thr-Leu-Asn Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O IMDMLDSVUSMAEJ-HJGDQZAQSA-N 0.000 description 1
- RRRRCRYTLZVCEN-HJGDQZAQSA-N Thr-Leu-Asp Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O RRRRCRYTLZVCEN-HJGDQZAQSA-N 0.000 description 1
- RFKVQLIXNVEOMB-WEDXCCLWSA-N Thr-Leu-Gly Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)O)N)O RFKVQLIXNVEOMB-WEDXCCLWSA-N 0.000 description 1
- MEJHFIOYJHTWMK-VOAKCMCISA-N Thr-Leu-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)[C@@H](C)O MEJHFIOYJHTWMK-VOAKCMCISA-N 0.000 description 1
- UJQVSMNQMQHVRY-KZVJFYERSA-N Thr-Met-Ala Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](C)C(O)=O UJQVSMNQMQHVRY-KZVJFYERSA-N 0.000 description 1
- 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 1
- YGZWVPBHYABGLT-KJEVXHAQSA-N Thr-Pro-Tyr Chemical compound C[C@@H](O)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 YGZWVPBHYABGLT-KJEVXHAQSA-N 0.000 description 1
- AHERARIZBPOMNU-KATARQTJSA-N Thr-Ser-Leu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(O)=O AHERARIZBPOMNU-KATARQTJSA-N 0.000 description 1
- ZMYCLHFLHRVOEA-HEIBUPTGSA-N Thr-Thr-Ser Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(O)=O ZMYCLHFLHRVOEA-HEIBUPTGSA-N 0.000 description 1
- GRIUMVXCJDKVPI-IZPVPAKOSA-N Thr-Thr-Tyr Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O GRIUMVXCJDKVPI-IZPVPAKOSA-N 0.000 description 1
- GJOBRAHDRIDAPT-NGTWOADLSA-N Thr-Trp-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)NC(=O)[C@H]([C@@H](C)O)N GJOBRAHDRIDAPT-NGTWOADLSA-N 0.000 description 1
- NLWDSYKZUPRMBJ-IEGACIPQSA-N Thr-Trp-Leu Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)N[C@@H](CC(C)C)C(=O)O)N)O NLWDSYKZUPRMBJ-IEGACIPQSA-N 0.000 description 1
- RPECVQBNONKZAT-WZLNRYEVSA-N Thr-Tyr-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)NC(=O)[C@H]([C@@H](C)O)N RPECVQBNONKZAT-WZLNRYEVSA-N 0.000 description 1
- KPMIQCXJDVKWKO-IFFSRLJSSA-N Thr-Val-Glu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O KPMIQCXJDVKWKO-IFFSRLJSSA-N 0.000 description 1
- CURFABYITJVKEW-QTKMDUPCSA-N Thr-Val-His Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N)O CURFABYITJVKEW-QTKMDUPCSA-N 0.000 description 1
- QNXZCKMXHPULME-ZNSHCXBVSA-N Thr-Val-Pro Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](C(C)C)C(=O)N1CCC[C@@H]1C(=O)O)N)O QNXZCKMXHPULME-ZNSHCXBVSA-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
- 241000723677 Tobacco ringspot virus Species 0.000 description 1
- 108700009124 Transcription Initiation Site Proteins 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- RSUXQZNWAOTBQF-XIRDDKMYSA-N Trp-Arg-Gln Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N RSUXQZNWAOTBQF-XIRDDKMYSA-N 0.000 description 1
- DTPARJBMONKGGC-IHPCNDPISA-N Trp-Cys-Phe Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H](CC2=CNC3=CC=CC=C32)N DTPARJBMONKGGC-IHPCNDPISA-N 0.000 description 1
- PTAWAMWPRFTACW-SZMVWBNQSA-N Trp-Gln-Lys Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CCCCN)C(=O)O)N PTAWAMWPRFTACW-SZMVWBNQSA-N 0.000 description 1
- KDWZQYUTMJSYRJ-BHYGNILZSA-N Trp-Glu-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CC2=CNC3=CC=CC=C32)N)C(=O)O KDWZQYUTMJSYRJ-BHYGNILZSA-N 0.000 description 1
- CSRCUZAVBSEDMB-FDARSICLSA-N Trp-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](CC1=CNC2=CC=CC=C21)N CSRCUZAVBSEDMB-FDARSICLSA-N 0.000 description 1
- MEZCXKYMMQJRDE-PMVMPFDFSA-N Trp-Leu-Tyr Chemical compound C([C@H](NC(=O)[C@@H](NC(=O)[C@@H](N)CC=1C2=CC=CC=C2NC=1)CC(C)C)C(O)=O)C1=CC=C(O)C=C1 MEZCXKYMMQJRDE-PMVMPFDFSA-N 0.000 description 1
- JDWUNEPOEZAZGD-BVSLBCMMSA-N Trp-Phe-Met Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(O)=O)NC(=O)[C@@H](N)CC=1C2=CC=CC=C2NC=1)C1=CC=CC=C1 JDWUNEPOEZAZGD-BVSLBCMMSA-N 0.000 description 1
- UHXOYRWHIQZAKV-SZMVWBNQSA-N Trp-Pro-Arg Chemical compound O=C([C@H](CC=1C2=CC=CC=C2NC=1)N)N1CCC[C@H]1C(=O)N[C@@H](CCCN=C(N)N)C(O)=O UHXOYRWHIQZAKV-SZMVWBNQSA-N 0.000 description 1
- VCGOTJGGBXEBFO-FDARSICLSA-N Trp-Pro-Ile Chemical compound [H]N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)CC)C(O)=O VCGOTJGGBXEBFO-FDARSICLSA-N 0.000 description 1
- LORJKYIPJIRIRT-BVSLBCMMSA-N Trp-Pro-Tyr Chemical compound C([C@H](NC(=O)[C@@H]1CCCN1C(=O)[C@H](CC=1C2=CC=CC=C2NC=1)N)C(O)=O)C1=CC=C(O)C=C1 LORJKYIPJIRIRT-BVSLBCMMSA-N 0.000 description 1
- 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 1
- SWSUXOKZKQRADK-FDARSICLSA-N Trp-Val-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)N SWSUXOKZKQRADK-FDARSICLSA-N 0.000 description 1
- BURPTJBFWIOHEY-UWJYBYFXSA-N Tyr-Ala-Asp Chemical compound OC(=O)C[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 BURPTJBFWIOHEY-UWJYBYFXSA-N 0.000 description 1
- LGEYOIQBBIPHQN-UWJYBYFXSA-N Tyr-Ala-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 LGEYOIQBBIPHQN-UWJYBYFXSA-N 0.000 description 1
- NOXKHHXSHQFSGJ-FQPOAREZSA-N Tyr-Ala-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 NOXKHHXSHQFSGJ-FQPOAREZSA-N 0.000 description 1
- HSVPZJLMPLMPOX-BPNCWPANSA-N Tyr-Arg-Ala 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](C)C(O)=O HSVPZJLMPLMPOX-BPNCWPANSA-N 0.000 description 1
- ZNFPUOSTMUMUDR-JRQIVUDYSA-N Tyr-Asn-Thr 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]([C@@H](C)O)C(O)=O ZNFPUOSTMUMUDR-JRQIVUDYSA-N 0.000 description 1
- NSTPFWRAIDTNGH-BZSNNMDCSA-N Tyr-Asn-Tyr 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=C(O)C=C1)C(O)=O NSTPFWRAIDTNGH-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
- LOOCQRRBKZTPKO-AVGNSLFASA-N Tyr-Glu-Asn Chemical compound NC(=O)C[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 LOOCQRRBKZTPKO-AVGNSLFASA-N 0.000 description 1
- JWGXUKHIKXZWNG-RYUDHWBXSA-N Tyr-Gly-Gln Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)NCC(=O)N[C@@H](CCC(=O)N)C(=O)O)N)O JWGXUKHIKXZWNG-RYUDHWBXSA-N 0.000 description 1
- IJUTXXAXQODRMW-KBPBESRZSA-N Tyr-Gly-His Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)NCC(=O)N[C@@H](CC2=CN=CN2)C(=O)O)N)O IJUTXXAXQODRMW-KBPBESRZSA-N 0.000 description 1
- JKUZFODWJGEQAP-KBPBESRZSA-N Tyr-Gly-Lys Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)O)N)O JKUZFODWJGEQAP-KBPBESRZSA-N 0.000 description 1
- NENACTSCXYHPOX-ULQDDVLXSA-N Tyr-His-Met Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CCSC)C(O)=O NENACTSCXYHPOX-ULQDDVLXSA-N 0.000 description 1
- USYGMBIIUDLYHJ-GVARAGBVSA-N Tyr-Ile-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 USYGMBIIUDLYHJ-GVARAGBVSA-N 0.000 description 1
- AXWBYOVVDRBOGU-SIUGBPQLSA-N Tyr-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](CC1=CC=C(C=C1)O)N AXWBYOVVDRBOGU-SIUGBPQLSA-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
- NKUGCYDFQKFVOJ-JYJNAYRXSA-N Tyr-Leu-Gln Chemical compound NC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 NKUGCYDFQKFVOJ-JYJNAYRXSA-N 0.000 description 1
- QHLIUFUEUDFAOT-MGHWNKPDSA-N Tyr-Leu-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC1=CC=C(C=C1)O)N QHLIUFUEUDFAOT-MGHWNKPDSA-N 0.000 description 1
- LMKKMCGTDANZTR-BZSNNMDCSA-N Tyr-Phe-Asp Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC(O)=O)C(O)=O)C1=CC=C(O)C=C1 LMKKMCGTDANZTR-BZSNNMDCSA-N 0.000 description 1
- FDKDGFGTHGJKNV-FHWLQOOXSA-N Tyr-Phe-Gln Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)O)NC(=O)[C@H](CC2=CC=C(C=C2)O)N FDKDGFGTHGJKNV-FHWLQOOXSA-N 0.000 description 1
- PHKQVWWHRYUCJL-HJOGWXRNSA-N Tyr-Phe-Tyr 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](CC1=CC=C(O)C=C1)C(O)=O PHKQVWWHRYUCJL-HJOGWXRNSA-N 0.000 description 1
- NZBSVMQZQMEUHI-WZLNRYEVSA-N Tyr-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](CC1=CC=C(C=C1)O)N NZBSVMQZQMEUHI-WZLNRYEVSA-N 0.000 description 1
- KUXCBJFJURINGF-PXDAIIFMSA-N Tyr-Trp-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)NC(=O)[C@H](CC3=CC=C(C=C3)O)N KUXCBJFJURINGF-PXDAIIFMSA-N 0.000 description 1
- MQUYPYFPHIPVHJ-MNSWYVGCSA-N Tyr-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](CC3=CC=C(C=C3)O)N)O MQUYPYFPHIPVHJ-MNSWYVGCSA-N 0.000 description 1
- JRMCISZDVLOTLR-BVSLBCMMSA-N Tyr-Trp-Val Chemical compound CC(C)[C@@H](C(=O)O)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)NC(=O)[C@H](CC3=CC=C(C=C3)O)N JRMCISZDVLOTLR-BVSLBCMMSA-N 0.000 description 1
- UUJHRSTVQCFDPA-UFYCRDLUSA-N Tyr-Tyr-Val Chemical compound C([C@@H](C(=O)N[C@@H](C(C)C)C(O)=O)NC(=O)[C@@H](N)CC=1C=CC(O)=CC=1)C1=CC=C(O)C=C1 UUJHRSTVQCFDPA-UFYCRDLUSA-N 0.000 description 1
- RGJZPXFZIUUQDN-BPNCWPANSA-N Tyr-Val-Ala 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](C)C(O)=O RGJZPXFZIUUQDN-BPNCWPANSA-N 0.000 description 1
- YKBUNNNRNZZUID-UFYCRDLUSA-N Tyr-Val-Tyr 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](CC1=CC=C(O)C=C1)C(O)=O YKBUNNNRNZZUID-UFYCRDLUSA-N 0.000 description 1
- ZMDCGGKHRKNWKD-LAEOZQHASA-N Val-Asn-Glu Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N ZMDCGGKHRKNWKD-LAEOZQHASA-N 0.000 description 1
- IQQYYFPCWKWUHW-YDHLFZDLSA-N Val-Asn-Tyr Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)O)N IQQYYFPCWKWUHW-YDHLFZDLSA-N 0.000 description 1
- IRLYZKKNBFPQBW-XGEHTFHBSA-N Val-Cys-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H](C(C)C)N)O IRLYZKKNBFPQBW-XGEHTFHBSA-N 0.000 description 1
- AAOPYWQQBXHINJ-DZKIICNBSA-N Val-Gln-Tyr Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)O)N AAOPYWQQBXHINJ-DZKIICNBSA-N 0.000 description 1
- VVZDBPBZHLQPPB-XVKPBYJWSA-N Val-Glu-Gly Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(O)=O VVZDBPBZHLQPPB-XVKPBYJWSA-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
- AGXGCFSECFQMKB-NHCYSSNCSA-N Val-Leu-Asp Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)O)NC(=O)[C@H](C(C)C)N AGXGCFSECFQMKB-NHCYSSNCSA-N 0.000 description 1
- HGJRMXOWUWVUOA-GVXVVHGQSA-N Val-Leu-Gln Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)O)NC(=O)[C@H](C(C)C)N HGJRMXOWUWVUOA-GVXVVHGQSA-N 0.000 description 1
- ZHQWPWQNVRCXAX-XQQFMLRXSA-N Val-Leu-Pro Chemical compound CC(C)C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](C(C)C)N ZHQWPWQNVRCXAX-XQQFMLRXSA-N 0.000 description 1
- BTWMICVCQLKKNR-DCAQKATOSA-N Val-Leu-Ser Chemical compound CC(C)[C@H]([NH3+])C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C([O-])=O BTWMICVCQLKKNR-DCAQKATOSA-N 0.000 description 1
- MGVYZTPLGXPVQB-CYDGBPFRSA-N Val-Met-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CCSC)NC(=O)[C@H](C(C)C)N MGVYZTPLGXPVQB-CYDGBPFRSA-N 0.000 description 1
- WMRWZYSRQUORHJ-YDHLFZDLSA-N Val-Phe-Asp Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(=O)O)C(=O)O)N WMRWZYSRQUORHJ-YDHLFZDLSA-N 0.000 description 1
- CKTMJBPRVQWPHU-JSGCOSHPSA-N Val-Phe-Gly Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)NCC(=O)O)N CKTMJBPRVQWPHU-JSGCOSHPSA-N 0.000 description 1
- NHXZRXLFOBFMDM-AVGNSLFASA-N Val-Pro-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)C(C)C NHXZRXLFOBFMDM-AVGNSLFASA-N 0.000 description 1
- 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 1
- WUFHZIRMAZZWRS-OSUNSFLBSA-N Val-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](C(C)C)N WUFHZIRMAZZWRS-OSUNSFLBSA-N 0.000 description 1
- RLVTVHSDKHBFQP-ULQDDVLXSA-N Val-Tyr-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)C(C)C)CC1=CC=C(O)C=C1 RLVTVHSDKHBFQP-ULQDDVLXSA-N 0.000 description 1
- 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 1
- 241000724701 Velvet tobacco mottle virus Species 0.000 description 1
- 241000219977 Vigna Species 0.000 description 1
- 241000726445 Viroids Species 0.000 description 1
- 235000009392 Vitis Nutrition 0.000 description 1
- 241000219095 Vitis Species 0.000 description 1
- 241000209149 Zea Species 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 108010081404 acein-2 Proteins 0.000 description 1
- 230000021736 acetylation Effects 0.000 description 1
- 238000006640 acetylation reaction Methods 0.000 description 1
- 150000008043 acidic salts Chemical class 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 244000193174 agave Species 0.000 description 1
- 238000012271 agricultural production Methods 0.000 description 1
- 230000009418 agronomic effect Effects 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- 108010041407 alanylaspartic acid Proteins 0.000 description 1
- 108010005233 alanylglutamic acid Proteins 0.000 description 1
- 108010047495 alanylglycine Proteins 0.000 description 1
- 150000001447 alkali salts Chemical class 0.000 description 1
- 239000002168 alkylating agent Substances 0.000 description 1
- 229910052782 aluminium Inorganic materials 0.000 description 1
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 1
- 230000006229 amino acid addition Effects 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 108010008355 arginyl-glutamine Proteins 0.000 description 1
- 108010052670 arginyl-glutamyl-glutamic acid Proteins 0.000 description 1
- 108010038850 arginyl-isoleucyl-tyrosine Proteins 0.000 description 1
- 108010084758 arginyl-tyrosyl-aspartic acid Proteins 0.000 description 1
- 108010068380 arginylarginine Proteins 0.000 description 1
- 108010062796 arginyllysine Proteins 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 108010010430 asparagine-proline-alanine Proteins 0.000 description 1
- 108010077245 asparaginyl-proline Proteins 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- 108010068265 aspartyltyrosine Proteins 0.000 description 1
- 238000005452 bending Methods 0.000 description 1
- MJVXAPPOFPTTCA-UHFFFAOYSA-N beta-Sistosterol Natural products CCC(CCC(C)C1CCC2C3CC=C4C(C)C(O)CCC4(C)C3CCC12C)C(C)C MJVXAPPOFPTTCA-UHFFFAOYSA-N 0.000 description 1
- LGJMUZUPVCAVPU-UHFFFAOYSA-N beta-Sitostanol Natural products C1CC2CC(O)CCC2(C)C2C1C1CCC(C(C)CCC(CC)C(C)C)C1(C)CC2 LGJMUZUPVCAVPU-UHFFFAOYSA-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
- 108010066270 beta-lactorphin Proteins 0.000 description 1
- NJKOMDUNNDKEAI-UHFFFAOYSA-N beta-sitosterol Natural products CCC(CCC(C)C1CCC2(C)C3CC=C4CC(O)CCC4C3CCC12C)C(C)C NJKOMDUNNDKEAI-UHFFFAOYSA-N 0.000 description 1
- 108091008324 binding proteins Proteins 0.000 description 1
- 238000012742 biochemical analysis Methods 0.000 description 1
- 238000002306 biochemical method Methods 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 229960001561 bleomycin Drugs 0.000 description 1
- OYVAGSVQBOHSSS-UAPAGMARSA-O bleomycin A2 Chemical compound N([C@H](C(=O)N[C@H](C)[C@@H](O)[C@H](C)C(=O)N[C@@H]([C@H](O)C)C(=O)NCCC=1SC=C(N=1)C=1SC=C(N=1)C(=O)NCCC[S+](C)C)[C@@H](O[C@H]1[C@H]([C@@H](O)[C@H](O)[C@H](CO)O1)O[C@@H]1[C@H]([C@@H](OC(N)=O)[C@H](O)[C@@H](CO)O1)O)C=1N=CNC=1)C(=O)C1=NC([C@H](CC(N)=O)NC[C@H](N)C(N)=O)=NC(N)=C1C OYVAGSVQBOHSSS-UAPAGMARSA-O 0.000 description 1
- 239000002981 blocking agent Substances 0.000 description 1
- 229910052796 boron Inorganic materials 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 239000001390 capsicum minimum Substances 0.000 description 1
- 150000004657 carbamic acid derivatives Chemical class 0.000 description 1
- 125000004432 carbon atom Chemical group C* 0.000 description 1
- 229910002091 carbon monoxide Inorganic materials 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- JSVPGVHCEQDJCZ-VGEHDTSWSA-N cathasterone Chemical compound C([C@@H]1C(=O)C2)[C@@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@H](C)[C@@H](O)C[C@@H](C)C(C)C)[C@@]2(C)CC1 JSVPGVHCEQDJCZ-VGEHDTSWSA-N 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000010307 cell transformation Effects 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 235000013339 cereals Nutrition 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 229930002868 chlorophyll a Natural products 0.000 description 1
- 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 1
- 229930002869 chlorophyll b Natural products 0.000 description 1
- NSMUHPMZFPKNMZ-VBYMZDBQSA-M chlorophyll b Chemical compound C1([C@@H](C(=O)OC)C(=O)C2=C3C)=C2N2C3=CC(C(CC)=C3C=O)=[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 NSMUHPMZFPKNMZ-VBYMZDBQSA-M 0.000 description 1
- 239000013611 chromosomal DNA Substances 0.000 description 1
- 235000020971 citrus fruits Nutrition 0.000 description 1
- 239000003184 complementary RNA Substances 0.000 description 1
- 239000000039 congener Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 238000012786 cultivation procedure Methods 0.000 description 1
- 125000001559 cyclopropyl group Chemical group [H]C1([H])C([H])([H])C1([H])* 0.000 description 1
- 108010016616 cysteinylglycine Proteins 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 229940104302 cytosine Drugs 0.000 description 1
- 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 1
- 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 1
- 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 1
- 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 1
- NHVNXKFIZYSCEB-XLPZGREQSA-N dTTP Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C1 NHVNXKFIZYSCEB-XLPZGREQSA-N 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000002939 deleterious effect Effects 0.000 description 1
- 238000010520 demethylation reaction Methods 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 239000005547 deoxyribonucleotide Substances 0.000 description 1
- 125000002637 deoxyribonucleotide group Chemical group 0.000 description 1
- 229960000633 dextran sulfate Drugs 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- QBSJHOGDIUQWTH-UHFFFAOYSA-N dihydrolanosterol Natural products CC(C)CCCC(C)C1CCC2(C)C3=C(CCC12C)C4(C)CCC(C)(O)C(C)(C)C4CC3 QBSJHOGDIUQWTH-UHFFFAOYSA-N 0.000 description 1
- 230000003292 diminished effect Effects 0.000 description 1
- 235000004879 dioscorea Nutrition 0.000 description 1
- 108010054813 diprotin B Proteins 0.000 description 1
- NAGJZTKCGNOGPW-UHFFFAOYSA-N dithiophosphoric acid Chemical class OP(O)(S)=S NAGJZTKCGNOGPW-UHFFFAOYSA-N 0.000 description 1
- 230000003828 downregulation Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 210000002257 embryonic structure Anatomy 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 210000002615 epidermis Anatomy 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 239000011888 foil Substances 0.000 description 1
- 239000004459 forage Substances 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 238000012215 gene cloning Methods 0.000 description 1
- 238000012252 genetic analysis Methods 0.000 description 1
- 238000003167 genetic complementation Methods 0.000 description 1
- 230000007614 genetic variation Effects 0.000 description 1
- 238000012268 genome sequencing Methods 0.000 description 1
- IXORZMNAPKEEDV-UHFFFAOYSA-N gibberellic acid GA3 Natural products OC(=O)C1C2(C3)CC(=C)C3(O)CCC2C2(C=CC3O)C1C3(C)C(=O)O2 IXORZMNAPKEEDV-UHFFFAOYSA-N 0.000 description 1
- IXORZMNAPKEEDV-OBDJNFEBSA-N gibberellin A3 Chemical class C([C@@]1(O)C(=C)C[C@@]2(C1)[C@H]1C(O)=O)C[C@H]2[C@]2(C=C[C@@H]3O)[C@H]1[C@]3(C)C(=O)O2 IXORZMNAPKEEDV-OBDJNFEBSA-N 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- 230000013595 glycosylation Effects 0.000 description 1
- 238000006206 glycosylation reaction Methods 0.000 description 1
- 108010028188 glycyl-histidyl-serine Proteins 0.000 description 1
- 108010045126 glycyl-tyrosyl-glycine Proteins 0.000 description 1
- 108010087823 glycyltyrosine Proteins 0.000 description 1
- 239000005090 green fluorescent protein Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 229910001385 heavy metal Inorganic materials 0.000 description 1
- 108010036413 histidylglycine Proteins 0.000 description 1
- 108010025306 histidylleucine Proteins 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 230000000640 hydroxylating effect Effects 0.000 description 1
- 230000033444 hydroxylation Effects 0.000 description 1
- 230000009618 hypocotyl growth Effects 0.000 description 1
- 238000001114 immunoprecipitation Methods 0.000 description 1
- 238000012744 immunostaining Methods 0.000 description 1
- 238000007901 in situ hybridization Methods 0.000 description 1
- 230000000415 inactivating effect Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- PZOUSPYUWWUPPK-UHFFFAOYSA-N indole Natural products CC1=CC=CC2=C1C=CN2 PZOUSPYUWWUPPK-UHFFFAOYSA-N 0.000 description 1
- RKJUIXBNRJVNHR-UHFFFAOYSA-N indolenine Natural products C1=CC=C2CC=NC2=C1 RKJUIXBNRJVNHR-UHFFFAOYSA-N 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- OSELKOCHBMDKEJ-WGMIZEQOSA-N isofucosterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CC/C(=C/C)C(C)C)[C@@]1(C)CC2 OSELKOCHBMDKEJ-WGMIZEQOSA-N 0.000 description 1
- CAHGCLMLTWQZNJ-RGEKOYMOSA-N lanosterol Chemical compound C([C@]12C)C[C@@H](O)C(C)(C)[C@H]1CCC1=C2CC[C@]2(C)[C@H]([C@H](CCC=C(C)C)C)CC[C@@]21C CAHGCLMLTWQZNJ-RGEKOYMOSA-N 0.000 description 1
- 229940058690 lanosterol Drugs 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 231100000518 lethal Toxicity 0.000 description 1
- 230000001665 lethal effect Effects 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 108010003700 lysyl aspartic acid Proteins 0.000 description 1
- 108010017391 lysylvaline Proteins 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 235000005739 manihot Nutrition 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- MYWUZJCMWCOHBA-VIFPVBQESA-N methamphetamine Chemical compound CN[C@@H](C)CC1=CC=CC=C1 MYWUZJCMWCOHBA-VIFPVBQESA-N 0.000 description 1
- 230000011987 methylation Effects 0.000 description 1
- 238000007069 methylation reaction Methods 0.000 description 1
- 229950009116 mevastatin Drugs 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 210000001589 microsome Anatomy 0.000 description 1
- 235000019713 millet Nutrition 0.000 description 1
- 102000035118 modified proteins Human genes 0.000 description 1
- 108091005573 modified proteins Proteins 0.000 description 1
- 238000001823 molecular biology technique Methods 0.000 description 1
- 230000003562 morphometric effect Effects 0.000 description 1
- 238000013425 morphometry Methods 0.000 description 1
- 231100000219 mutagenic Toxicity 0.000 description 1
- 230000003505 mutagenic effect Effects 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 230000031787 nutrient reservoir activity Effects 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 239000003921 oil Substances 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000008447 perception Effects 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 108010065135 phenylalanyl-phenylalanyl-phenylalanine Proteins 0.000 description 1
- 150000004713 phosphodiesters Chemical group 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 230000027874 photomorphogenesis Effects 0.000 description 1
- 238000000053 physical method Methods 0.000 description 1
- 229930195732 phytohormone Natural products 0.000 description 1
- 229930000184 phytotoxin Natural products 0.000 description 1
- 238000003976 plant breeding Methods 0.000 description 1
- 230000037039 plant physiology Effects 0.000 description 1
- 235000002378 plant sterols Nutrition 0.000 description 1
- 239000003123 plant toxin Substances 0.000 description 1
- 229920000729 poly(L-lysine) polymer Polymers 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000002953 preparative HPLC Methods 0.000 description 1
- 239000000186 progesterone Substances 0.000 description 1
- 229960003387 progesterone Drugs 0.000 description 1
- 108060006633 protein kinase Proteins 0.000 description 1
- 235000014774 prunus Nutrition 0.000 description 1
- 239000012521 purified sample Substances 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
- 238000009790 rate-determining step (RDS) Methods 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 238000012340 reverse transcriptase PCR Methods 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 239000002336 ribonucleotide Substances 0.000 description 1
- 125000002652 ribonucleotide group Chemical group 0.000 description 1
- 108020004418 ribosomal RNA Proteins 0.000 description 1
- 238000007363 ring formation reaction Methods 0.000 description 1
- 230000002786 root growth Effects 0.000 description 1
- 238000004626 scanning electron microscopy Methods 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000005204 segregation Methods 0.000 description 1
- 108010071207 serylmethionine Proteins 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- HBMJWWWQQXIZIP-UHFFFAOYSA-N silicon carbide Chemical compound [Si+]#[C-] HBMJWWWQQXIZIP-UHFFFAOYSA-N 0.000 description 1
- 229910010271 silicon carbide Inorganic materials 0.000 description 1
- KZJWDPNRJALLNS-VJSFXXLFSA-N sitosterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CC[C@@H](CC)C(C)C)[C@@]1(C)CC2 KZJWDPNRJALLNS-VJSFXXLFSA-N 0.000 description 1
- 235000015500 sitosterol Nutrition 0.000 description 1
- 229950005143 sitosterol Drugs 0.000 description 1
- NLQLSVXGSXCXFE-UHFFFAOYSA-N sitosterol Natural products CC=C(/CCC(C)C1CC2C3=CCC4C(C)C(O)CCC4(C)C3CCC2(C)C1)C(C)C NLQLSVXGSXCXFE-UHFFFAOYSA-N 0.000 description 1
- 238000005549 size reduction Methods 0.000 description 1
- 230000027772 skotomorphogenesis Effects 0.000 description 1
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 1
- 159000000000 sodium salts Chemical class 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000009331 sowing Methods 0.000 description 1
- 125000002328 sterol group Chemical group 0.000 description 1
- BFDNMXAIBMJLBB-UHFFFAOYSA-N stigmasterol Natural products CCC(C=CC(C)C1CCCC2C3CC=C4CC(O)CCC4(C)C3CCC12C)C(C)C BFDNMXAIBMJLBB-UHFFFAOYSA-N 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 108010078552 syringomycin Proteins 0.000 description 1
- SBSXXCCMIWEPEE-GZKYLSGOSA-N teasterone Chemical compound C([C@@H]1C(=O)C2)[C@@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@H](C)[C@@H](O)[C@H](O)[C@@H](C)C(C)C)[C@@]2(C)CC1 SBSXXCCMIWEPEE-GZKYLSGOSA-N 0.000 description 1
- 238000007861 thermal asymmetric interlaced PCR Methods 0.000 description 1
- 229950003937 tolonium Drugs 0.000 description 1
- HNONEKILPDHFOL-UHFFFAOYSA-M tolonium chloride Chemical compound [Cl-].C1=C(C)C(N)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 HNONEKILPDHFOL-UHFFFAOYSA-M 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 108700012359 toxins Proteins 0.000 description 1
- 230000014621 translational initiation Effects 0.000 description 1
- 230000032258 transport Effects 0.000 description 1
- 125000000026 trimethylsilyl group Chemical group [H]C([H])([H])[Si]([*])(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 239000001226 triphosphate Substances 0.000 description 1
- 235000011178 triphosphate Nutrition 0.000 description 1
- 125000002264 triphosphate group Chemical class [H]OP(=O)(O[H])OP(=O)(O[H])OP(=O)(O[H])O* 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
- SBSXXCCMIWEPEE-SELDZKRUSA-N typhasterol Chemical compound C([C@@H]1C(=O)C2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@H](C)[C@@H](O)[C@H](O)[C@@H](C)C(C)C)[C@@]2(C)CC1 SBSXXCCMIWEPEE-SELDZKRUSA-N 0.000 description 1
- 108010003137 tyrosyltyrosine Proteins 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- 229940035893 uracil Drugs 0.000 description 1
- 108010009962 valyltyrosine Proteins 0.000 description 1
- 230000006459 vascular development Effects 0.000 description 1
- 108700026220 vif Genes Proteins 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
- 230000001018 virulence Effects 0.000 description 1
- 108010034306 xylene monooxygenase Proteins 0.000 description 1
- 108010069678 xyloglucan endotransglycosylase Proteins 0.000 description 1
- 210000005253 yeast cell Anatomy 0.000 description 1
Images
Classifications
-
- 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
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
- C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
-
- 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
-
- 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
- C12N15/8291—Hormone-influenced development
- C12N15/8298—Brassinosteroids
-
- 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
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
- C12Y114/19—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with oxidation of a pair of donors resulting in the reduction of molecular oxygen to two molecules of water (1.14.19)
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
- Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants
Definitions
- Sterols are known to play at least two critical roles in plants: as bulk components of membranes regulating stability and permeability (Bach et al. (1997) Prog. Lipid Res. 36:197-226) and as precursors of growth-promoting brassinosteroids (BRs; Fujioka and Sakurai (1997) Nat. Prod. Rep. 14: 1-10). Lesions in brassinosteroid (BR) biosynthetic genes result in characteristic dwarf phenotypes in plants. Understanding the regulation of BR biosynthesis demands continued isolation and characterization of mutants corresponding to the genes involved in BR biosynthesis.
- photosynthetic organisms require somewhat different biosynthetic enzymes, such as cycloartol synthase (Corey et al. (1993) Proc. Natl. Acad. Sci. USA 90:11628-11632) and cycloeucalenol-obtusifoliol isomerase, which are required to open the cyclopropane ring in cycloartol (FIG. 1).
- biosynthetic enzymes such as cycloartol synthase (Corey et al. (1993) Proc. Natl. Acad. Sci. USA 90:11628-11632) and cycloeucalenol-obtusifoliol isomerase, which are required to open the cyclopropane ring in cycloartol (FIG. 1).
- cycloartol synthase Corey et al. (1993) Proc. Natl. Acad. Sci. USA 90:11628-11632
- steros are subject to a series of modifications before conversion to BL.
- Different sterols such as 24-methylenecholesterol (24-MC), campesterol (CR), isofucosterol, and sitosterol, are converted to the BL congeners dolicholide, BL, 28-homodolicholide, and 28-homoBL, respectively, in a species-specific manner (Fujioka et al. (1997) Plant Cell 9:1951-1962; Sasse (1997) Physiol. Plant. 100:696-701).
- the BR-specific pathway diverges into the early and the late C-6 oxidation pathways.
- BR biosynthetic dwarfs share a characteristic dwarf phenotype, which includes short robust stems, reduced fertility, prolonged life cycle, and dark-green, round, and curled leaves when grown in the light. In the dark, these mutants exhibit short hypocotyls and expanded cotyledons, cpd (dwf3) mutants are only rescued by 23 ⁇ -hydroxylated compounds (Szekeres et al. (1996) Cell 85:171-182). The CPD gene was shown to encode a cytochrome P450 steroid hydroxylating enzyme (CYP90A1). In addition, Li et al. (1996) Science 272:398-401 and Li et al. (1997) Proc. Natl. Acad. Sci.
- DET2 was found to be homologous to steroid 5 ⁇ -reductases. Like its animal equivalents, DET2 successfully converted progesterone (3-oxo- ⁇ 4.5 steroid) to 4,5-dihydroprogesterone in a human cell line. In addition, the human 5 ⁇ -reductase gene effectively complemented det2 mutants (Li et al. (1997) Proc. Natl. Acad. Sci. USA 94:3554-3559).
- DWF4 encodes a cytochrome P450 whose amino acid sequence is 43% identical to CPD; DWF4 has been named CYP90B1 (Choe et al. (1998) Plant Cell 10:231-243). Based on results from feeding studies using BR biosynthetic intermediates, the proposed rate-limiting step of BR biosynthesis, 22 ⁇ -hydroxylation, is now known to be blocked in dwf4 mutants.
- ERGOSTEROL25 (ERG25) homolog for Arabidopsis (C-4 demethylase) also has been discovered in the genome sequencing project (GenBank accession number AL021635).
- a sterol C-7 reductase has been cloned by heterologous expression of an Arabidopsis cDNA in yeast (Lecain et al. (1996) J. Biol. Chem. 271:10866-10873).
- the present invention is based on the discovery of various mutants of a BR biosynthetic locus, designated dwarf7 (dwf7).
- the STE1 locus in dwf7 mutants contain loss-of-function mutations.
- Two allelic variants of dwf7 have been characterized, dwf7-1 and dwf7-2, also designated ste1-2 and ste1-3, respectively.
- Feeding studies with BR biosynthetic intermediates and analysis of endogenous levels of BR and sterol biosynthetic intermediates indicate that the defective step in the dwf7 mutants resides before the production of 24-methylenecholesterol in the sterol biosynthetic pathway. Furthermore, results from feeding studies with 13 C-labeled mevalonic acid and compactin show that the defective step is specifically the ⁇ 7 sterol C-5 desaturation. Sequencing of the STE1 locus in the two dwf7 variants shows premature stop codons in the first (dwf7-2) and the third (dwf7-1) exons. Thus, the reduction of BRs in dwf7 is due to a shortage of substrate sterols and is the direct cause of the dwarf phenotype in dwf7.
- the present invention is directed to an isolated dwf7 polynucleotide that imparts at least one dwf7 mutant phenotype when expressed in a plant.
- the polynucleotide is selected from the group consisting of (a) a polynucleotide comprising the nucleotide sequence depicted at positions 143 to 322 , inclusive, of FIGS. 8 A- 8 D; (b) a polynucleotide comprising the nucleotide sequence depicted at positions 143 to 1552, inclusive, of FIGS.
- a polynucleotide comprising a nucleotide sequence having at least about 70% identity to the nucleotide sequence of (a) or (b); (d) a fragment of (a), (b) or (c) comprising at least about 15 contiguous nucleotides; and (e) complements of (a), (b), (c), (d) or (e).
- the present invention is directed to an isolated dwf7 polynucleotide that imparts at least one dwf7 mutant phenotype when expressed in a plant.
- the polynucleotide is selected from the group consisting of (a) a polynucleotide comprising the nucleotide sequence depicted at positions 1506 to 2720, inclusive, of FIGS.
- the present invention is directed to recombinant vectors comprising the isolated dwf7 polynucleotides described above, and control elements that are operably linked to the polynucleotides whereby a coding sequence within the polynucleotides can be transcribed and translated in a host cell, and at least one of the control elements is heterologous to the coding sequence.
- host cells transformed with the recombinant vectors, and methods of producing a DWF7 polypeptide comprising providing a population of host cells as described above and culturing the population of cells under conditions whereby the DWF7 polypeptide encoded by the coding sequence present in the recombinant vector is expressed.
- the subject invention is directed to a transgenic plant comprising a polynucleotide described above, as well as methods of producing a transgenic plant comprising the steps of introducing a polynucleotide into a plant cell to produce a transformed plant cell; and producing a transgenic plant from the transformed plant cell.
- the invention is directed to a method for altering the sterol composition of a plant relative to the wild-type plant comprising introducing a polynucleotide as described above into a plant cell to produce a transformed plant cell and producing a transgenic plant from the transformed plant cell, wherein the transgenic plant has an altered sterol composition relative to the wild-type plant, such as an altered cholesterol composition relative to the wild-type plant.
- the invention is directed to isolated DWF7 polypeptides encoded by the polynucleotides as described above.
- the polypeptide consists of the amino acid sequence depicted at positions 1-60, inclusive, of FIG. 9 or the amino acid sequence depicted at positions 1-230, inclusive, of FIG. 9. In other embodiments, the polypeptide consists of the amino acid sequence depicted at positions 1-279, inclusive, of FIG. 11.
- the subject invention is directed to an isolated control element having at least about 70% identity to a control element found within nucleotide positions 43-142 of FIGS. 8 A- 8 D, or 1-1505 of FIGS. 10 A- 10 F, a recombinant vector comprising the control element and a polynucleotide comprising a coding sequence which is heterologous to the control element, host cells transformed with the recombinant vector, and methods of producing a recombinant polypeptide comprising providing a population of the host cells and culturing the population of cells under conditions whereby the recombinant polypeptide encoded by the coding sequence present in the recombinant vector is expressed.
- FIG. 1 shows the proposed BL biosynthetic pathway from squalene to BL.
- the BL biosynthetic pathway is divided into the sterol-specific pathway, squalene to campesterol, and the BR-specific pathway, campesterol to brassinolide.
- Common names for the compounds are labeled, and proposed enzymes involved in each reaction are boxed and labeled. Genes identified by mutants are marked. The acronyms for some compounds are in parentheses. In the inset, the carbon atoms of the sterol core rings and side chain are numbered.
- FIG. 2 is a bar graph of measurements of gynoecia and stamens of wild-type, (ecotype Wassilewskija-2 [Ws-2]), dwf7-1, and dwf4-3 plants.
- the dwf7-1 plant displays a concomitant reduction in the length of gynoecia and stamens, whereas dwf4-3 displays a greater reduction in stamen length.
- Each data point represents the average length for five flowers. Standard errors are shown at each data point. Solid bars indicate the gynoecium and white bars denote the stamen.
- FIG. 3 compares the response of light-grown wild-type and dwf7-1 hypocotyls to different concentrations of BL. Black bars indicate results using the Wassilewskij a-2 (Ws-2) wild type and white bars dwf7-1 plants. The dwf7-1 plant responds to 10 ⁇ 9 M BL and is completely rescued by 10 ⁇ 8 M BL. Error bars indicate ⁇ SE.
- FIG. 4 is a bar graph comparing wild-type and dwf7-1 inflorescences treated with BR intermediates.
- the lengths of pedicels treated with water, 6-deoxoCT, 22-OHCR, and BL were measured to the nearest millimeter (n>15).
- the pedicels elongated greater than twofold in response to all the BRs tested, suggesting that the biosynthetic defect in dwf7-1 resides before the production of CR. Error bars indicate ⁇ SE.
- FIG. 5 shows GC-MS analysis of wild-type and dwf7-1 seedlings fed with 13 C-MVA in the presence of compactin, an inhibitor of MVA biosynthesis.
- the units are in micrograms per 5 g fresh weight of tissue.
- the designation ND (not detected) means that the quantity is lower than the detection limit.
- Ws-2 is the Wassilewskija-2 wild type.
- FIG. 6 is a schematic representation of the STE1 gene. Comparison of cDNA and genomic DNA sequences revealed three exons (thick boxes) and two introns (horizontal bars). The single open reading frame encodes a protein of 281 amino acids.
- the dwf7-2 (ste1 -3) mutation is located in the first exon, changing a tryptophan to a stop codon.
- the dwf7-1 (ste1-2) mutation also changes a tryptophan to a stop codon (amino acid position 230).
- FIG. 7 depicts a multiple sequence alignment of DWF7/STE1 with known sequences for ⁇ 7 sterol C-5 desaturases.
- GenBank accession numbers for the sequences are M62623 ( S. cerevisiae ) (SEQ ID NO:______), AB004539 ( Schizosaccharomyces pombe ) (SEQ ID NO:______), L40390 ( C. glabrata ) (SEQ ID NO:_______), and AF105034 (DWF7/STE1, Arabidopsis) (SEQ ID NO:_______).
- the conserved transmembrane domains and histidine clusters are boxed and labeled.
- the positions of the premature stop codons in dwf7-1 and dwf7-2 are indicated with filled circles. Histidine residues in each conserved histidine box are identified with filled triangles.
- a consensus sequence (SEQ ID NO:_______) is shown in the bottom row of the alignment.
- Capital letters stand for residues conserved among all sequences, whereas lowercase letters mean ⁇ 50% identical. Dashes indicate gaps introduced to maximize alignment. Multiple sequence alignment was performed using PILEUP in the Genetics Computer Group software (Madison, Wis.) with a gap creation penalty of 4 and a gap extension parameter of 1.
- the annotation of the aligned sequences was performed using the ALSCRIPT software (Barton (1993) Protein Eng. 6:37-40).
- FIGS. 8 A- 8 D depict the complete gene sequence of dwf7, denoted by a dark grey bar.
- the premature stop codons for dwf7-1 and dwf7-2 are shown with triangles at nucleotide positions 1552 and 322, respectively.
- the coding sequence and corresponding amino acid sequence are represented by a light grey bar.
- the mRNA sequence is represented by a black bar and is shown in three segments.
- the gene includes two introns (positions 369-735 and 1042-1395) and three exons.
- FIG. 9 shows the amino acid sequence corresponding to the coding sequence designated in FIGS. 8 A- 8 D.
- the polypeptide sequences corresponding to the dwf7-2 and dwf7-1 alleles occur at positions 1-60 and 1-230, respectively.
- FIGS. 10 A- 10 F show the gene sequence of the dwf7 homologue, HDF7.
- the coding sequence and corresponding amino acid sequence are shown in three segments (exons), occurring at positions 1506-1734, 2024-2329 and 2416-2720 of the figure.
- the 5′ UTR is shown at positions 1-1505 and the 3′ UTR occurs at positions 2721-2925.
- FIG. 11 shows the amino acid sequence corresponding to the coding sequence designated in FIGS. 10 A- 10 F.
- the polypeptide sequence corresponding to the HDF7 dwf7 polypeptide occurs at positions 1-230 of the figure.
- nucleic acid molecule and “polynucleotide” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. This term refers only to the primary structure of the molecule and thus includes double- and single-stranded DNA and RNA.
- internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties, such as, for example proteins (including e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelates (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylators, those with modified linkages (e.g., alpha
- Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown.
- Nonlimiting examples of polynucleotides include a gene, a gene fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.
- mRNA messenger RNA
- transfer RNA transfer RNA
- ribosomal RNA ribozymes
- cDNA recombinant polynucleotides
- branched polynucleotides plasmids
- vectors isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.
- a polynucleotide is typically composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); and thymine (T) (uracil (U) for thymine (T) when the polynucleotide is RNA).
- A adenine
- C cytosine
- G guanine
- T thymine
- U uracil
- T thymine
- sequence identity refers to an exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively.
- Two or more sequences can be compared by determining their “percent identity.”
- the percent identity of two sequences, whether nucleic acid or amino acid sequences is the number of exact matches between two aligned sequences divided by the length of the shorter sequences and multiplied by 100.
- An approximate alignment for nucleic acid sequences is provided by the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981). This algorithm can be applied to amino acid sequences by using the scoring matrix developed by Dayhoff, Atlas of Protein Sequences and Structure, M. O. Dayhoff ed., 5 suppl.
- the degree of sequence similarity between polynucleotides can be determined by hybridization of polynucleotides under conditions that form stable duplexes between homologous regions, followed by digestion with single-stranded-specific nuclease(s), and size determination of the digested fragments.
- Two DNA, or two polypeptide sequences are “substantially homologous” to each other when the sequences exhibit at least about 70%-85%, preferably at least about 85%-90%, more preferably at least about 90%-95%, and most preferably at least about 95%-98% sequence identity over a defined length of the molecules, or any percentage between the above-specified ranges, as determined using the methods above.
- substantially homologous also refers to sequences showing complete identity to the specified DNA or polypeptide sequence.
- DNA sequences that are substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Sambrook et al., supra; DNA Cloning, supra; Nucleic Acid Hybridization, supra.
- the degree of sequence identity between two nucleic acid molecules affects the efficiency and strength of hybridization events between such molecules.
- a partially identical nucleic acid sequence will at least partially inhibit a completely identical sequence from hybridizing to a target molecule. Inhibition of hybridization of the completely identical sequence can be assessed using hybridization assays that are well known in the art (e.g., Southern blot, Northern blot, solution hybridization, or the like, see Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, (1989) Cold Spring Harbor, N.Y.). Such assays can be conducted using varying degrees of selectivity, for example, using conditions varying from low to high stringency.
- a nucleic acid probe is chosen that is complementary to a target nucleic acid sequence, and then by selection of appropriate conditions the probe and the target sequence “selectively hybridize,” or bind, to each other to form a hybrid molecule.
- a nucleic acid molecule that is capable of hybridizing selectively to a target sequence under “moderately stringent” typically hybridizes under conditions that allow detection of a target nucleic acid sequence of at least about 10-14 nucleotides in length having at least approximately 70% sequence identity with the sequence of the selected nucleic acid probe.
- Stringent hybridization conditions typically allow detection of target nucleic acid sequences of at least about 10-14 nucleotides in length having a sequence identity of greater than about 90-95% with the sequence of the selected nucleic acid probe.
- Hybridization conditions useful for probe/target hybridization where the probe and target have a specific degree of sequence identity can be determined as is known in the art (see, for example, Nucleic Acid Hybridization: A Practical Approach, editors B. D. Hames and S. J. Higgins, (1985) Oxford; Washington, D.C.; IRL Press).
- stringency conditions for hybridization it is well known in the art that numerous equivalent conditions can be employed to establish a particular stringency by varying, for example, the following factors: the length and nature of probe and target sequences, base composition of the various sequences, concentrations of salts and other hybridization solution components, the presence or absence of blocking agents in the hybridization solutions (e.g., formamide, dextran sulfate, and polyethylene glycol), hybridization reaction temperature and time parameters, as well as, varying wash conditions.
- the selection of a particular set of hybridization conditions is selected following standard methods in the art (see, for example, Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, (1989) Cold Spring Harbor, N.Y.).
- a “gene” as used in the context of the present invention is a sequence of nucleotides in a genetic nucleic acid (chromosome, plasmid, etc.) with which a genetic function is associated.
- a gene is a hereditary unit, for example of an organism, comprising a polynucleotide sequence that occupies a specific physical location (a “gene locus” or “genetic locus”) within the genome of an organism.
- a gene can encode an expressed product, such as a polypeptide or a polynucleotide (e.g., tRNA).
- a gene may define a genomic location for a particular event/function, such as the binding of proteins and/or nucleic acids, wherein the gene does not encode an expressed product.
- a gene includes coding sequences, such as, polypeptide encoding sequences, and non-coding sequences, such as, promoter sequences, polyadenlyation sequences, transcriptional regulatory sequences (e.g., enhancer sequences).
- coding sequences such as, polypeptide encoding sequences
- non-coding sequences such as, promoter sequences, polyadenlyation sequences, transcriptional regulatory sequences (e.g., enhancer sequences).
- Many eucaryotic genes have “exons”(coding sequences) interrupted by “introns”(non-coding sequences).
- a gene may share sequences with another gene(s) (e.g., overlapping genes).
- a “coding sequence” or a sequence which “encodes” a selected polypeptide is a nucleic acid molecule which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide, for example, in vivo when placed under the control of appropriate regulatory sequences (or “control elements”).
- the boundaries of the coding sequence are typically determined by a start codon at the 5′(amino) terminus and a translation stop codon at the 3′(carboxy) terminus.
- a coding sequence can include, but is not limited to, cDNA from viral, procaryotic or eucaryotic mRNA, genomic DNA sequences from viral or procaryotic DNA, and even synthetic DNA sequences.
- a transcription termination sequence may be located 3′ to the coding sequence.
- Other “control elements” may also be associated with a coding sequence.
- a DNA sequence encoding a polypeptide can be optimized for expression in a selected cell by using the codons preferred by the selected cell to represent the DNA copy of the desired polypeptide coding sequence.
- “Encoded by” refers to a nucleic acid sequence which codes for a polypeptide sequence, wherein the polypeptide sequence or a portion thereof contains an amino acid sequence of at least 3 to 5 amino acids, more preferably at least 8 to 10 amino acids, and even more preferably at least 15 to 20 amino acids from a polypeptide encoded by the nucleic acid sequence. Also encompassed are polypeptide sequences which are immunologically identifiable with a polypeptide encoded by the sequence.
- control elements include, but are not limited to, transcription promoters, transcription enhancer elements, transcription termination signals, polyadenylation sequences (located 3′ to the translation stop codon), sequences for optimization of initiation of translation (located 5′ to the coding sequence), translation enhancing sequences, and translation termination sequences.
- Transcription promoters can include inducible promoters (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), repressible promoters (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), and constitutive promoters.
- control elements for the dwf7 gene are found in the 5′ and 3′ UTRs shown in FIGS. 8 A- 8 B, particularly at positions 43-142 and 1710-1890, respectively, of the figure.
- Control elements for HDF7 are found within the 5′ and 3′ UTRs shown in FIGS. 10 A- 10 F, particularly within the region between positions 1-1505 and 2721-2925, respectively.
- a control element such as a promoter, “directs the transcription” of a coding sequence in a cell when RNA polymerase will bind the promoter and transcribe the coding sequence into mRNA, which is then translated into the polypeptide encoded by the coding sequence.
- “Expression enhancing sequences” typically refer to control elements that improve transcription or translation of a polynucleotide relative to the expression level in the absence of such control elements (for example, promoters, promoter enhancers, enhancer elements, and translational enhancers (e.g., Shine and Delagarno sequences).
- “Operably linked” refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended maimer.
- a control sequence “operably linked” to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.
- the control elements need not be contiguous with the coding sequence, so long as they function to direct the expression thereof.
- intervening untranslated yet transcribed sequences can be present between a promoter and the coding sequence and the promoter can still be considered “operably linked” to the coding sequence.
- a “heterologous sequence” as used herein typically refers to a nucleic acid sequence that is not normally found in the cell or organism of interest.
- a DNA sequence encoding a polypeptide can be obtained from a plant cell and introduced into a bacterial cell.
- the plant DNA sequence is “heterologous” to the native DNA of the bacterial cell.
- the “native sequence” or “wild-type sequence” of a gene is the polynucleotide sequence that comprises the genetic locus corresponding to the gene, e.g., all regulatory and open-reading frame coding sequences required for expression of a completely functional gene product as they are present in the wild-type genome of an organism.
- the native sequence of a gene can include, for example, transcriptional promoter sequences, translation enhancing sequences, introns, exons, and poly-A processing signal sites. It is noted that in the general population, wild-type genes may include multiple prevalent versions that contain alterations in sequence relative to each other and yet do not cause a discernible pathological effect. These variations are designated “polymorphisms” or “allelic variations.”
- “Recombinant” as used herein to describe a nucleic acid molecule means a polynucleotide of genomic, cDNA, semisynthetic, or synthetic origin which, by virtue of its origin or manipulation: (1) is not associated with all or a portion of the polynucleotide with which it is associated in nature; and/or (2) is linked to a polynucleotide other than that to which it is linked in nature.
- the term “recombinant” as used with respect to a protein or polypeptide means a polypeptide produced by expression of a recombinant polynucleotide.
- vector is meant any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, virus etc., which is capable of transferring gene sequences to target cells.
- a vector is capable of replication when associated with the proper control elements.
- the term includes cloning and expression vehicles, as well as viral vectors and integrating vectors.
- expression cassette refers to a molecule comprising at least one coding sequence operably linked to a control sequence which includes all nucleotide sequences required for the transcription of cloned copies of the coding sequence and the translation of the mRNAs in an appropriate host cell.
- Such expression cassettes can be used to express eukaryotic genes in a variety of hosts such as bacteria, blue-green algae, plant cells, yeast cells, insect cells and animal cells.
- expression cassettes can include, but are not limited to, cloning vectors, specifically designed plasmids, viruses or virus particles.
- the cassettes may further include an origin of replication for autonomous replication in host cells, selectable markers, various restriction sites, a potential for high copy number and strong promoters.
- a cell has been “transformed” by an exogenous polynucleotide when the polynucleotide has been introduced inside the cell.
- the exogenous polynucleotide may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell.
- the exogenous DNA may be maintained on an episomal element, such as a plasmid.
- a stably transformed cell is one in which the exogenous DNA has become integrated into the chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eucaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the exogenous DNA.
- “Recombinant host cells,” “host cells,” “cells,” “cell lines,” “cell cultures,” and other such terms denoting procaryotic microorganisms or eucaryotic cell lines cultured as unicellular entities, are used interchangeably, and refer to cells which can be, or have been, used as recipients for recombinant vectors or other transfer DNA, and include the progeny of the original cell which has been transfected. It is understood that the progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total DNA complement to the original parent, due to accidental or deliberate mutation.
- Progeny of the parental cell which are sufficiently similar to the parent to be characterized by the relevant property, such as the presence of a nucleotide sequence encoding a desired peptide, are included in the progeny intended by this definition, and are covered by the above terms.
- dwf7 polynucleotide refers to a polynucleotide derived from, or homologous to, the dwf7 gene.
- the gene encodes the protein variously referred to herein as DWF7, STE1 and DWF7/STE1.
- DWF7 is a ⁇ 7 sterol C-5 desaturase that functions in the brassinolide (BL) biosynthetic pathway from squalene to BL (see, FIG. 1).
- the dwf7 polynucleotide sequence and corresponding amino acid sequence are known and have been described in, e.g., Gachotte et al. (1996) Plant J. 9:391-398 and GenBank accession No.
- FIGS. 8 A- 8 D depicting the dwf7 gene sequence and the corresponding DWF7 amino acid sequence.
- the dwf7 gene spans the region from nucleotide positions 1-1889; the upstream 5′ UTR, including the promoter region, spans nucleotide positions 1-142; the downstream 3′ UTR is present from nucleotide position 1710-1889.
- the term as used herein encompasses a polynucleotide including a native sequence depicted in FIGS. 8 A- 8 D, as well as modifications and fragments thereof.
- the term encompasses alterations to the polynucleotide sequence, so long as the alteration results in a plant displaying one or more dwf7 phenotypic traits (described below) when the polynucleotide is expressed in a plant.
- modifications typically include deletions, additions and substitutions, to the native dwf7 sequence, so long as the mutation results in a plant displaying a dwf7 phenotype as defined below.
- These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of plants which express the dwf7 polynucleotide or errors due to PCR amplification.
- allelic variants of the wild-type dwf7 sequence which may occur by normal genetic variation or are produced by genetic engineering methods and which result in a detectable change in the wild-type dwf7 phenotype.
- Two particular dwf7 allelic variants described herein are dwf7-1 and dwf7-2.
- Polypeptides corresponding to these variants include about amino acids 1-60 and 1-230, respectively, of FIG. 9. However, the boundaries of these polypeptides may vary by 1 to 10 or more amino acids, or any integer therebetween.
- dwf7-1 and dwf7-2 polypeptides may include, for example, amino acids 1-59 and 1-229, respectively, or 3-62 and 3-232, respectively, and so on.
- dwf7 phenotype refers to any microscopic or macroscopic change in structure or morphology of a plant, such as a transgenic plant, as well as biochemical differences, which are characteristic of a dwf7 plant, compared to a progenitor, wild-type plant cultivated under the same conditions.
- morphological differences include short robust stems, reduced fertility, prolonged life cycle, dark-green, round, and curled leaves when grown in the light. In the dark, these plants exhibit short hypocotyls and expanded cotyledons, as compared to the wild-type plant.
- a “polypeptide” is used in it broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs, or other peptidomimetics. The subunits may be linked by peptide bonds or by other bonds, for example ester, ether, etc.
- amino acid refers to either natural and/or unnatural or synthetic amino acids, including glycine and both the D or L optical isomers, and amino acid analogs and peptidomimetics.
- a peptide of three or more amino acids is commonly called an oligopeptide if the peptide chain is short. If the peptide chain is long, the peptide is typically called a polypeptide or a protein.
- Full-length proteins, analogs, and fragments thereof are encompassed by the definition.
- the terms also include postexpression modifications of the polypeptide, for example, glycosylation, acetylation, phosphorylation and the like.
- a particular polypeptide may be obtained as an acidic or basic salt, or in neutral form.
- a polypeptide may be obtained directly from the source organism, or may be recombinantly or synthetically produced (see further below).
- a “DWF7” polypeptide is a polypeptide as defined above, which is derived from a ⁇ 7 sterol C-5 desaturase that functions in the brassinolide (BL) biosynthetic pathway from squalene to BL (see, FIG. 1).
- the native sequence of full-length DWF7 is shown in FIG. 9. However, the term encompasses analogs and fragments of the native sequence so long as the protein functions for its intended purpose.
- the term “DWF7 polypeptide” is intended to encompass the HDF7 polypeptidc and analogs thereof.
- DWF7 analog refers to derivatives of DWF7 and HDF7, or fragments of such derivatives, that retain desired function, e.g., as measured in assays as described further below.
- analog refers to compounds having a native polypeptide sequence and structure with one or more amino acid additions, substitutions (generally conservative in nature) and/or deletions, relative to the native molecule, so long as the modifications do not destroy desired activity.
- the analog has at least the same activity as the native molecule. Methods for making polypeptide analogs are known in the art and are described further below.
- amino acids are generally divided into four families: (1) acidic—aspartate and glutamate; (2) basic—lysine, arginine, histidine; (3) non-polar—alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar—glycine, asparagine, glutamine, cysteine, serine threonine, tyrosine.
- Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids.
- an isolated replacement of leucine with isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar conservative replacement of an amino acid with a structurally related amino acid will not have a major effect on the biological activity.
- the terms include the various sequence polymorphisms that exist, wherein amino acid substitutions in the protein sequence do not affect the essential functions of the protein.
- purified and isolated is meant, when referring to a polypeptide or polynucleotide, that the molecule is separate and discrete from the whole organism with which the molecule is found in nature; or devoid, in whole or part, of sequences normally associated with it in nature; or a sequence, as it exists in nature, but having heterologous sequences (as defined below) in association therewith. It is to be understood that the term “isolated” with reference to a polynucleotide intends that the polynucleotide is separate and discrete from the chormosome from which the polynucleotide may derive.
- isolated polynucleotide which encodes a particular polypeptide refers to a nucleic acid molecule which is substantially free of other nucleic acid molecules that do not encode the subject polypeptide; however, the molecule may include some additional bases or moieties which do not deleteriously affect the basic characteristics of the composition.
- fragment is intended a polypeptide or polynucleotide consisting of only a part of the intact sequence and structure of the reference polypeptide or polynucleotide, respectively.
- the fragment can include a 3′ or C-terminal deletion or a 5′ or N-terminal deletion, or even an internal deletion, of the native molecule.
- a polynucleotide fragment of a dwf7 sequence will generally include at least about 15 contiguous bases of the molecule in question, more preferably 18-25 contiguous bases, even more preferably 30-50 or more contiguous bases of the dwf7 molecule, or any integer between 15 bases and the full-length sequence of the molecule.
- Fragments which provide at least one dwf7 phenotype as defined above are useful in the production of transgenic plants. Fragments are also useful as oligonucleotide probes, to find additional dwf7 sequences.
- a polypeptide fragment of a DWF7 molecule will generally include at least about 10 contiguous amino acid residues of the full-length molecule, preferably at least about 15-25 contiguous amino acid residues of the full-length molecule, and most preferably at least about 20-50 or more contiguous amino acid residues of the full-length DWF7 molecule, or any integer between 10 amino acids and the full-length sequence of the molecule.
- Such fragments are useful for the production of antibodies and the like.
- transgenic plant is meant a plant into which one or more exogenous polynucleotides have been introduced. Examples of means by which this can be accomplished are described below, and include Agrobacterium-mediated transformation, biolistic methods, electroporation, and the like.
- the transgenic plant contains a polynucleotide which is not normally present in the corresponding wild-type plant and which confers at least one dwf7 phenotypic trait to the plant.
- the transgenic plant therefore exhibits altered structure, morphology or biochemistry as compared with a progenitor plant which does not contain the transgene, when the transgenic plant and the progenitor plant are cultivated under similar or equivalent growth conditions.
- Transgenic plants may also arise from sexual cross or by selfing of transgenic plants into which exogenous polynucleotides have been introduced.
- Such a plant containing the exogenous nucleic acid is also referred to here as an R 1 generation transgenic plant.
- Transgenic plants which arise from a sexual cross with another parent line or by selfing are “descendants or the progeny” of a R 1 plant and are generally called F n plants or S n plants, respectively, n meaning the number of generations.
- the present invention is based on the morphological, biochemical, and molecular analysis of Arabidopsis dwf7 mutants. Morphologically, dwf7 plants display a dramatic reduction in the length of many different organs examined, and this size reduction is attributable to a defect in cell elongation. Biochemically, dwf7 hypocotyls are converted to wild-type length with the application of BL, suggesting a deficiency in BRs.
- BR intermediate feeding analysis accompanied by analysis of endogenous levels of BRs and sterols by using GC-SIM, indicates that dwf7 is defective specifically in the ⁇ 7 sterol C-5 desaturase step of the sterol biosynthetic pathway.
- dwf7-1 and dwf7-2 revealed premature stop codons, suggesting loss-of-function mutations.
- the molecules of the present invention are therefore useful in the production of transgenic plants which display at least one dwf7 phenotype, so that the resulting plants have altered structure or morphology.
- the present invention particularly provides for altered structure or morphology such as reduced cell length, extended flowering periods, increased size of leaves or fruit, increased branching, increased seed production and altered sterol composition relative wild-type plants.
- the DWF7 polypeptides can be expressed to engineer a plant with desirable properties. The engineering is accomplished by transforming plants with nucleic acid constructs described herein which may also comprise promoters and secretion signal peptides. The transformed plants or their progenies are screened for plants that express the desired polypeptide.
- Engineered plants exhibiting the desired altered structure or morphology can be used in plant breeding or directly in agricultural production or industrial applications. Plants having the altered polypeptide can be crossed with other altered plants engineered with alterations in other growth modulation enzymes, proteins or polypeptides to produce lines with even further enhanced altered structural morphology characteristics compared to the parents or progenitor plants.
- oligonucleotide probes based on the sequences disclosed here can be used to identify the desired gene in a cDNA or genomic DNA library from a desired plant species.
- genomic libraries large segments of genomic DNA are generated by random fragmentation, e.g. using restriction endonucleases, and are ligated with vector DNA to form concatemers that can be packaged into the appropriate vector.
- tissue-specific cDNAs mRNA is isolated from tissues and a cDNA library which contains the gene transcripts is prepared from the mRNA.
- the cDNA or genomic library can then be screened using a probe based upon the sequence of a cloned gene such as the polynucleotides disclosed here. Probes may be used to hybridize with genomic DNA or cDNA sequences to isolate homologous genes in the same or different plant species.
- the nucleic acids of interest can be amplified from nucleic acid samples using amplification techniques. For instance, polymerase chain reaction (PCR) technology to amplify the sequences of the genes directly from mRNA, from cDNA, from genomic libraries or cDNA libraries.
- PCR polymerase chain reaction
- 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 MRNA in samples, for nucleic acid sequencing, or for other purposes.
- Appropriate primers and probes for identifying dwf7-specific genes from plant tissues are generated from comparisons of the sequences provided herein.
- Appropriate primers for this invention include, for instance, those primers described in the Examples and Sequence Listings, as well as other primers derived from the dwf sequences disclosed herein.
- Suitable amplifications conditions may be readily determined by one of skill in the art in view of the teachings herein, for example, including reaction components and amplification conditions as follows: 10 mM Tris-HCl, pH 8.3, 50 mM potassium chloride, 1.5 mM magnesium chloride, 0.001% gelatin, 200 ⁇ M dATP, 200 ⁇ M dCTP, 200 ⁇ M dGTP, 200 ⁇ M dTTP, 0.4 ⁇ M primers, and 100 units per mL Taq polymerase; 96° C. for 3 min., 30 cycles of 96° C. for 45 seconds, 50° C. for 60 seconds, 72° C. for 60 seconds, followed by 72° C. for 5 min.
- Polynucleotides may also be synthesized by well-known techniques as described in the technical literature. See, e.g., Carruthers, et al. (1982) Cold Spring Harbor Symp. Quant. Biol. 47:411-418, and Adams, et al. (1983) J. Am. Chem. Soc. 105:661. Double stranded DNA fragments may then be obtained either by synthesizing the complementary strand and annealing the strands together under appropriate conditions, or by adding the complementary strand using DNA polymerase with an appropriate primer sequence.
- the polynucleotides of the present invention may also be used to isolate or create other mutant cell gene alleles.
- Mutagenesis consists primarily of site-directed mutagenesis followed by phenotypic testing of the altered gene product. Some of the more commonly employed site-directed mutagenesis protocols take advantage of vectors that can provide single stranded as well as double stranded DNA, as needed. Generally, the mutagenesis protocol with such vectors is as follows.
- a mutagenic primer i.e., a primer complementary to the sequence to be changed, but consisting of one or a small number of altered, added, or deleted bases, is synthesized.
- the primer is extended in vitro by a DNA polymerase and, after some additional manipulations, the now double-stranded DNA is transfected into bacterial cells.
- the desired mutated DNA is identified, and the desired protein is purified from clones containing the mutated sequence.
- additional cloning steps are often required because long inserts (longer than 2 kilobases) are unstable in those vectors. Protocols are known to one skilled in the art and kits for site-directed mutagenesis are widely available from biotechnology supply companies, for example from Amersham Life Science, Inc. (Arlington Heights, Ill.) and Stratagene Cloning Systems (La Jolla, Calif.).
- Regulatory regions can be isolated from the dwf7 gene and used in recombinant constructs for modulating the expression of the dwf7 gene or a heterologous gene in vitro and/or in vivo.
- the coding region of the dwf7 gene begins at nucleotide position 143.
- the region of the gene spanning nucleotide positions 1-142 of FIGS. 8 A- 8 D includes the dwf7 promoter. This region may be used in its entirety or fragments of the region may be isolated which provide the ability to direct expression of a coding sequence linked thereto.
- promoters can be identified by analyzing the 5′ sequences of a genomic clone corresponding to the dwf7-specific genes described here. Sequences characteristic of promoter sequences can be used to identify the promoter. Sequences controlling eukaryotic gene expression have been extensively studied. For instance, promoter sequence elements include the TATA box consensus sequence (TATAAT), which is usually 20 to 30 base pairs upstream of the transcription start site. In most instances the TATA box is required for accurate transcription initiation. In plants, further upstream from the TATA box, at positions ⁇ 80 to ⁇ 100, there is typically a promoter element with a series of adenines surrounding the trinucleotide G (or T) N G. (See, J.
- the promoter region may include nucleotide substitutions, insertions or deletions that do not substantially affect the binding of relevant DNA binding proteins and hence the promoter function. It may, at times, be desirable to decrease the binding of relevant DNA binding proteins to “silence” or “down-regulate” a promoter, or conversely to increase the binding of relevant DNA binding proteins to “enhance” or “up-regulate” a promoter.
- the nucleotide sequence of the promoter region may be modified by, e.g., inserting additional nucleotides, changing the identity of relevant nucleotides, including use of chemically-modified bases, or by deleting one or more nucleotides.
- Promoter function can be assayed by methods known in the art:, preferably by measuring activity of a reporter gene operatively linked to the sequence being tested for promoter function.
- reporter genes include those encoding luciferase, green fluorescent protein, GUS, neo, cat and bar.
- UTR sequences include introns and 5′ or 3′ untranslated regions (5′ UTRs or 3′ UTRS).
- the dwf7 gene sequence includes three exons (thick boxes) and two introns (horizontal bars). See, also, FIGS. 8 A- 8 D for the 5′ and 3′ UTRs.
- the HDF7 gene includes three exons (at positions 1506-1734, 2024-2329 and 2416-2720, denoted by the corresponding protein sequence indicated) and two introns (between these exons) and 5 40 and 3′ UTRs.
- portions of the dwf7 and HDF7 genes can have regulatory functions related to, for example, translation rate and MRNA stability.
- these portions of the gene can be isolated for use as elements of gene constructs for expression of polynucleotides encoding desired polypeptides.
- Introns of genomic DNA segments may also have regulatory functions. Sometimes promoter elements, especially transcription enhancer or suppressor elements, are found within introns. Also, elements related to stability of heteronuclear RNA and efficiency of transport to the cytoplasm for translation can be found in intron elements. Thus, these segments can also find use as elements of expression vectors intended for use to transform plants.
- the introns, UTR sequences and intron/exon junctions can vary from the native sequence. Such changes from those sequences preferably will not affect the regulatory activity of the UTRs or intron or intron/exon junction sequences on expression, transcription, or translation. However, in some instances, down-regulation of such activity may be desired to modulate traits or phenotypic or in vitro activity.
- expression cassettes of the invention can be used to suppress endogenous dwf7 gene expression.
- Inhibiting expression can be useful, for instance, in suppressing the phenotype (e.g., dwarf appearance, the ⁇ 7 sterol C-5 desaturase activity) exhibited by dwf7 plants.
- a number of methods can be used to inhibit gene expression in plants.
- antisense technology can be conveniently used.
- a nucleic acid segment from the desired gene is cloned and operably linked to a promoter such that the antisense strand of RNA will be transcribed.
- the expression cassette is then transformed into plants and the antisense strand of RNA is produced.
- antisense RNA inhibits gene expression by preventing the accumulation of mRNA which encodes the enzyme of interest, see, e.g., Sheehy et al. (1988) Proc. Nat. Acad. Sci. USA 85:8805-8809, and Hiatt et al., U.S. Pat. No. 4,801,340.
- the nucleic acid segment to be introduced generally will be substantially identical to at least a portion of the endogenous gene or genes to be repressed.
- the sequence need not be perfectly identical to inhibit expression.
- the vectors of the present invention can be designed such that the inhibitory effect applies to other proteins within a family of genes exhibiting homology or substantial homology to the target gene.
- the introduced sequence also need not be full length relative to either the primary transcription product or fully processed mRNA. Generally, higher homology can be used to compensate for the use of a shorter sequence. Furthermore, the introduced sequence need not have the same intron or exon pattern, and homology of non-coding segments may be equally effective. Normally, a sequence of between about 30 or 40 nucleotides and about full length nucleotides should be used, though a sequence of at least about 100 nucleotides is preferred, a sequence of at least about 200 nucleotides is more preferred, and a sequence of at least about 500 nucleotides is especially preferred. It is to be understood that any integer between the above-recited ranges is intended to be captured herein.
- RNA molecules or ribozymes can also be used to inhibit expression of dwf7 genes. It is possible to design ribozymes that specifically pair with virtually any target RNA and cleave the phosphodiester backbone at a specific location, thereby functionally inactivating the target RNA. In carrying out this cleavage, the ribozyme is not itself altered, and is thus capable of recycling and cleaving other molecules, making it a true enzyme. The inclusion of ribozyme sequences within antisense RNAs confers RNA-cleaving activity upon them, thereby increasing the activity of the constructs.
- RNAs A number of classes of ribozymes have been identified.
- One class of ribozymes is derived from a number of small circular RNAs which are capable of self-cleavage and replication in plants.
- the RNAs replicate either alone (viroid RNAs) or with a helper virus (satellite RNAs). Examples include RNAs from avocado sunblotch viroid and the satellite RNAs from tobacco ringspot virus, lucerne transient streak virus, velvet tobacco mottle virus, solanum nodiflorum mottle virus and subterranean clover mottle virus.
- the design and use of target RNA-specific ribozymes is described in Haseloff et al. (1988) Nature 334:585-591.
- Another method of suppression is sense suppression.
- Introduction of expression cassettes in which a nucleic acid is configured in the sense orientation with respect to the promoter has been shown to be an effective means by which to block the transcription of target genes.
- this method to modulate expression of endogenous genes see, Napoli et al. (1990) The Plant Cell 2:279-289 and U.S. Pat. Nos. 5,034,323, 5,231,020, and 5,283,184.
- the introduced sequence generally will be substantially identical to the endogenous sequence intended to be repressed. This minimal identity will typically be greater than about 65%, but a higher identity might exert a more effective repression of expression of the endogenous sequences. Substantially greater identity of more than about 80% is preferred, though about 95% to absolute identity would be most preferred. It is to be understood that any integer between the above-recited ranges is intended to be captured herein. As with antisense regulation, the effect should apply to any other proteins within a similar family of genes exhibiting homology or substantial homology.
- the introduced sequence in the expression cassette needing less than absolute identity, also need not be full length, relative to either the primary transcription product or fully processed mRNA. This may be preferred to avoid concurrent production of some plants which are overexpressers. A higher identity in a shorter than full length sequence compensates for a longer, less identical sequence. Furthermore, the introduced sequence need not have the same intron or exon pattern, and identity of non-coding segments will be equally effective. Normally, a sequence of the size ranges noted above for antisense regulation is used.
- the polynucleotides of the invention can be used to increase certain features such as extending flowering, producing larger leaves or fruit, producing increased branching and increasing seed production. This can be accomplished by the overexpression of dwf7 polynucleotides.
- the exogenous dwf7 polynucleotides do not have to code for exact copies of the endogenous DWF7 and HDF7 proteins.
- Modified DWF7 and HDF7 protein chains can also be readily designed utilizing various recombinant DNA techniques well known to those skilled in the art and described for instance, in Sambrook et al., supra. Hydroxylamine can also be used to introduce single base mutations into the coding region of the gene (Sikorski et al. (1991) Meth. Enzymol. 194: 302-318).
- the chains can vary from the naturally occurring sequence at the primary structure level by amino acid substitutions, additions, deletions, and the like. These modifications can be used in a number of combinations to produce the final modified protein chain.
- DNA sequence coding for the desired polypeptide for example a cDNA sequence encoding the full length DWF7 protein, will preferably be combined with transcriptional and translational initiation regulatory sequences which will direct the transcription of the sequence from the gene in the intended tissues of the transgenic plant.
- Such regulatory elements include but are not limited to the promoters derived from the genome of plant cells (e.g., heat shock promoters such as soybean hspl7.5-E or hsp17.3-B (Gurley et al. (1986) Mol. Cell. Biol. 6:559-565); the promoter for the small subunit of RUBISCO (Coruzzi et al. (1984) EMBO J. 3:1671-1680; Broglie et al. (1984) Science 224:838-843); the promoter for the chlorophyll a/b binding protein) or from plant viruses viral promoters such as the 35S RNA and 19S RNA promoters of CaMV (Brisson et al.
- promoters derived from the genome of plant cells e.g., heat shock promoters such as soybean hspl7.5-E or hsp17.3-B (Gurley et al. (1986) Mol. Cell. Biol. 6:559-565
- a plant promoter fragment may be employed which will direct expression of the gene in all tissues of a regenerated plant.
- Such promoters are referred to herein as “constitutive” promoters and are active under most environmental conditions and states of development or cell differentiation.
- constitutive promoters include the cauliflower mosaic virus (CaMV) 35S transcription initiation region, the T-DNA mannopine synthetase promoter (e.g., the 1′- or 2′-promoter derived from T-DNA of Agrobacterium tumafaciens), and other transcription initiation regions from various plant genes known to those of skill.
- the plant promoter may direct expression of the polynucleotide of the invention in a specific tissue (tissue-specific promoters) or may be otherwise under more precise environmental control (inducible promoters).
- tissue-specific promoters under developmental control include promoters that initiate transcription only in certain tissues, such as fruit, seeds, or flowers such as tissue- or developmental-specific promoter, such as, but not limited to the cell promoter, the CHS promoter, the PATATIN promoter, etc.
- the tissue specific E8 promoter from tomato is particularly useful for directing gene expression so that a desired gene product is located in fruits.
- Suitable promoters include those from genes encoding embryonic storage proteins. Examples of environmental conditions that may affect transcription by inducible promoters include anaerobic conditions, elevated temperature, or the presence of light. If proper polypeptide expression is desired, a polyadenylation region at the 3′-end of the coding region should be included.
- the polyadenylation region can be derived from the natural gene, from a variety of other plant genes, or from T-DNA.
- the promoter itself can be derived from the dwf7 or HDF7 genes, as described above.
- the vector comprising the sequences (e.g., promoters or coding regions) from genes of the invention will typically comprise a marker gene which confers a selectable phenotype on plant cells.
- the marker may encode biocide resistance, particularly antibiotic resistance, such as resistance to kanamycin, G418, bleomycin, hygromycin, or herbicide resistance, such as resistance to chlorosluforon or Basta.
- DNA constructs of the invention may be introduced into the genome of the desired plant host by a variety of conventional techniques. For reviews of such techniques see, for example, Weissbach & Weissbach Methods for Plant Molecular Biology (1988, Academic Press, N.Y.) Section VIII, pp. 421-463; and Grierson & Corey, Plant Molecular Biology (1988, 2d Ed.), Blackie, London, Ch. 7-9.
- the DNA construct may be introduced directly into the genomic DNA of the plant cell using techniques such as electroporation and microinjection of plant cell protoplasts, or the DNA constructs can be introduced directly to plant tissue using biolistic methods, such as DNA particle bombardment (see, e.g., Klein et al.
- the DNA constructs may be combined with suitable T-DNA flanking regions and introduced into a conventional Agrobacterium tumefaciens host vector.
- Agrobacterium tumefaciens-mediated transformation techniques including disarming and use of binary vectors, are well described in the scientific literature. See, for example Horsch et al. (1984) Science 233:496-498, and Fraley et al. (1983) Proc. Nat'l. Acad. Sci. USA 80:4803.
- the virulence functions of the Agrobacterium tumefaciens host will direct the insertion of the construct and adjacent marker into the plant cell DNA when the cell is infected by the bacteria using binary T DNA vector (Bevan (1984) Nuc. Acid Res. 12:8711-8721) or the co-cultivation procedure (Horsch et al. (1985) Science 227:1229-1231).
- binary T DNA vector Bevan (1984) Nuc. Acid Res. 12:8711-8721
- the co-cultivation procedure Horsch et al. (1985) Science 227:1229-1231.
- the Agrobacterium transformation system is used to engineer dicotyledonous plants (Bevan et al. (1982) Ann. Rev. Genet 16:357-384; Rogers et al. (1986) Methods Enzymol. 118:627-641).
- the Agrobacterium transformation system may also be used to transform, as well as transfer, DNA to monocotyledonous plants and plant cells.
- EMBO J 3:3039-3041 Hooykass-Van Slogteren et al. (1984) Nature 311:763-764; Grimsley et al. (1987) Nature 325:1677-179; Boulton et al. (1989) Plant Mol. Biol. 12:31-40.; and Gould et al. (1991) Plant Physiol. 95:426-434).
- Alternative gene transfer and transformation methods include, but are not limited to, protoplast transformation through calcium-, polyethylene glycol (PEG)- or electroporation-mediated uptake of naked DNA (see Paszkowski et al. (1984) EMBO J 3:2717-2722, Potrykus et al. (1985) Molec. Gen. Genet. 199:169-177; Fromm et al. (1985) Proc. Nat. Acad. Sci. USA 82:5824-5828; and Shimamoto (1989) Nature 338:274-276) and electroporation of plant tissues (D'Halluin et al. (1992) Plant Cell 4:1495-1505).
- PEG polyethylene glycol
- Additional methods for plant cell transformation include microinjection, silicon carbide mediated DNA uptake (Kaeppler et al. (1990) Plant Cell Reporter 9:415-418), and microprojectile bombardment (see Klein et al. (1988) Proc. Nat. Acad. Sci. USA 85:4305-4309; and Gordon-Kamm et al. (1990) Plant Cell 2:603-618).
- Transformed plant cells which are produced 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 desired nucleotide sequences.
- Plant regeneration from cultured protoplasts is described in Evans, et al., “Protoplasts Isolation and Culture” in Handbook of Plant Cell Culture, pp. 124-176, Macmillian Publishing Company, New York, 1983; and Binding, Regeneration of Plants, Plant Protoplasts, pp. 21-73, CRC Press, Boca Raton, 1985.
- Regeneration can also be obtained from plant callus, explants, organs, pollens, embryos or parts thereof.
- Such regeneration techniques are described generally in Klee et al. (1987) Ann. Rev. of Plant Phys. 38:467-486
- nucleic acids of the invention can be used to confer desired traits on essentially any plant.
- a wide variety of plants and plant cell systems may be engineered for the desired physiological and agronomic characteristics described herein using the nucleic acid constructs of the present invention and the various transformation methods mentioned above.
- target plants and plant cells for engineering include, but are not limited to, those monocotyledonous and dicotyledonous plants, such as crops including grain crops (e.g., wheat, maize, rice, millet, barley), fruit crops (e.g., tomato, apple, pear, strawberry, orange), forage crops (e.g., alfalfa), root vegetable crops (e.g., carrot, potato, sugar beets, yam), leafy vegetable crops (e.g., lettuce, spinach); flowering plants (e.g., petunia, rose, chrysanthemum), conifers and pine trees (e.g., pine fir, spruce); plants used in phytoremediation (e.g., heavy metal accumulating plants); oil crops (e.g., sunflower, rape seed) and plants used for experimental purposes (e.g., Arabidopsis).
- crops including grain crops e.g., wheat, maize, rice, millet, barley
- the invention has use over a broad range of plants, including, but not limited to, species from the genera Asparagus, Avena, Brassica, Citrus, Citrullus, Capsicum, Cucurbita, Daucus, Glycine, Hordeum, Lactuca, Lycopersicon, Malus, Manihot, Nicotiana, Oryza, Persea, Pisum, Pyrus, Prunus, Raphanus, Secale, Solanum, Sorghum, Triticum, Vitis, Vigna, and Zea.
- a transformed plant cell, callus, tissue or plant may be identified and isolated by selecting or screening the engineered plant material for traits encoded by the marker genes present on the transforming DNA. For instance, selection may be performed by growing the engineered plant material on media containing an inhibitory amount of the antibiotic or herbicide to which the transforming gene construct confers resistance. Further, transformed plants and plant cells may also be identified by screening for the activities of any visible marker genes (e.g., the ⁇ -glucuronidase, luciferase, B or C1 genes) that may be present on the recombinant nucleic acid constructs of the present invention. Such selection and screening methodologies are well known to those skilled in the art.
- any visible marker genes e.g., the ⁇ -glucuronidase, luciferase, B or C1 genes
- Physical and biochemical methods also may be used to identify plant or plant cell transformants containing the gene constructs of the present invention. These methods include but are not limited to: 1) Southern analysis or PCR amplification for detecting and determining the structure of the recombinant DNA insert; 2) Northern blot, S1 RNase protection, primer-extension or reverse transcriptase-PCR amplification for detecting and examining RNA transcripts of the gene constructs; 3) enzymatic assays for detecting enzyme or ribozyme activity, where such gene products are encoded by the gene construct; 4) protein gel electrophoresis, Western blot techniques, immunoprecipitation, or enzyme-linked immunoassays, where the gene construct products are proteins.
- RNA e.g., mRNA
- mRNA RNA isolated from the tissues of interest.
- mRNA RNA isolated from the tissues of interest.
- dwf7 dwf7 gene is being expressed at a greater rate than before.
- Other methods of measuring DWF7 activity can be used. For example, cell length can be measured at specific times. Because dwf7 affects the BR biosynthetic pathway, an assay that measures the amount of BL can also be used. Such assays are known in the art.
- enzymatic assays can be used, depending on the substrate used and the method of detecting the increase or decrease of a reaction product or by-product.
- the levels of DWF7 protein expressed can be measured immunochemically, i.e., ELISA, RIA, EIA and other antibody based assays well known to those of skill in the art, by electrophoretic detection assays (either with staining or western blotting), and sterol (BL) detection assays.
- the present invention also encompasses seeds of the transgenic plants described above wherein the seed has the transgene or gene construct.
- the present invention further encompasses the progeny, clones, cell lines or cells of the transgenic plants described above wherein said progeny, clone, cell line or cell has the transgene or gene construct.
- the present invention also includes DWF7 polypeptides, including such polypeptides as a fusion, or chimeric protein product (comprising the protein, fragment, analog, mutant or derivative joined via a peptide bond to a heterologous protein sequence (of a different protein)).
- a chimeric product can be made by ligating the appropriate nucleic acid sequences encoding the desired amino acid sequences to each other by methods known in the art, in the proper coding frame, and expressing the chimeric product by methods commonly known in the art.
- DWF7 polypeptides, derivatives (including fragments and chimeric proteins), mutants and analogues can be chemically synthesized. See, e.g., Clark-Lewis et al. (1991) Biochem. 30:3128-3135 and Merrifield (1963) J. Amer. Chem. Soc. 85:2149-2156.
- DWF7, derivatives, mutants and analogs can be synthesized by solid phase techniques, cleaved from the resin, and purified by preparative high performance liquid chromatography (e.g., see Creighton, 1983, Proteins, Structures and Molecular Principles, W. H. Freeman and Co., N.Y., pp. 50-60).
- the polynucleotide sequences may additionally be used to isolate mutant dwf7 gene alleles. Such mutant alleles may be isolated from plant species either known or proposed to have a genotype which contributes to altered plant morphology.
- plant dwf7 gene sequences can be used to detect plant dwf7 gene regulatory (e.g., promoter or promotor/enhancer) defects which can affect plant growth.
- plant dwf7 gene regulatory e.g., promoter or promotor/enhancer
- the molecules of the present invention can be used to provide plants with increased seed and/fruit production, extended flowering periods and increased branching.
- the molecules described herein can be used to alter the sterol composition of a plant, thereby increasing or reducing cholesterol content in the plant.
- a still further utility of the molecules of the present invention is to provide a tool for studying the biosynthesis of brassinosteriods, both in vitro and in vivo.
- the dwf7 gene of the invention also has utility as a transgene encoding a the ⁇ 7 sterol C-5 desaturation protein that mediates one or more steps in brassinosteriod biosynthesis which results in a transgenic plant to alter plant structure or morphology.
- the dwf7 gene also has utility for encoding the DWF7 protein in recombinant vectors which may be inserted into host cells to express the DWF7 protein.
- the dwf7 polynucleotides of the invention may be utilized (1) as nucleic acid probes to screen nucleic acid libraries to identify other enzymatic genes or mutants; (2) as nucleic acid sequences to be mutated or modified to produce DWF7 protein variants or derivatives; (3) as nucleic acids encoding the ⁇ 7 sterol C-5 desaturases in molecular biology techniques or industrial applications commonly known to those skilled in the art.
- the dwf7 nucleic acid molecules may be used to design antisense molecules, useful, for example, in gene regulation or as antisense primers in amplification reactions of dwf7 gene nucleic acid sequences.
- antisense molecules useful, for example, in gene regulation or as antisense primers in amplification reactions of dwf7 gene nucleic acid sequences.
- dwf7 gene regulation such techniques can be used to regulate, for example, plant growth, development or gene expression.
- sequences may be used as part of ribozyme and/or triple helix sequences, also useful for dwf7 gene regulation.
- the dwf7 control element (e.g., promoter) of the present invention may be utilized as a plant promoter to express any protein, polypeptide or peptide of interest in a transgenic plant.
- the dwf7 promoter may be used to express a protein involved in brassinosteriod biosynthesis.
- the Arabidopsis DWF7 protein of the invention can be used in any biochemical applications (experimental or industrial) where ⁇ 7 sterol C-5 desaturation activity is desired, for example, but not limited to, regulation of BL synthesis, regulation of other sterol synthesis, modification of elongating plant structures, and experimental or industrial biochemical applications known to those skilled in the art.
- Restriction and modifying enzymes as well as PCR reagents were purchased from commercial sources, and used according to the manufacturers' directions. In the cloning of DNA fragments, except where noted, all DNA manipulations were done according to standard procedures. See, e.g., Sambrook et al., supra. Restriction enzymes, T 4 DNA ligase, E. coli, DNA polymerase I, Klenow fragment, and other biological reagents were purchased from commercial suppliers and used according to the manufacturers' directions.
- GC-SIM gas chromatography-selective ion monitoring
- dwf7 was performed using simple sequence length polymorphism (SSLP) markers (Bell and Ecker (1994) Genomics 19:137-144). Briefly, dwf7-1 mutants (Wassilewskija-2 [Ws-2] background) were crossed to Columbia wild-type plants. Genomic DNA was isolated (Dellaporta et al. 1983) from individual F 2 dwarf plants. To locate the mutation to one of the five chromosomes, 20 individual plants were tested with at least two SSLP markers per chromosome. The polymerase chain reaction (PCR) amplified products were analyzed on 4% agarose gels in 1 ⁇ TAE buffer (40 mM Tris-acetate and 10 mM EDTA).
- PCR polymerase chain reaction
- PCR products amplified using primer sets derived from the cDNA sequence of STEROL1 were subjected to sequencing.
- primer sets derived from the cDNA sequence of STEROL1 were subjected to sequencing.
- To design sets of primers that do not fall in exon-intron junctions we predicted possible splice sites by using the RNASPL program available at the internet site of Baylor College of Medicine (Houston, Tex.; http://dot.imgen.bcm.tmc.edu:9331/seq-search/gene-search.html).
- Primers were designed using the Primer Selection software of DNAstar (DNASTAR Inc., Madison, Wis.).
- Oligonucleotide sequences 5′ to 3′ are CAGTGTGAGTAAT T TAGCAT TACTA (S5D_FF), GGAAAGATCATC-AAACAT T TACATGT (S5D_LR), GCGCAATCT TCT T TCGT T T (S5D — 1F), TGGACAACAACAACACAAGA (S5D — 1R), GATGCACAGAGAGCT-TCATGAC (S5D — 2F), CCGGCAAATGGAGAGAGTGTAT (S5D — 2R), CACCCATCATATCTACAACAA (S5D — 3F), and CATCT T TGCCG-GCGAATCTAT (S5D — 4F) (underlines were added to distinguish forward or reverse primers from the gene acronym S5D).
- Primers were purchased from Genosys Biotechnologies, Inc. (The Woodlands, Tex.).
- genomic DNA was isolated from two or three leaves of dwf7-1 and wild-type plants according to the method described by Krysan et al. (1996) Proc. Natl. Acad. Sci. USA 93:8145-8150.
- Amplification of the DNA fragment spanning the whole coding region was performed with the S5D — 4F and S5D — 1R primer set with Taq polymerase (Boehringer Mannheim).
- Standard PCR reaction mixtures 1 ⁇ PCR buffer (10 mM Tris-HCl, 1.5 mM MgCl 2 , and 50 mM KCl, pH 8.3), 0.2 ⁇ M each of forward and reverse primer, 0.2 mM each deoxynucleotide triphosphates, 1 ng of genomic DNA, and 2 units of Taq polymerase were subjected to a PCR program consisting of an initial denaturation at 95° C. for 2 min and then for 35 cycles (95° C. for 30 sec, 56° C. for 30 sec, and 72° C. for 2.5 min), with a final elongation step of 7 min at 72° C.
- PCR-amplified DNA was size-separated on 0.8% agarose gels in 1 ⁇ TAE, and the resulting DNA bands were gel-purified using a DNA purification kit (Bio-Rad). The concentration of the extracted DNA was measured by comparing the band intensity with a DNA mass standard (Bethesda Research Laboratories). Sequencing of the DNA was performed at the Arizona Research Laboratory (University of Arizona, Arlington). DNA sequence analysis was conducted using software packages, including one from Genetics Computer Group (Madison, Wis.) and other database search tools available on the Internet.
- the base change in dwf7-1 eliminated the recognition site for a restriction enzyme HaeIII by converting the sequence from GGCC to AGCC.
- This polymorphism to test the co segregation of the dwarf phenotype with the mutation.
- the 0.8 kb of DNA spanning the mutation was amplified using S5D — 3F and S5D — 1R primers from 17 different dwarf plants from the mapping lines.
- Two microliters from each 20 ⁇ L of PCR-amplified DNA was digested with the restriction enzyme HaeIII (Boehringer Mannheim). After complete digestion, the samples were resolved on a 2% agarose gel in 1 ⁇ TAE buffer.
- Genomic DNA sequence flanking the CDNA was identified by sequencing the products obtained from thermal asymmetric interlaced PCR (TAIL PCR) (Liu et al. (1995) Plant J. 8:457-463). Two sets of primers were used to amplify the 5′ and 3′ flanking DNA.
- TAIL PCR thermal asymmetric interlaced PCR
- Oligonucleotide sequences 5′ to 3′ are GTAGAAGCACCAGAGGAAACCGGAGATGAAGT (D7-5-1; melting temperature of 69° C.), AAGTATAGTAGGGT TCCGGCGAGG-TA (D7-5-2; melting temperature of 64° C.), ATAGAT TCGCCG-GCAAAAGATGACTC (D7-5-3; melting temperature of 63° C.), TGC-AGGATACCATACGATACACCACACGACAT (D7-3-1; melting temperature of 68° C.), CATACGATACACCACACGACATACAAGCAT-AACTA (D7-3-2; melting temperature of 67° C.), and ATATGGATG-GAT TGGATGT T TGGCTCTC (D7-3-3; melting temperature of 63° C.).
- each primer was calculated with the formula 69.3+0.41 (%GC) 650/L (Mazars et al. (1991) Nucleic Acids Res. 19:4783), where L is length of primer.
- Arbitrary degenerate primers AD1, AD2, and AD3 were synthesized according to the sequence described by Liu et al. (1995) Plant J. 8:457-463.
- TAIL PCR was performed according to the program originally described by Liu et al. 1995. TAIL PCR-amplified DNA was separated on 1% agarose gels and gel extracted for sequencing.
- the seedlings ( ⁇ 5 g fresh weight of both Ws-2 and dwf7-1 plant materials) were extracted with methanol (250 mL), and the extract was partitioned between CHCl 3 and H 2 O.
- the CHCl 3 -soluble fraction was purified with a silica cartridge column (Sep-Pak Vac 12 cc; Waters, Milford, Mass.), which was eluted with 20 mL of CHCl 3 .
- the eluate was purified with an octadecylsilane (ODS) cartridge column (Sep-Pak PLUS C18; Waters), which was eluted with 20 mL of methanol.
- ODS octadecylsilane
- the fraction was subjected to HPLC on an ODS column as follows: column, Senshu Pak ODS 4150-N (150 ⁇ 10 mm); solvent, methanol; flow rate, 2 mL /min; and detection, UV 205 nm. Fractions were collected every 0.5 min (between retention times of 10 to 20 min). Main fractions of each sterol were as follows: 5-dehydroepisterol (retention time of 11.5 to 12 min), episterol (retention time of 12.5 to 13 min), 24-methylenecholesterol (24-MC; retention time of 13 to 13.5 min), 7-dehydrocampestanol (retention time of 14.5 to 15 min), and campesterol (CR; retention time of 15.5 to 16 min).
- GC-MS analyses were performed on a JEOL Automass JMS-AM 150 mass spectrometer (Tokyo, Japan) connected to a Hewlett-Packard 5890A-II gas chromatograph with a capillary column DB-5 (0.25 mm ⁇ 15 m; 0.25- ⁇ m film thickness). The analytical conditions were the same as previously described (Fujioka et al. 1997).
- the dwf7-1 mutant originally was identified in a screen of 14,000 T-DNA-transformed lines of Arabidopsis. Genetic complementation tests with other dwf loci indicated that dwf7 belongs to a unique complementation group. dwf7-1 segregated as a monogenic recessive mutation; progeny from a heterozygote segregated 325 (wild-type): 98 (dwf7-1). Although dwf7-1 originated from a T-DNA mutant population, it failed to cosegregate with the kanamycin resistance marker in the T-DNA, suggesting that dwf7-1 was an untagged mutant.
- mapping the dwf7-1 mutation to the Arabidopsis genome by using simple sequence length polymorphisms (SSLPs; Bell and Ecker (1994) Genomics 19:137-144) confirmed that dwf7 maps to a location different from previously isolated dwarfs.
- the meiotic recombination ratio between dwf7 and the SSLP marker ngal 72 on chromosome 3 was scored as 0/86, indicating tight linkage of dwf7 to ngal72.
- ngal72 is located 2.2 centimorgans from the top of chromosome 3.
- a second allele of dwf7 was identified among 43 dwarf mutants isolated by screening >50,000 M2 seeds of an EMS mutant population. Similar to dw7-1, the new allele was biochemically complemented by early BR biosynthetic intermediates, including 22 ⁇ -hydroxycampesterol (22-OHCR) and cathasterone, and mapped near ngal72. Sequencing revealed a premature stop codon in exon 1 (see below).
- dwf7 displays many of the characteristics of other BR dwarfs.
- the characteristic dwarf phenotype such as short robust stems, reduced fertility, and dark-green, round, and curled leaves are found in the plants.
- dwf7-1 plants grown for 5 weeks in the light possess short robust inflorescences, dark-green, round leaves, reduced fertility, and short pedicels and siliques.
- the wild-type generally terminates flowering before 7 weeks of age; however, dwf7-1 continues to produce flowers at this age. At 7 weeks of age, wild-type plants had ceased growing, whereas dwf7-1 plants continued to grow, indicating a prolonged life span.
- null mutations in the BR pathway result in a dwarf phenotype, as well as defects in skotomorphogenesis
- dwf7-1 displays a less severe phenotype (35% that of wild-type hypocotyl length) than do other BR dwarfs (e.g., 7% of wild type in dwf4; Choe et al. (1998) Plant Cell 10:231-243). Furthermore, dwf7-1 frequently displayed closed cotyledons and hooks similar to those of the wild type, whereas severe dwarfs, including bril/dwf2, cpd/dwf3, and dwf4, showed expanded cotyledons and open hooks.
- dwf7-1 mutants are not mechanically sterile.
- Scanning electron microscopy demonstrated a relationship between fertility and floral structure.
- the length of stamens was greater than or similar to that of the gynoecium (quantified in FIG. 2), facilitating dehiscence of pollen on the stigmatic surface.
- the fertile dwf7-1 flower had a concomitant reduction in the size of the gynoecium and the stamen.
- dwf7-1 flowers possess stamens and gynoecia that are shorter than those in the wild type, the fertility of dwf7-1 flowers is possible through the concomitant reduction in the length of both organs.
- stamen elongation was affected more severely in dwf4-3 flowers (FIG. 2). Because sterile dwf4-3 flowers have shorter filaments than the gynoecium, pollen dehiscence on the stigmatic surface is prevented.
- dwf4 The short stamen length in dwf4 is likely to cause dehiscence of pollen on the ovary wall rather than on the stigmatic surface. In fact, when dwf4 pollen is transferred to either wild-type or dwf7-1 stigmas, viable seeds are made.
- the common denominator for the various phenotypes found in dwf7-1 mutants is a reduction in longitudinal growth, which could be due to either a reduced number of cells or a failure in cell elongation.
- Observations made with other BR dwarf mutants suggest that the number of cells is comparable in the wild type and mutants (Kauschmann et al. (1996) Plant J. 9:701-713; Nomura et al. (1997) Plant Physiol. 113:31-37; Azpiroz et al. (1998) Plant Cell 10:219-230).
- the length of cells in the epidermis, cortex, and xylem of dwf7-1 was greatly reduced ( ⁇ 30% of wild type). This reduced cell size was converted to the length of the wild type in response to daily application of 10 ⁇ 7 M BL for 1 week.
- the reduced organ length in dwf7-1 also is due to a failure of cell elongation.
- vascular bundles in wild-type and dwf7-1 mutants were also examined. Wild-type inflorescences possessed eight vascular bundles. However, the number of vascular bundles was reduced to six in dwf7-1. Furthermore, the spacing between the vascular bundles in dwf7-1 was irregular. In the wild type, interfascicular parenchyma cells alternated regularly with vascular bundles; however, cross-sections of dwf7-1 showed that two vascular bundles were joined without being separated by parenchyma cells.
- FIG. 3 demonstrates that dwf7-1 seedlings grown in BL-supplemented liquid media were remarkably sensitive to BL. Growth in 1 nM BL induced significant elongation of dwf7-1 hypocotyls (160% increase), whereas the wild-type increase was marginal (5%). Treatment with 10 and 100 nM BL completely rescued dwf7-1 hypocotyls to wild-type length. The strongest response of the wild type to BL was obtained at 100 nM (FIG. 3). Higher concentrations of BL (1 ⁇ M) caused a stressed morphology, including inhibition of root growth and swollen, twisted, and fragile hypocotyls in both dwf7-1 and wild-type plants. After BL treatment of dwf7-1, cells in the treated region of the stem were similar in length to wild-type cells.
- the overall morphology of plants is dependent on three factors: cell size, shape, and number (Cosgrove (1997) Plant Cell 9:1031-1041). Various signals modulate these factors.
- Environmental signals such as water, temperature, and light, are transduced to invoke internal hormone signals, including auxins, gibberellins, and BRs. These signals then trigger the cell elongation process, including but not limited to cell wall loosening by xyloglucan endotransglycosylases and expansins.
- a block in any of the signal transduction cascades from the environmental signals to the cell elongation process could result in dwarfism.
- auxin e.g., auxin resistant2[axr2]; Timpte (1992) Genetics 138:1239-1249
- gibberellin [ga1 to ga5 and gai]; Koornneef and van der Veen (1980) Theor. Appl. Genet. 58:257-263; Koornneef et al. (1985) Physiol. Plant. 65:33-39
- dwf7 is either rescued by or resistant to exogenous application of these hormones.
- dwf7-1 hypocotyls The length of dwf7-1 hypocotyls was increased 160% in response to 1 nM BL as compared with the untreated control, whereas the wild-type, responded marginally (5%).
- application of BRs to 3-week-old dwf7-1 plants induced the growth of many different organs, including stems, leaves, siliques, petioles, and pedicels, suggesting that the major defect in dwf7-1 is a deficiency of BL.
- Auxins also are known to be a major factor affecting differentiation of the vascular system (Aloni (1987) Annu. Rev. Plant Physiol. 38:179-204). Lincoln et al. (1990) Plant Cell 2:1071-1080 showed that stem cross-sections of axr1 displayed altered development of the vascular system. The vascular bundles in axr1 mutants are located peripherally and are not as regularly spaced as compared with those in wild-type plants (Lincoln et al. (1990) Plant Cell 2:1071-1080).
- auxins and BRs play opposing roles in determining the number of vascular bundles.
- Two other assays in which auxin and BR interactions have been demonstrated are the rice lamina bending assay and hypocotyl hook opening bioassay. Results from these assays include the fact that the degree of effect caused by the combined application of auxin and BR was greater than was the sum of the effect of each, indicative of a synergistic effect of the two hormones (Yopp et al. (1981) Physiol. Plant.
- hypocotyl growth in darkness is accomplished through both GA- and BR-dependent cell elongation processes.
- One piece of evidence for dependence on both GA and BR is that dwf7-1 hypocotyls elongated fivefold in response to darkness as compared with light-grown hypocotyls, although they are still shorter than those of the wild-type.
- BL levels are not detectable in dwf7-1 plants (Table 2), growth of dwf7-1 in the dark could be accomplished mostly by GA-dependent cell elongation processes. Peng and Harberd (1997) Plant Physiol. 113:105 1-1058 and Azpiroz et al.
- sterols were extracted and purified by silica and octadecylsilane (ODS) cartridge columns and ODS-HPLC. Purified samples were derivatized and analyzed by gas chromatography-mass spectrometry (GC-MS). As shown in FIG. 5, 13 C-MVA was converted to 13 C 5 -episterol and subsequent sterols, such as 13 C 5 -24-MC and 13 C 5 -CR in the wild-type. However, the 13 C 5 -5-dehydroepisterol and downstream compounds were not detected in dwf7-1 mutants, whereas the precursor 13 C 5 -episterol accumulated fourfold as compared with the wild-type.
- ODS octadecylsilane
- a defect either in a biosynthetic enzyme or a factor modulating an enzymatic activity could lead to deficiency of endogenous BRs.
- dwf7 To place dwf7 at a specific step in the proposed BR biosynthetic pathway, we first chose to perform feeding studies with BR biosynthetic intermediates. Rescue of dwf7-1 by exogenous application of 22-OHCR suggests that the biosynthetic defect likely resides before the production of CR. Consistent with the results from feeding studies, the endogenous levels of 24-MC, CR, and CN were extremely reduced in dwf7-1 (Table 2).
- HDF7 is 80% identical in amino acid sequence with STE1.
- Fujioka et al. 1997 reported that the endogenous level of CN in det2, which is defective in a step between CR and CN, is ⁇ 10% that of the wild-type amount. The authors hypothesized that the 10% leakage through the defective step in det2 mutants, even in a null allele, could be associated with a second copy of DET2 that lightly hybridizes in DNA gel blot analyses.
- dwf7 Placing dwf7 at a single sterol biosynthetic step was accomplished through feeding studies with 13 C-MVA and compactin. A greater than fourfold accumulation of episterol accompanying the absence of downstream intermediates in dwf7-1 indicates that the ⁇ 7 sterol C-5 desaturase step is blocked in dwf7. In addition, the feeding studies identified an accumulation of 7-dehydrocampestanol, which is an uncommon sterol in plants (FIG. 5). Accumulation of this compound only in dwf7-1 suggests that sterol biosynthesis in dwf7-1 could proceed to a C-24 reduction step, skipping C-5 desaturation as well as the next immediate C-7 reduction.
- dwf7-1 and dwf7-2 were located in the third and the first exons, respectively. Both of the dwf7 alleles contained a base change from a guanine to an adenine, converting tryptophan (TGG) to a stop codon (TAG in dwf7-1 and TGA in dwf7-2).
- the mutation in dwf7-1 eliminated a HaellI restriction enzyme recognition site (GGCC to AGCC). Taking advantage of this restriction enzyme site change, we tested the linkage of this mutation to the dwf7-1 phenotype.
- DNAs isolated from 17 different dwarf plants from a segregating F 2 population were subjected to polymerase chain reaction (PCR) analysis by using S5D — 3F and S5D — 1R primers (underlines were used to distinguish forward or reverse primers from the gene acronym S5D), and the PCR products were digested with HaeIII.
- PCR polymerase chain reaction
- STE1 in relation to other C-5 desaturase proteins isolated from fungi.
- the STE1 protein is composed of 281 predicted amino acids with a theoretical pI of 6.39 and molecular mass of 33 kD.
- yeast ERG3 (38% identical; Arthington et al. (1991) Gene 107:173-174; GenBank accession number M62623) is predicted to contain four transmembrane domains
- STE1 possesses three putative transmembrane domains.
- the nonsense mutations are located in the first exon (dwf7-2) and the third exon, immediately before the third histidine box (dwf7-1), indicating that at least one histidine domain is deleted in each of the dwf7 mutants as a result of the premature stop codons.
- HX 3 H Closely spaced histidine residues, HX 3 H in helices, serve as typical metal binding motifs in many proteins (Regan (1993) Annu. Rev. Biophys. Biomol. Struct. 22:257-281). Shanklin et al. (1994) Biochemistry 33:12787-12794 showed that three membrane-associated bacterial enzymes, fatty acid desaturase, alkane hydroxylase, and xylene monooxygenase, possess eight histidine residues that are conserved in three regions dispersed in these enzymes, HX (3-4) H, HX (2-3) HH, and HX (2-3) HH (where X stands for any amino acid).
Landscapes
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Biophysics (AREA)
- Cell Biology (AREA)
- Endocrinology (AREA)
- Nutrition Science (AREA)
- Medicinal Chemistry (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
- This application is related to provisional patent application Serial No. 60/179,901, filed Feb. 2, 2000, from which priority is claimed under 35 USC §119(e)(1) and which is incorporated herein by reference in its entirety.
- The present invention relates generally to plants that display altered structure or morphology and to the genes imparting such pheontypes. In particular, the present invention pertains to Dwarf7 (dwf7) mutants and methods of using the same.
- Sterols are known to play at least two critical roles in plants: as bulk components of membranes regulating stability and permeability (Bach et al. (1997) Prog. Lipid Res. 36:197-226) and as precursors of growth-promoting brassinosteroids (BRs; Fujioka and Sakurai (1997) Nat. Prod. Rep. 14: 1-10). Lesions in brassinosteroid (BR) biosynthetic genes result in characteristic dwarf phenotypes in plants. Understanding the regulation of BR biosynthesis demands continued isolation and characterization of mutants corresponding to the genes involved in BR biosynthesis.
- Sterol biosynthesis in plants has been studied extensively through enzyme purification or gene cloning (Grunwald (1975) Annu. Rev. Plant Physiol. 26:209-236; Goodwin (1979) Annu. Rev. Plant Physiol. 30:369-404; Benveniste (1986) Annu. Rev. Plant Physiol. 37:275-308; Bach and Benveniste (1997) Prog. Lipid Res. 36:197-226). FIG. 1 shows the proposed biosynthetic pathway from squalene to brassinolide (BL). A major difference between photosynthetic and nonphotosynthetic organisms is that cyclization of
squalene 2,3-oxide is bifurcated to a different route for each system (Benveniste (1986) Annu. Rev. Plant Physiol. 37:275-308). In animals and yeast,squalene 2,3-oxide is cyclized to lanosterol, whereas in photosynthetic organisms it is cyclized to cycloartenol (Ncs and McKean (1977) Biochemistry of Steroids and Other Isopentenoids. (Baltimore, Md.: University Park Press)). Accordingly, photosynthetic organisms require somewhat different biosynthetic enzymes, such as cycloartenol synthase (Corey et al. (1993) Proc. Natl. Acad. Sci. USA 90:11628-11632) and cycloeucalenol-obtusifoliol isomerase, which are required to open the cyclopropane ring in cycloartenol (FIG. 1). However, most of the enzymatic steps are shared between the two different pathways. - In plants, steros are subject to a series of modifications before conversion to BL. Different sterols, such as 24-methylenecholesterol (24-MC), campesterol (CR), isofucosterol, and sitosterol, are converted to the BL congeners dolicholide, BL, 28-homodolicholide, and 28-homoBL, respectively, in a species-specific manner (Fujioka et al. (1997) Plant Cell 9:1951-1962; Sasse (1997) Physiol. Plant. 100:696-701). The BR-specific pathway diverges into the early and the late C-6 oxidation pathways. In the early C-6 oxidation pathway, introduction of a 6-oxo group occurs before the vicinal hydroxylation reactions at the side chain, whereas it occurs after these hydroxylations in the late C-6 oxidation pathway (FIG. 1; Choi et al. (1997) Phytochemistry 44:609-613).
- Several mutants, such as constitutive photomorphogenesis and dwarfism (cpd), deetiolated2 (det2), and dwarf4 (dwf4), have been shown to be defective in the BR-specific pathway (Li et al. (1996) Science 272:398-401; Li et al. (1997) Proc. Natl. Acad. Sci. USA 94:3554-3559; Szekeres et al. (1996) Cell 85:171-182; Choe et al. (1998) Plant Cell 10:231-243). These BR biosynthetic dwarfs share a characteristic dwarf phenotype, which includes short robust stems, reduced fertility, prolonged life cycle, and dark-green, round, and curled leaves when grown in the light. In the dark, these mutants exhibit short hypocotyls and expanded cotyledons, cpd (dwf3) mutants are only rescued by 23α-hydroxylated compounds (Szekeres et al. (1996) Cell 85:171-182). The CPD gene was shown to encode a cytochrome P450 steroid hydroxylating enzyme (CYP90A1). In addition, Li et al. (1996) Science 272:398-401 and Li et al. (1997) Proc. Natl. Acad. Sci. USA 94:3554-3559 showed that det2/dwf6 is blocked in the C-5 reduction step. DET2 was found to be homologous to steroid 5α-reductases. Like its animal equivalents, DET2 successfully converted progesterone (3-oxo-Δ4.5 steroid) to 4,5-dihydroprogesterone in a human cell line. In addition, the human 5α-reductase gene effectively complemented det2 mutants (Li et al. (1997) Proc. Natl. Acad. Sci. USA 94:3554-3559). Recently, it has been shown that DWF4 encodes a cytochrome P450 whose amino acid sequence is 43% identical to CPD; DWF4 has been named CYP90B1 (Choe et al. (1998) Plant Cell 10:231-243). Based on results from feeding studies using BR biosynthetic intermediates, the proposed rate-limiting step of BR biosynthesis, 22α-hydroxylation, is now known to be blocked in dwf4 mutants.
- In the plant sterol biosynthetic pathway, several of the genes have been cloned or identified based on heterologous expression or sequence similarity. First, Corey et al. (1993) Proc. Natl. Acad. Sci. USA 90:11628-11632 isolated a cycloartenol synthase cDNA by heterologous complementation of yeast mutants lacking lanosterol synthase. In addition, two types of cDNAs encoding sterol methyltransferases have been isolated from soybean (Shi et al. (1996) J. Biol. Chem. 271:9384-9389) and Arabidopsis (Husselstein et al. (1996) FEBS Lett. 381:87-92). The Arabidopsis cDNA has been shown to mediate a second methyltransferase step leading to C29 sterols (Bouvier-Nave et al. (1997) Eur. J. Biochem. 246:518-529). For the 14α-demethylation reaction, Bak et al. (1997) Plant J. 11:191-201 cloned the cDNA encoding the 14-ademethylase cytochrome P450 enzyme (CYP51) from Sorghum bicolor. Based on sequence similarity, Grebenok et al. (1997) Plant Mol. Biol. 34:891-896 identified an Arabidopsis sterol C-8 isomerase (GenBank accession number AF030357). Furthermore, an ERGOSTEROL25 (ERG25) homolog for Arabidopsis (C-4 demethylase) also has been discovered in the genome sequencing project (GenBank accession number AL021635). Finally, a sterol C-7 reductase has been cloned by heterologous expression of an Arabidopsis cDNA in yeast (Lecain et al. (1996) J. Biol. Chem. 271:10866-10873).
- As compared with the wealth of cloned genes in sterol biosynthesis, only one mutant has been found in these genes. Gachotte et al. (1995) Plant J. 8:407-416 screened an ethyl methanesulfonate (EMS)-induced mutant population (22,000 M2 plants) for mutants displaying an altered sterol profile. The screen yielded one mutant, sterol1 (ste1), whose endogenous level of C-5-desaturated sterols is reduced to 30% of that of the wild type. Expression of the yeast gene ERG3 (the gene for Δ7 sterol C-5 desaturase) in the ste1-1 mutant increased the level of C-5-desaturated sterols 1.7- to 2.8-fold compared with the ste1-1 control, suggesting functional conservation of the enzymes from yeast and plants. However, visible phenotypes were not found in ste1-1 plants. Thus, the authors hypothesized that the residual 30% level of C-5-desaturated sterols was sufficient for the growth of plants.
- A large collection of BR dwarf mutants have been characterized. Of the eight dwf loci identified to date, dwf3 (cpd; Szekeres et al. (1996) Cell 85:171-182), dwf4 (Choe et al. (1998) Plant Cell 10:231-243), and dwf6 (det2; Li et al. (1996) Science 272:398-401) have been shown to act in the BR biosynthetic pathway, whereas dwf2 (bri1) probably is involved in BR perception (Clouse et al. (1996) Plant Physiol. 111:671-678; Li and Chory (1997) Cell 90:929-938).
- Disclosure of the Invention
- The present invention is based on the discovery of various mutants of a BR biosynthetic locus, designated dwarf7 (dwf7). The STE1 locus in dwf7 mutants contain loss-of-function mutations. Two allelic variants of dwf7 have been characterized, dwf7-1 and dwf7-2, also designated ste1-2 and ste1-3, respectively. A homologue of the dwf7 mutants, HDF7, is also described herein. Feeding studies with BR biosynthetic intermediates and analysis of endogenous levels of BR and sterol biosynthetic intermediates indicate that the defective step in the dwf7 mutants resides before the production of 24-methylenecholesterol in the sterol biosynthetic pathway. Furthermore, results from feeding studies with13C-labeled mevalonic acid and compactin show that the defective step is specifically the Δ7 sterol C-5 desaturation. Sequencing of the STE1 locus in the two dwf7 variants shows premature stop codons in the first (dwf7-2) and the third (dwf7-1) exons. Thus, the reduction of BRs in dwf7 is due to a shortage of substrate sterols and is the direct cause of the dwarf phenotype in dwf7.
- Accordingly, in one embodiment, the present invention is directed to an isolated dwf7 polynucleotide that imparts at least one dwf7 mutant phenotype when expressed in a plant. The polynucleotide is selected from the group consisting of (a) a polynucleotide comprising the nucleotide sequence depicted at positions 143 to 322 , inclusive, of FIGS.8A-8D; (b) a polynucleotide comprising the nucleotide sequence depicted at positions 143 to 1552, inclusive, of FIGS. 8A-8D; (c) a polynucleotide comprising a nucleotide sequence having at least about 70% identity to the nucleotide sequence of (a) or (b); (d) a fragment of (a), (b) or (c) comprising at least about 15 contiguous nucleotides; and (e) complements of (a), (b), (c), (d) or (e).
- In other embodiments, the present invention is directed to an isolated dwf7 polynucleotide that imparts at least one dwf7 mutant phenotype when expressed in a plant. The polynucleotide is selected from the group consisting of (a) a polynucleotide comprising the nucleotide sequence depicted at positions 1506 to 2720, inclusive, of FIGS.10A-10F; (b) a polynucleotide comprising a nucleotide sequence having at least 70% identity to the nucleotide sequence of (b); (c) a fragment of (a) or (b) comprising at least 15 contiguous nucleotides; and (d) complements of (a), (b), (c) or (d).
- In additional embodiments, the present invention is directed to recombinant vectors comprising the isolated dwf7 polynucleotides described above, and control elements that are operably linked to the polynucleotides whereby a coding sequence within the polynucleotides can be transcribed and translated in a host cell, and at least one of the control elements is heterologous to the coding sequence. Also provided are host cells transformed with the recombinant vectors, and methods of producing a DWF7 polypeptide comprising providing a population of host cells as described above and culturing the population of cells under conditions whereby the DWF7 polypeptide encoded by the coding sequence present in the recombinant vector is expressed.
- In yet further embodiments, the subject invention is directed to a transgenic plant comprising a polynucleotide described above, as well as methods of producing a transgenic plant comprising the steps of introducing a polynucleotide into a plant cell to produce a transformed plant cell; and producing a transgenic plant from the transformed plant cell.
- In an additional embodiment, the invention is directed to a method for altering the sterol composition of a plant relative to the wild-type plant comprising introducing a polynucleotide as described above into a plant cell to produce a transformed plant cell and producing a transgenic plant from the transformed plant cell, wherein the transgenic plant has an altered sterol composition relative to the wild-type plant, such as an altered cholesterol composition relative to the wild-type plant.
- In still further embodiments, the invention is directed to isolated DWF7 polypeptides encoded by the polynucleotides as described above. In certain embodiments, the polypeptide consists of the amino acid sequence depicted at positions 1-60, inclusive, of FIG. 9 or the amino acid sequence depicted at positions 1-230, inclusive, of FIG. 9. In other embodiments, the polypeptide consists of the amino acid sequence depicted at positions 1-279, inclusive, of FIG. 11.
- In other embodiments, the subject invention is directed to an isolated control element having at least about 70% identity to a control element found within nucleotide positions 43-142 of FIGS.8A-8D, or 1-1505 of FIGS. 10A-10F, a recombinant vector comprising the control element and a polynucleotide comprising a coding sequence which is heterologous to the control element, host cells transformed with the recombinant vector, and methods of producing a recombinant polypeptide comprising providing a population of the host cells and culturing the population of cells under conditions whereby the recombinant polypeptide encoded by the coding sequence present in the recombinant vector is expressed.
- These and other embodiments of the present invention will readily occur to those of ordinary skill in the art in view of the disclosure herein.
- FIG. 1 shows the proposed BL biosynthetic pathway from squalene to BL. The BL biosynthetic pathway is divided into the sterol-specific pathway, squalene to campesterol, and the BR-specific pathway, campesterol to brassinolide. Common names for the compounds are labeled, and proposed enzymes involved in each reaction are boxed and labeled. Genes identified by mutants are marked. The acronyms for some compounds are in parentheses. In the inset, the carbon atoms of the sterol core rings and side chain are numbered.
- FIG. 2 is a bar graph of measurements of gynoecia and stamens of wild-type, (ecotype Wassilewskija-2 [Ws-2]), dwf7-1, and dwf4-3 plants. The dwf7-1 plant displays a concomitant reduction in the length of gynoecia and stamens, whereas dwf4-3 displays a greater reduction in stamen length. Each data point represents the average length for five flowers. Standard errors are shown at each data point. Solid bars indicate the gynoecium and white bars denote the stamen.
- FIG. 3 compares the response of light-grown wild-type and dwf7-1 hypocotyls to different concentrations of BL. Black bars indicate results using the Wassilewskij a-2 (Ws-2) wild type and white bars dwf7-1 plants. The dwf7-1 plant responds to 10−9 M BL and is completely rescued by 10−8 M BL. Error bars indicate ±SE.
- FIG. 4 is a bar graph comparing wild-type and dwf7-1 inflorescences treated with BR intermediates. The lengths of pedicels treated with water, 6-deoxoCT, 22-OHCR, and BL were measured to the nearest millimeter (n>15). The pedicels elongated greater than twofold in response to all the BRs tested, suggesting that the biosynthetic defect in dwf7-1 resides before the production of CR. Error bars indicate ±SE.
- FIG. 5 shows GC-MS analysis of wild-type and dwf7-1 seedlings fed with13C-MVA in the presence of compactin, an inhibitor of MVA biosynthesis. Accumulation of episterol with a simultaneous decrease of downstream intermediates, including 24-MC and CR, predicts that the C-5 desaturation step is blocked in dwf7-1 plants. The units are in micrograms per 5 g fresh weight of tissue. The designation ND (not detected) means that the quantity is lower than the detection limit. Ws-2 is the Wassilewskija-2 wild type.
- FIG. 6 is a schematic representation of the STE1 gene. Comparison of cDNA and genomic DNA sequences revealed three exons (thick boxes) and two introns (horizontal bars). The single open reading frame encodes a protein of 281 amino acids. The dwf7-2 (ste1 -3) mutation is located in the first exon, changing a tryptophan to a stop codon. The dwf7-1 (ste1-2) mutation also changes a tryptophan to a stop codon (amino acid position 230). The three white boxes indicate the transmembrane domains, and the three histidine boxes are lightly shadowed. The figure is drawn to scale by using the GCK software (Textco, Inc., West Lebanon, N.H.). Bar=120 bp.
- FIG. 7 depicts a multiple sequence alignment of DWF7/STE1 with known sequences for Δ7 sterol C-5 desaturases. The GenBank accession numbers for the sequences are M62623 (S. cerevisiae) (SEQ ID NO:______), AB004539 (Schizosaccharomyces pombe) (SEQ ID NO:______), L40390 (C. glabrata) (SEQ ID NO:______), and AF105034 (DWF7/STE1, Arabidopsis) (SEQ ID NO:______). The conserved transmembrane domains and histidine clusters are boxed and labeled. The positions of the premature stop codons in dwf7-1 and dwf7-2 are indicated with filled circles. Histidine residues in each conserved histidine box are identified with filled triangles. A consensus sequence (SEQ ID NO:______) is shown in the bottom row of the alignment. Capital letters stand for residues conserved among all sequences, whereas lowercase letters mean ≧50% identical. Dashes indicate gaps introduced to maximize alignment. Multiple sequence alignment was performed using PILEUP in the Genetics Computer Group software (Madison, Wis.) with a gap creation penalty of 4 and a gap extension parameter of 1. The annotation of the aligned sequences was performed using the ALSCRIPT software (Barton (1993) Protein Eng. 6:37-40).
- FIGS.8A-8D depict the complete gene sequence of dwf7, denoted by a dark grey bar. The premature stop codons for dwf7-1 and dwf7-2 are shown with triangles at nucleotide positions 1552 and 322, respectively. The coding sequence and corresponding amino acid sequence are represented by a light grey bar. The mRNA sequence is represented by a black bar and is shown in three segments. The gene includes two introns (positions 369-735 and 1042-1395) and three exons.
- FIG. 9 shows the amino acid sequence corresponding to the coding sequence designated in FIGS.8A-8D. The polypeptide sequences corresponding to the dwf7-2 and dwf7-1 alleles occur at positions 1-60 and 1-230, respectively.
- FIGS.10A-10F show the gene sequence of the dwf7 homologue, HDF7. The coding sequence and corresponding amino acid sequence are shown in three segments (exons), occurring at positions 1506-1734, 2024-2329 and 2416-2720 of the figure. The 5′ UTR is shown at positions 1-1505 and the 3′ UTR occurs at positions 2721-2925.
- FIG. 11 shows the amino acid sequence corresponding to the coding sequence designated in FIGS.10A-10F. The polypeptide sequence corresponding to the HDF7 dwf7 polypeptide occurs at positions 1-230 of the figure.
- The practice of the present invention will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Evans, et al.,Handbook of Plant Cell Culture (1983, Macmillan Publishing Co.); Binding, Regeneration of Plants, Plant Protoplasts (1985, CRC Press); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990).
- All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.
- It must be noted that, as used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a polypeptide” includes a mixture of two or more polypeptides, and the like.
- The following amino acid abbreviations are used throughout the text:
Alanine: Ala (A) Arginine: Arg (R) Asparagine: Asn (N) Aspartic acid: Asp (D) Cysteine: Cys (C) Glutamine: Gln (Q) Glutamic acid: Glu (E) Glycine: Gly (G) Histidine: His (H) Isoleucine: Ile (I) Leucine: Leu (L) Lysine: Lys (K) Methionine: Met (M) Phenylalanine: Phe (F) Proline: Pro (P) Serine: Ser (S) Threonine: Thr (T) Tryptophan: Trp (W) Tyrosine: Tyr (Y) Valine: Val (V) - I. Definitions
- In describing the present invention, the following terms will be employed, and are intended to be defined as indicated below.
- The terms “nucleic acid molecule” and “polynucleotide” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. This term refers only to the primary structure of the molecule and thus includes double- and single-stranded DNA and RNA. It also includes known types of modifications, for example, labels which are known in the art, methylation, “caps”, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties, such as, for example proteins (including e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelates (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotide. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown. Nonlimiting examples of polynucleotides include a gene, a gene fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.
- A polynucleotide is typically composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); and thymine (T) (uracil (U) for thymine (T) when the polynucleotide is RNA). Thus, the term polynucleotide sequence is the alphabetical representation of a polynucleotide molecule. This alphabetical representation can be input into databases in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searching.
- Techniques for determining nucleic acid and amino acid “sequence identity” are known in the art. Typically, such techniques include determining the nucleotide sequence of the mRNA for a gene and/or determining the amino acid sequence encoded thereby, and comparing these sequences to a second nucleotide or amino acid sequence. In general, “identity” refers to an exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Two or more sequences (polynucleotide or amino acid) can be compared by determining their “percent identity.” The percent identity of two sequences, whether nucleic acid or amino acid sequences, is the number of exact matches between two aligned sequences divided by the length of the shorter sequences and multiplied by 100. An approximate alignment for nucleic acid sequences is provided by the local homology algorithm of Smith and Waterman,Advances in Applied Mathematics 2:482-489 (1981). This algorithm can be applied to amino acid sequences by using the scoring matrix developed by Dayhoff, Atlas of Protein Sequences and Structure, M. O. Dayhoff ed., 5 suppl. 3:353-358, National Biomedical Research Foundation, Washington, D.C., USA, and normalized by Gribskov, Nucl. Acids Res. 14(6):6745-6763 (1986). An exemplary implementation of this algorithm to determine percent identity of a sequence is provided by the Genetics Computer Group (Madison, Wis.) in the “BestFit” utility application. The default parameters for this method are described in the Wisconsin Sequence Analysis Package Program Manual, Version 8 (1995) (available from Genetics Computer Group, Madison, Wis.). A preferred method of establishing percent identity in the context of the present invention is to use the MPSRCH package of programs copyrighted by the University of Edinburgh, developed by John F. Collins and Shane S. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View, Calif.). From this suite of packages the Smith-Waterman algorithm can be employed where default parameters are used for the scoring table (for example, gap open penalty of 12, gap extension penalty of one, and a gap of six). From the data generated the “Match” value reflects “sequence identity.” Other suitable programs for calculating the percent identity or similarity between sequences are generally known in the art, for example, another alignment program is BLAST, used with default parameters. For example, BLASTN and BLASTP can be used using the following default parameters: genetic code=standard; filter=none; srand=both; cutoff =60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swiss protein+Spupdate+PIR. Details of these programs can be found at the following internet address: http://www.ncbi.nlm.gov/cgi-bin/BLAST.
- Alternatively, the degree of sequence similarity between polynucleotides can be determined by hybridization of polynucleotides under conditions that form stable duplexes between homologous regions, followed by digestion with single-stranded-specific nuclease(s), and size determination of the digested fragments. Two DNA, or two polypeptide sequences are “substantially homologous” to each other when the sequences exhibit at least about 70%-85%, preferably at least about 85%-90%, more preferably at least about 90%-95%, and most preferably at least about 95%-98% sequence identity over a defined length of the molecules, or any percentage between the above-specified ranges, as determined using the methods above. As used herein, substantially homologous also refers to sequences showing complete identity to the specified DNA or polypeptide sequence. DNA sequences that are substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Sambrook et al., supra;DNA Cloning, supra; Nucleic Acid Hybridization, supra.
- The degree of sequence identity between two nucleic acid molecules affects the efficiency and strength of hybridization events between such molecules. A partially identical nucleic acid sequence will at least partially inhibit a completely identical sequence from hybridizing to a target molecule. Inhibition of hybridization of the completely identical sequence can be assessed using hybridization assays that are well known in the art (e.g., Southern blot, Northern blot, solution hybridization, or the like, see Sambrook, et al.,Molecular Cloning: A Laboratory Manual, Second Edition, (1989) Cold Spring Harbor, N.Y.). Such assays can be conducted using varying degrees of selectivity, for example, using conditions varying from low to high stringency. If conditions of low stringency are employed, the absence of non-specific binding can be assessed using a secondary probe that lacks even a partial degree of sequence identity (for example, a probe having less than about 30% sequence identity with the target molecule), such that, in the absence of non-specific binding events, the secondary probe will not hybridize to the target.
- When utilizing a hybridization-based detection system, a nucleic acid probe is chosen that is complementary to a target nucleic acid sequence, and then by selection of appropriate conditions the probe and the target sequence “selectively hybridize,” or bind, to each other to form a hybrid molecule. A nucleic acid molecule that is capable of hybridizing selectively to a target sequence under “moderately stringent” typically hybridizes under conditions that allow detection of a target nucleic acid sequence of at least about 10-14 nucleotides in length having at least approximately 70% sequence identity with the sequence of the selected nucleic acid probe. Stringent hybridization conditions typically allow detection of target nucleic acid sequences of at least about 10-14 nucleotides in length having a sequence identity of greater than about 90-95% with the sequence of the selected nucleic acid probe. Hybridization conditions useful for probe/target hybridization where the probe and target have a specific degree of sequence identity, can be determined as is known in the art (see, for example,Nucleic Acid Hybridization: A Practical Approach, editors B. D. Hames and S. J. Higgins, (1985) Oxford; Washington, D.C.; IRL Press).
- With respect to stringency conditions for hybridization, it is well known in the art that numerous equivalent conditions can be employed to establish a particular stringency by varying, for example, the following factors: the length and nature of probe and target sequences, base composition of the various sequences, concentrations of salts and other hybridization solution components, the presence or absence of blocking agents in the hybridization solutions (e.g., formamide, dextran sulfate, and polyethylene glycol), hybridization reaction temperature and time parameters, as well as, varying wash conditions. The selection of a particular set of hybridization conditions is selected following standard methods in the art (see, for example, Sambrook, et al.,Molecular Cloning: A Laboratory Manual, Second Edition, (1989) Cold Spring Harbor, N.Y.).
- A “gene” as used in the context of the present invention is a sequence of nucleotides in a genetic nucleic acid (chromosome, plasmid, etc.) with which a genetic function is associated. A gene is a hereditary unit, for example of an organism, comprising a polynucleotide sequence that occupies a specific physical location (a “gene locus” or “genetic locus”) within the genome of an organism. A gene can encode an expressed product, such as a polypeptide or a polynucleotide (e.g., tRNA). Alternatively, a gene may define a genomic location for a particular event/function, such as the binding of proteins and/or nucleic acids, wherein the gene does not encode an expressed product. Typically, a gene includes coding sequences, such as, polypeptide encoding sequences, and non-coding sequences, such as, promoter sequences, polyadenlyation sequences, transcriptional regulatory sequences (e.g., enhancer sequences). Many eucaryotic genes have “exons”(coding sequences) interrupted by “introns”(non-coding sequences). In certain cases, a gene may share sequences with another gene(s) (e.g., overlapping genes).
- A “coding sequence” or a sequence which “encodes” a selected polypeptide, is a nucleic acid molecule which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide, for example, in vivo when placed under the control of appropriate regulatory sequences (or “control elements”). The boundaries of the coding sequence are typically determined by a start codon at the 5′(amino) terminus and a translation stop codon at the 3′(carboxy) terminus. A coding sequence can include, but is not limited to, cDNA from viral, procaryotic or eucaryotic mRNA, genomic DNA sequences from viral or procaryotic DNA, and even synthetic DNA sequences. A transcription termination sequence may be located 3′ to the coding sequence. Other “control elements” may also be associated with a coding sequence. A DNA sequence encoding a polypeptide can be optimized for expression in a selected cell by using the codons preferred by the selected cell to represent the DNA copy of the desired polypeptide coding sequence. “Encoded by” refers to a nucleic acid sequence which codes for a polypeptide sequence, wherein the polypeptide sequence or a portion thereof contains an amino acid sequence of at least 3 to 5 amino acids, more preferably at least 8 to 10 amino acids, and even more preferably at least 15 to 20 amino acids from a polypeptide encoded by the nucleic acid sequence. Also encompassed are polypeptide sequences which are immunologically identifiable with a polypeptide encoded by the sequence.
- Typical “control elements”, include, but are not limited to, transcription promoters, transcription enhancer elements, transcription termination signals, polyadenylation sequences (located 3′ to the translation stop codon), sequences for optimization of initiation of translation (located 5′ to the coding sequence), translation enhancing sequences, and translation termination sequences. Transcription promoters can include inducible promoters (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), repressible promoters (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), and constitutive promoters. For purposes of the present invention, control elements for the dwf7 gene are found in the 5′ and 3′ UTRs shown in FIGS.8A-8B, particularly at positions 43-142 and 1710-1890, respectively, of the figure. Control elements for HDF7 are found within the 5′ and 3′ UTRs shown in FIGS. 10A-10F, particularly within the region between positions 1-1505 and 2721-2925, respectively.
- A control element, such as a promoter, “directs the transcription” of a coding sequence in a cell when RNA polymerase will bind the promoter and transcribe the coding sequence into mRNA, which is then translated into the polypeptide encoded by the coding sequence.
- “Expression enhancing sequences” typically refer to control elements that improve transcription or translation of a polynucleotide relative to the expression level in the absence of such control elements (for example, promoters, promoter enhancers, enhancer elements, and translational enhancers (e.g., Shine and Delagarno sequences).
- “Operably linked” refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended maimer. A control sequence “operably linked” to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences. The control elements need not be contiguous with the coding sequence, so long as they function to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter and the coding sequence and the promoter can still be considered “operably linked” to the coding sequence.
- A “heterologous sequence” as used herein typically refers to a nucleic acid sequence that is not normally found in the cell or organism of interest. For example, a DNA sequence encoding a polypeptide can be obtained from a plant cell and introduced into a bacterial cell. In this case the plant DNA sequence is “heterologous” to the native DNA of the bacterial cell.
- The “native sequence” or “wild-type sequence” of a gene is the polynucleotide sequence that comprises the genetic locus corresponding to the gene, e.g., all regulatory and open-reading frame coding sequences required for expression of a completely functional gene product as they are present in the wild-type genome of an organism. The native sequence of a gene can include, for example, transcriptional promoter sequences, translation enhancing sequences, introns, exons, and poly-A processing signal sites. It is noted that in the general population, wild-type genes may include multiple prevalent versions that contain alterations in sequence relative to each other and yet do not cause a discernible pathological effect. These variations are designated “polymorphisms” or “allelic variations.”
- “Recombinant” as used herein to describe a nucleic acid molecule means a polynucleotide of genomic, cDNA, semisynthetic, or synthetic origin which, by virtue of its origin or manipulation: (1) is not associated with all or a portion of the polynucleotide with which it is associated in nature; and/or (2) is linked to a polynucleotide other than that to which it is linked in nature. The term “recombinant” as used with respect to a protein or polypeptide means a polypeptide produced by expression of a recombinant polynucleotide.
- By “vector” is meant any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, virus etc., which is capable of transferring gene sequences to target cells. Generally, a vector is capable of replication when associated with the proper control elements. Thus, the term includes cloning and expression vehicles, as well as viral vectors and integrating vectors.
- As used herein, the term “expression cassette” refers to a molecule comprising at least one coding sequence operably linked to a control sequence which includes all nucleotide sequences required for the transcription of cloned copies of the coding sequence and the translation of the mRNAs in an appropriate host cell. Such expression cassettes can be used to express eukaryotic genes in a variety of hosts such as bacteria, blue-green algae, plant cells, yeast cells, insect cells and animal cells. Under the invention, expression cassettes can include, but are not limited to, cloning vectors, specifically designed plasmids, viruses or virus particles. The cassettes may further include an origin of replication for autonomous replication in host cells, selectable markers, various restriction sites, a potential for high copy number and strong promoters.
- A cell has been “transformed” by an exogenous polynucleotide when the polynucleotide has been introduced inside the cell. The exogenous polynucleotide may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell. In procaryotes and yeasts, for example, the exogenous DNA may be maintained on an episomal element, such as a plasmid. With respect to eucaryotic cells, a stably transformed cell is one in which the exogenous DNA has become integrated into the chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eucaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the exogenous DNA.
- “Recombinant host cells,” “host cells,” “cells,” “cell lines,” “cell cultures,” and other such terms denoting procaryotic microorganisms or eucaryotic cell lines cultured as unicellular entities, are used interchangeably, and refer to cells which can be, or have been, used as recipients for recombinant vectors or other transfer DNA, and include the progeny of the original cell which has been transfected. It is understood that the progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total DNA complement to the original parent, due to accidental or deliberate mutation. Progeny of the parental cell which are sufficiently similar to the parent to be characterized by the relevant property, such as the presence of a nucleotide sequence encoding a desired peptide, are included in the progeny intended by this definition, and are covered by the above terms.
- The term “dwf7 polynucleotide” refers to a polynucleotide derived from, or homologous to, the dwf7 gene. The gene encodes the protein variously referred to herein as DWF7, STE1 and DWF7/STE1. DWF7 is a Δ7sterol C-5 desaturase that functions in the brassinolide (BL) biosynthetic pathway from squalene to BL (see, FIG. 1). The dwf7 polynucleotide sequence and corresponding amino acid sequence are known and have been described in, e.g., Gachotte et al. (1996) Plant J. 9:391-398 and GenBank accession No. AF105034. See, also, FIGS. 8A-8D depicting the dwf7 gene sequence and the corresponding DWF7 amino acid sequence. As shown in FIGS. 8A-8D, the dwf7 gene spans the region from nucleotide positions 1-1889; the upstream 5′ UTR, including the promoter region, spans nucleotide positions 1-142; the downstream 3′ UTR is present from nucleotide position 1710-1889. The term as used herein encompasses a polynucleotide including a native sequence depicted in FIGS. 8A-8D, as well as modifications and fragments thereof.
- The term encompasses alterations to the polynucleotide sequence, so long as the alteration results in a plant displaying one or more dwf7 phenotypic traits (described below) when the polynucleotide is expressed in a plant. Such modifications typically include deletions, additions and substitutions, to the native dwf7 sequence, so long as the mutation results in a plant displaying a dwf7 phenotype as defined below. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of plants which express the dwf7 polynucleotide or errors due to PCR amplification. The term encompasses expressed allelic variants of the wild-type dwf7 sequence which may occur by normal genetic variation or are produced by genetic engineering methods and which result in a detectable change in the wild-type dwf7 phenotype. Two particular dwf7 allelic variants described herein are dwf7-1 and dwf7-2. Polypeptides corresponding to these variants include about amino acids 1-60 and 1-230, respectively, of FIG. 9. However, the boundaries of these polypeptides may vary by 1 to 10 or more amino acids, or any integer therebetween. Thus, dwf7-1 and dwf7-2 polypeptides may include, for example, amino acids 1-59 and 1-229, respectively, or 3-62 and 3-232, respectively, and so on. Also described herein is a dwf7 polynucleotide termed “HDF7. ” The term “dwf7 polynucleotide” as used herein, is intended to encompass the HDF7 polynucleotide. This polynucleotide is shown in FIGS10A-10F herein. The polypeptide encoded by HDF7 is depicted at about positions 1-279 of FIG. 11. As with the dwf7-1 and dwf7-2 polypeptides, the boundaries of the HDF7 polypeptide may also vary by 1 to 10 or more amino acids, or any integer therebetween. These molecules are discussed in detail below.
- The term “dwf7 phenotype” as used herein refers to any microscopic or macroscopic change in structure or morphology of a plant, such as a transgenic plant, as well as biochemical differences, which are characteristic of a dwf7 plant, compared to a progenitor, wild-type plant cultivated under the same conditions. Generally, morphological differences include short robust stems, reduced fertility, prolonged life cycle, dark-green, round, and curled leaves when grown in the light. In the dark, these plants exhibit short hypocotyls and expanded cotyledons, as compared to the wild-type plant. The height of such plants will typically be 75% or less of the wild-type plant, more typically 50% or less of the wild-type plant, and even more typically 25% or less of the wild-type plant, or any integer in between. Additional phenotypic morphological attributes of the dwf7 mutant are summarized in Table 1 of the examples. Biochemically, dwf7 hypocotyls are converted to wild-type length with the application of BL.
- A “polypeptide” is used in it broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs, or other peptidomimetics. The subunits may be linked by peptide bonds or by other bonds, for example ester, ether, etc. As used herein, the term “amino acid” refers to either natural and/or unnatural or synthetic amino acids, including glycine and both the D or L optical isomers, and amino acid analogs and peptidomimetics. A peptide of three or more amino acids is commonly called an oligopeptide if the peptide chain is short. If the peptide chain is long, the peptide is typically called a polypeptide or a protein. Full-length proteins, analogs, and fragments thereof are encompassed by the definition. The terms also include postexpression modifications of the polypeptide, for example, glycosylation, acetylation, phosphorylation and the like. Furthermore, as ionizable amino and carboxyl groups are present in the molecule, a particular polypeptide may be obtained as an acidic or basic salt, or in neutral form. A polypeptide may be obtained directly from the source organism, or may be recombinantly or synthetically produced (see further below).
- A “DWF7” polypeptide is a polypeptide as defined above, which is derived from a Δ7sterol C-5 desaturase that functions in the brassinolide (BL) biosynthetic pathway from squalene to BL (see, FIG. 1). The native sequence of full-length DWF7 is shown in FIG. 9. However, the term encompasses analogs and fragments of the native sequence so long as the protein functions for its intended purpose. Moreover, the term “DWF7 polypeptide” is intended to encompass the HDF7 polypeptidc and analogs thereof.
- The term “DWF7 analog” refers to derivatives of DWF7 and HDF7, or fragments of such derivatives, that retain desired function, e.g., as measured in assays as described further below. In general, the term “analog” refers to compounds having a native polypeptide sequence and structure with one or more amino acid additions, substitutions (generally conservative in nature) and/or deletions, relative to the native molecule, so long as the modifications do not destroy desired activity. Preferably, the analog has at least the same activity as the native molecule. Methods for making polypeptide analogs are known in the art and are described further below.
- Particularly preferred analogs include substitutions that are conservative in nature, i.e., those substitutions that take place within a family of amino acids that are related in their side chains. Specifically, amino acids are generally divided into four families: (1) acidic—aspartate and glutamate; (2) basic—lysine, arginine, histidine; (3) non-polar—alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar—glycine, asparagine, glutamine, cysteine, serine threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids. For example, it is reasonably predictable that an isolated replacement of leucine with isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar conservative replacement of an amino acid with a structurally related amino acid, will not have a major effect on the biological activity. It is to be understood that the terms include the various sequence polymorphisms that exist, wherein amino acid substitutions in the protein sequence do not affect the essential functions of the protein.
- By “purified” and “isolated” is meant, when referring to a polypeptide or polynucleotide, that the molecule is separate and discrete from the whole organism with which the molecule is found in nature; or devoid, in whole or part, of sequences normally associated with it in nature; or a sequence, as it exists in nature, but having heterologous sequences (as defined below) in association therewith. It is to be understood that the term “isolated” with reference to a polynucleotide intends that the polynucleotide is separate and discrete from the chormosome from which the polynucleotide may derive. The term “purified” as used herein preferably means at least 75% by weight, more preferably at least 85% by weight, more preferably still at least 95% by weight, and most preferably at least 98% by weight, of biological macromolecules of the same type are present. An “isolated polynucleotide which encodes a particular polypeptide” refers to a nucleic acid molecule which is substantially free of other nucleic acid molecules that do not encode the subject polypeptide; however, the molecule may include some additional bases or moieties which do not deleteriously affect the basic characteristics of the composition.
- By “fragment” is intended a polypeptide or polynucleotide consisting of only a part of the intact sequence and structure of the reference polypeptide or polynucleotide, respectively. The fragment can include a 3′ or C-terminal deletion or a 5′ or N-terminal deletion, or even an internal deletion, of the native molecule. A polynucleotide fragment of a dwf7 sequence will generally include at least about 15 contiguous bases of the molecule in question, more preferably 18-25 contiguous bases, even more preferably 30-50 or more contiguous bases of the dwf7 molecule, or any integer between 15 bases and the full-length sequence of the molecule. Fragments which provide at least one dwf7 phenotype as defined above are useful in the production of transgenic plants. Fragments are also useful as oligonucleotide probes, to find additional dwf7 sequences.
- Similarly, a polypeptide fragment of a DWF7 molecule will generally include at least about 10 contiguous amino acid residues of the full-length molecule, preferably at least about 15-25 contiguous amino acid residues of the full-length molecule, and most preferably at least about 20-50 or more contiguous amino acid residues of the full-length DWF7 molecule, or any integer between 10 amino acids and the full-length sequence of the molecule. Such fragments are useful for the production of antibodies and the like.
- By “transgenic plant” is meant a plant into which one or more exogenous polynucleotides have been introduced. Examples of means by which this can be accomplished are described below, and include Agrobacterium-mediated transformation, biolistic methods, electroporation, and the like. In the context of the present invention, the transgenic plant contains a polynucleotide which is not normally present in the corresponding wild-type plant and which confers at least one dwf7 phenotypic trait to the plant. The transgenic plant therefore exhibits altered structure, morphology or biochemistry as compared with a progenitor plant which does not contain the transgene, when the transgenic plant and the progenitor plant are cultivated under similar or equivalent growth conditions. Such a plant containing the exogenous polynucleotide is referred to here as an R1 generation transgenic plant. Transgenic plants may also arise from sexual cross or by selfing of transgenic plants into which exogenous polynucleotides have been introduced. Such a plant containing the exogenous nucleic acid is also referred to here as an R1 generation transgenic plant. Transgenic plants which arise from a sexual cross with another parent line or by selfing are “descendants or the progeny” of a R1 plant and are generally called Fn plants or Sn plants, respectively, n meaning the number of generations.
- II. Modes of Carrying Out the Invention
- Before describing the present invention in detail, it is to be understood that this invention is not limited to particular formulations or process parameters as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting.
- Although a number of compositions and methods similar or equivalent to those described herein can be used in the practice of the present invention, The preferred materials and methods are described herein.
- The present invention is based on the morphological, biochemical, and molecular analysis of Arabidopsis dwf7 mutants. Morphologically, dwf7 plants display a dramatic reduction in the length of many different organs examined, and this size reduction is attributable to a defect in cell elongation. Biochemically, dwf7 hypocotyls are converted to wild-type length with the application of BL, suggesting a deficiency in BRs. In agreement with this, BR intermediate feeding analysis, accompanied by analysis of endogenous levels of BRs and sterols by using GC-SIM, indicates that dwf7 is defective specifically in the Δ7 sterol C-5 desaturase step of the sterol biosynthetic pathway. Sequencing of the Δ7 sterol C-5 desaturase gene in two allelic variants, dwf7-1 and dwf7-2, revealed premature stop codons, suggesting loss-of-function mutations. Thus, it appears that a shortage of sterols leads to a drastic reduction of BR levels in dwf7 mutants and to the characteristic dwarf phenotype.
- The molecules of the present invention are therefore useful in the production of transgenic plants which display at least one dwf7 phenotype, so that the resulting plants have altered structure or morphology. The present invention particularly provides for altered structure or morphology such as reduced cell length, extended flowering periods, increased size of leaves or fruit, increased branching, increased seed production and altered sterol composition relative wild-type plants. The DWF7 polypeptides can be expressed to engineer a plant with desirable properties. The engineering is accomplished by transforming plants with nucleic acid constructs described herein which may also comprise promoters and secretion signal peptides. The transformed plants or their progenies are screened for plants that express the desired polypeptide.
- Engineered plants exhibiting the desired altered structure or morphology can be used in plant breeding or directly in agricultural production or industrial applications. Plants having the altered polypeptide can be crossed with other altered plants engineered with alterations in other growth modulation enzymes, proteins or polypeptides to produce lines with even further enhanced altered structural morphology characteristics compared to the parents or progenitor plants.
- Isolation of Nucleic Acid Sequences from Plants
- The isolation of dwf7 sequences from the polynucleotides of the invention may be accomplished by a number of techniques. For instance, oligonucleotide probes based on the sequences disclosed here can be used to identify the desired gene in a cDNA or genomic DNA library from a desired plant species. To construct genomic libraries, large segments of genomic DNA are generated by random fragmentation, e.g. using restriction endonucleases, and are ligated with vector DNA to form concatemers that can be packaged into the appropriate vector. To prepare a library of tissue-specific cDNAs, mRNA is isolated from tissues and a cDNA library which contains the gene transcripts is prepared from the mRNA.
- The cDNA or genomic library can then be screened using a probe based upon the sequence of a cloned gene such as the polynucleotides disclosed here. Probes may be used to hybridize with genomic DNA or cDNA sequences to isolate homologous genes in the same or different plant species. Alternatively, the nucleic acids of interest can be amplified from nucleic acid samples using amplification techniques. For instance, polymerase chain reaction (PCR) technology to amplify the sequences of the genes directly from mRNA, 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 MRNA in samples, for nucleic acid sequencing, or for other purposes.
- Appropriate primers and probes for identifying dwf7-specific genes from plant tissues are generated from comparisons of the sequences provided herein. For a general overview of PCR see Innis et al. eds, PCT Protocols:A Guide to Methods and Applications, Academic Press, San Diego (1990). Appropriate primers for this invention include, for instance, those primers described in the Examples and Sequence Listings, as well as other primers derived from the dwf sequences disclosed herein. Suitable amplifications conditions may be readily determined by one of skill in the art in view of the teachings herein, for example, including reaction components and amplification conditions as follows: 10 mM Tris-HCl, pH 8.3, 50 mM potassium chloride, 1.5 mM magnesium chloride, 0.001% gelatin, 200 μM dATP, 200 μM dCTP, 200 μM dGTP, 200 μM dTTP, 0.4 μM primers, and 100 units per mL Taq polymerase; 96° C. for 3 min., 30 cycles of 96° C. for 45 seconds, 50° C. for 60 seconds, 72° C. for 60 seconds, followed by 72° C. for 5 min.
- Polynucleotides may also be synthesized by well-known techniques as described in the technical literature. See, e.g., Carruthers, et al. (1982)Cold Spring Harbor Symp. Quant. Biol. 47:411-418, and Adams, et al. (1983) J. Am. Chem. Soc. 105:661. Double stranded DNA fragments may then be obtained either by synthesizing the complementary strand and annealing the strands together under appropriate conditions, or by adding the complementary strand using DNA polymerase with an appropriate primer sequence.
- The polynucleotides of the present invention may also be used to isolate or create other mutant cell gene alleles. Mutagenesis consists primarily of site-directed mutagenesis followed by phenotypic testing of the altered gene product. Some of the more commonly employed site-directed mutagenesis protocols take advantage of vectors that can provide single stranded as well as double stranded DNA, as needed. Generally, the mutagenesis protocol with such vectors is as follows. A mutagenic primer, i.e., a primer complementary to the sequence to be changed, but consisting of one or a small number of altered, added, or deleted bases, is synthesized. The primer is extended in vitro by a DNA polymerase and, after some additional manipulations, the now double-stranded DNA is transfected into bacterial cells. Next, by a variety of methods, the desired mutated DNA is identified, and the desired protein is purified from clones containing the mutated sequence. For longer sequences, additional cloning steps are often required because long inserts (longer than 2 kilobases) are unstable in those vectors. Protocols are known to one skilled in the art and kits for site-directed mutagenesis are widely available from biotechnology supply companies, for example from Amersham Life Science, Inc. (Arlington Heights, Ill.) and Stratagene Cloning Systems (La Jolla, Calif.).
- Control Elements
- Regulatory regions can be isolated from the dwf7 gene and used in recombinant constructs for modulating the expression of the dwf7 gene or a heterologous gene in vitro and/or in vivo. As shown in FIGS.8A-8D, the coding region of the dwf7 gene (designated by the light grey bar) begins at nucleotide position 143. The region of the gene spanning nucleotide positions 1-142 of FIGS. 8A-8D includes the dwf7 promoter. This region may be used in its entirety or fragments of the region may be isolated which provide the ability to direct expression of a coding sequence linked thereto.
- Thus, promoters can be identified by analyzing the 5′ sequences of a genomic clone corresponding to the dwf7-specific genes described here. Sequences characteristic of promoter sequences can be used to identify the promoter. Sequences controlling eukaryotic gene expression have been extensively studied. For instance, promoter sequence elements include the TATA box consensus sequence (TATAAT), which is usually 20 to 30 base pairs upstream of the transcription start site. In most instances the TATA box is required for accurate transcription initiation. In plants, further upstream from the TATA box, at positions −80 to −100, there is typically a promoter element with a series of adenines surrounding the trinucleotide G (or T) N G. (See, J. Messing et al., inGenetic Engineering in Plants, pp. 221-227 (Kosage, Meredith and Hollaender, eds. (1983)). Methods for identifying and characterizing promoter regions in plant genomic DNA are described, for example, in Jordano et al. (1989) Plant Cell 1:855-866; Bustos et al. (1989) Plant Cell 1:839-854; Green et al. (1988) EMBO J. 7:4035-4044; Meier et al. (1991) Plant Cell 3:309-316; and Zhang et al. (1996) Plant Physiology 110:1069-1079).
- Additionally, the promoter region may include nucleotide substitutions, insertions or deletions that do not substantially affect the binding of relevant DNA binding proteins and hence the promoter function. It may, at times, be desirable to decrease the binding of relevant DNA binding proteins to “silence” or “down-regulate” a promoter, or conversely to increase the binding of relevant DNA binding proteins to “enhance” or “up-regulate” a promoter. In such instances, the nucleotide sequence of the promoter region may be modified by, e.g., inserting additional nucleotides, changing the identity of relevant nucleotides, including use of chemically-modified bases, or by deleting one or more nucleotides.
- Promoter function can be assayed by methods known in the art:, preferably by measuring activity of a reporter gene operatively linked to the sequence being tested for promoter function. Examples of reporter genes include those encoding luciferase, green fluorescent protein, GUS, neo, cat and bar.
- Polynucleotides comprising untranslated (UTR) sequences and intron/exon junctions are also within the scope of the invention. UTR sequences include introns and 5′ or 3′ untranslated regions (5′ UTRs or 3′ UTRS). As shown in FIG. 6, the dwf7 gene sequence includes three exons (thick boxes) and two introns (horizontal bars). See, also, FIGS.8A-8D for the 5′ and 3′ UTRs. Similarly, the HDF7 gene includes three exons (at positions 1506-1734, 2024-2329 and 2416-2720, denoted by the corresponding protein sequence indicated) and two introns (between these exons) and 540 and 3′ UTRs. These portions of the dwf7 and HDF7 genes especially UTRs, can have regulatory functions related to, for example, translation rate and MRNA stability. Thus, these portions of the gene can be isolated for use as elements of gene constructs for expression of polynucleotides encoding desired polypeptides.
- Introns of genomic DNA segments may also have regulatory functions. Sometimes promoter elements, especially transcription enhancer or suppressor elements, are found within introns. Also, elements related to stability of heteronuclear RNA and efficiency of transport to the cytoplasm for translation can be found in intron elements. Thus, these segments can also find use as elements of expression vectors intended for use to transform plants.
- The introns, UTR sequences and intron/exon junctions can vary from the native sequence. Such changes from those sequences preferably will not affect the regulatory activity of the UTRs or intron or intron/exon junction sequences on expression, transcription, or translation. However, in some instances, down-regulation of such activity may be desired to modulate traits or phenotypic or in vitro activity.
- Use of Nucleic Acids of the Invention to Inhibit Gene Expression
- The isolated sequences prepared as described herein, can be used to prepare expression cassettes useful in a number of techniques. For example, expression cassettes of the invention can be used to suppress endogenous dwf7 gene expression. Inhibiting expression can be useful, for instance, in suppressing the phenotype (e.g., dwarf appearance, the Δ7 sterol C-5 desaturase activity) exhibited by dwf7 plants.
- A number of methods can be used to inhibit gene expression in plants. For instance, antisense technology can be conveniently used. To accomplish this, a nucleic acid segment from the desired gene is cloned and operably linked to a promoter such that the antisense strand of RNA will be transcribed. The expression cassette is then transformed into plants and the antisense strand of RNA is produced. In plant cells, it has been suggested that antisense RNA inhibits gene expression by preventing the accumulation of mRNA which encodes the enzyme of interest, see, e.g., Sheehy et al. (1988)Proc. Nat. Acad. Sci. USA 85:8805-8809, and Hiatt et al., U.S. Pat. No. 4,801,340.
- The nucleic acid segment to be introduced generally will be substantially identical to at least a portion of the endogenous gene or genes to be repressed. The sequence, however, need not be perfectly identical to inhibit expression. The vectors of the present invention can be designed such that the inhibitory effect applies to other proteins within a family of genes exhibiting homology or substantial homology to the target gene.
- For antisense suppression, the introduced sequence also need not be full length relative to either the primary transcription product or fully processed mRNA. Generally, higher homology can be used to compensate for the use of a shorter sequence. Furthermore, the introduced sequence need not have the same intron or exon pattern, and homology of non-coding segments may be equally effective. Normally, a sequence of between about 30 or 40 nucleotides and about full length nucleotides should be used, though a sequence of at least about 100 nucleotides is preferred, a sequence of at least about 200 nucleotides is more preferred, and a sequence of at least about 500 nucleotides is especially preferred. It is to be understood that any integer between the above-recited ranges is intended to be captured herein.
- Catalytic RNA molecules or ribozymes can also be used to inhibit expression of dwf7 genes. It is possible to design ribozymes that specifically pair with virtually any target RNA and cleave the phosphodiester backbone at a specific location, thereby functionally inactivating the target RNA. In carrying out this cleavage, the ribozyme is not itself altered, and is thus capable of recycling and cleaving other molecules, making it a true enzyme. The inclusion of ribozyme sequences within antisense RNAs confers RNA-cleaving activity upon them, thereby increasing the activity of the constructs.
- A number of classes of ribozymes have been identified. One class of ribozymes is derived from a number of small circular RNAs which are capable of self-cleavage and replication in plants. The RNAs replicate either alone (viroid RNAs) or with a helper virus (satellite RNAs). Examples include RNAs from avocado sunblotch viroid and the satellite RNAs from tobacco ringspot virus, lucerne transient streak virus, velvet tobacco mottle virus, solanum nodiflorum mottle virus and subterranean clover mottle virus. The design and use of target RNA-specific ribozymes is described in Haseloff et al. (1988)Nature 334:585-591.
- Another method of suppression is sense suppression. Introduction of expression cassettes in which a nucleic acid is configured in the sense orientation with respect to the promoter has been shown to be an effective means by which to block the transcription of target genes. For an example of the use of this method to modulate expression of endogenous genes see, Napoli et al. (1990)The Plant Cell 2:279-289 and U.S. Pat. Nos. 5,034,323, 5,231,020, and 5,283,184.
- Generally, where inhibition of expression is desired, some transcription of the introduced sequence occurs. The effect may occur where the introduced sequence contains no coding sequence per se, but only intron or untranslated sequences homologous to sequences present in the primary transcript of the endogenous sequence. The introduced sequence generally will be substantially identical to the endogenous sequence intended to be repressed. This minimal identity will typically be greater than about 65%, but a higher identity might exert a more effective repression of expression of the endogenous sequences. Substantially greater identity of more than about 80% is preferred, though about 95% to absolute identity would be most preferred. It is to be understood that any integer between the above-recited ranges is intended to be captured herein. As with antisense regulation, the effect should apply to any other proteins within a similar family of genes exhibiting homology or substantial homology.
- For sense suppression, the introduced sequence in the expression cassette, needing less than absolute identity, also need not be full length, relative to either the primary transcription product or fully processed mRNA. This may be preferred to avoid concurrent production of some plants which are overexpressers. A higher identity in a shorter than full length sequence compensates for a longer, less identical sequence. Furthermore, the introduced sequence need not have the same intron or exon pattern, and identity of non-coding segments will be equally effective. Normally, a sequence of the size ranges noted above for antisense regulation is used.
- Use of Nucleic Acids of the Invention to Enhance Gene Expression
- In addition to inhibiting certain features of a plant, the polynucleotides of the invention can be used to increase certain features such as extending flowering, producing larger leaves or fruit, producing increased branching and increasing seed production. This can be accomplished by the overexpression of dwf7 polynucleotides.
- The exogenous dwf7 polynucleotides do not have to code for exact copies of the endogenous DWF7 and HDF7 proteins. Modified DWF7 and HDF7 protein chains can also be readily designed utilizing various recombinant DNA techniques well known to those skilled in the art and described for instance, in Sambrook et al., supra. Hydroxylamine can also be used to introduce single base mutations into the coding region of the gene (Sikorski et al. (1991)Meth. Enzymol. 194: 302-318). For example, the chains can vary from the naturally occurring sequence at the primary structure level by amino acid substitutions, additions, deletions, and the like. These modifications can be used in a number of combinations to produce the final modified protein chain.
- Preparation of Recombinant Vectors
- To use isolated sequences in the above techniques, recombinant DNA vectors suitable for transformation of plant cells are prepared. Techniques for transforming a wide variety of higher plant species are well known and described further below as well as in the technical and scientific literature. See, for example, Weising et al. (1988)Ann. Rev. Genet. 22:421-477. A DNA sequence coding for the desired polypeptide, for example a cDNA sequence encoding the full length DWF7 protein, will preferably be combined with transcriptional and translational initiation regulatory sequences which will direct the transcription of the sequence from the gene in the intended tissues of the transgenic plant.
- Such regulatory elements include but are not limited to the promoters derived from the genome of plant cells (e.g., heat shock promoters such as soybean hspl7.5-E or hsp17.3-B (Gurley et al. (1986)Mol. Cell. Biol. 6:559-565); the promoter for the small subunit of RUBISCO (Coruzzi et al. (1984) EMBO J. 3:1671-1680; Broglie et al. (1984) Science 224:838-843); the promoter for the chlorophyll a/b binding protein) or from plant viruses viral promoters such as the 35S RNA and 19S RNA promoters of CaMV (Brisson et al. (1984) Nature 310:511-514), or the coat protein promoter of TMV (Takamatsu et al. (1987) EMBO J. 6:307-311), cytomegalovirus hCMV immediate early gene, the early or late promoters of SV40 adenovirus, the lac system, the trp system, the TAC system, the TRC system, the major operator and promoter regions of phage A, the control regions of fd coat protein, the promoter for 3-phosphoglycerate kinase, the promoters of acid phosphatase, heat shock promoters (e.g., as described above) and the promoters of the yeast alpha-mating factors.
- In construction of recombinant expression cassettes of the invention, a plant promoter fragment may be employed which will direct expression of the gene in all tissues of a regenerated plant. Such promoters are referred to herein as “constitutive” promoters and 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 T-DNA mannopine synthetase promoter (e.g., the 1′- or 2′-promoter derived from T-DNA of Agrobacterium tumafaciens), and other transcription initiation regions from various plant genes known to those of skill.
- Alternatively, the plant promoter may direct expression of the polynucleotide of the invention in a specific tissue (tissue-specific promoters) or may be otherwise under more precise environmental control (inducible promoters). Examples of tissue-specific promoters under developmental control include promoters that initiate transcription only in certain tissues, such as fruit, seeds, or flowers such as tissue- or developmental-specific promoter, such as, but not limited to the cell promoter, the CHS promoter, the PATATIN promoter, etc. The tissue specific E8 promoter from tomato is particularly useful for directing gene expression so that a desired gene product is located in fruits.
- Other suitable promoters include those from genes encoding embryonic storage proteins. Examples of environmental conditions that may affect transcription by inducible promoters include anaerobic conditions, elevated temperature, or the presence of light. If proper polypeptide expression is desired, a polyadenylation region at the 3′-end of the coding region should be included. The polyadenylation region can be derived from the natural gene, from a variety of other plant genes, or from T-DNA. In addition, the promoter itself can be derived from the dwf7 or HDF7 genes, as described above.
- The vector comprising the sequences (e.g., promoters or coding regions) from genes of the invention will typically comprise a marker gene which confers a selectable phenotype on plant cells. For example, the marker may encode biocide resistance, particularly antibiotic resistance, such as resistance to kanamycin, G418, bleomycin, hygromycin, or herbicide resistance, such as resistance to chlorosluforon or Basta.
- Production of Transgenic Plants
- DNA constructs of the invention may be introduced into the genome of the desired plant host by a variety of conventional techniques. For reviews of such techniques see, for example, Weissbach & WeissbachMethods for Plant Molecular Biology (1988, Academic Press, N.Y.) Section VIII, pp. 421-463; and Grierson & Corey, Plant Molecular Biology (1988, 2d Ed.), Blackie, London, Ch. 7-9. For example, the DNA construct may be introduced directly into the genomic DNA of the plant cell using techniques such as electroporation and microinjection of plant cell protoplasts, or the DNA constructs can be introduced directly to plant tissue using biolistic methods, such as DNA particle bombardment (see, e.g., Klein et al. (1987) Nature 327:-70-73). Alternatively, the DNA constructs may be combined with suitable T-DNA flanking regions and introduced into a conventional Agrobacterium tumefaciens host vector. Agrobacterium tumefaciens-mediated transformation techniques, including disarming and use of binary vectors, are well described in the scientific literature. See, for example Horsch et al. (1984) Science 233:496-498, and Fraley et al. (1983) Proc. Nat'l. Acad. Sci. USA 80:4803. The virulence functions of the Agrobacterium tumefaciens host will direct the insertion of the construct and adjacent marker into the plant cell DNA when the cell is infected by the bacteria using binary T DNA vector (Bevan (1984) Nuc. Acid Res. 12:8711-8721) or the co-cultivation procedure (Horsch et al. (1985) Science 227:1229-1231). Generally, the Agrobacterium transformation system is used to engineer dicotyledonous plants (Bevan et al. (1982) Ann. Rev. Genet 16:357-384; Rogers et al. (1986) Methods Enzymol. 118:627-641). The Agrobacterium transformation system may also be used to transform, as well as transfer, DNA to monocotyledonous plants and plant cells. (see Hemalsteen et al. (1984) EMBO J 3:3039-3041; Hooykass-Van Slogteren et al. (1984) Nature 311:763-764; Grimsley et al. (1987) Nature 325:1677-179; Boulton et al. (1989) Plant Mol. Biol. 12:31-40.; and Gould et al. (1991) Plant Physiol. 95:426-434).
- Alternative gene transfer and transformation methods include, but are not limited to, protoplast transformation through calcium-, polyethylene glycol (PEG)- or electroporation-mediated uptake of naked DNA (see Paszkowski et al. (1984)EMBO J 3:2717-2722, Potrykus et al. (1985) Molec. Gen. Genet. 199:169-177; Fromm et al. (1985) Proc. Nat. Acad. Sci. USA 82:5824-5828; and Shimamoto (1989) Nature 338:274-276) and electroporation of plant tissues (D'Halluin et al. (1992) Plant Cell 4:1495-1505). Additional methods for plant cell transformation include microinjection, silicon carbide mediated DNA uptake (Kaeppler et al. (1990) Plant Cell Reporter 9:415-418), and microprojectile bombardment (see Klein et al. (1988) Proc. Nat. Acad. Sci. USA 85:4305-4309; and Gordon-Kamm et al. (1990) Plant Cell 2:603-618).
- Transformed plant cells which are produced 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 desired nucleotide sequences. Plant regeneration from cultured protoplasts is described in Evans, et al., “Protoplasts Isolation and Culture” inHandbook of Plant Cell Culture, pp. 124-176, Macmillian Publishing Company, New York, 1983; and Binding, Regeneration of Plants, Plant Protoplasts, pp. 21-73, CRC Press, Boca Raton, 1985. Regeneration can also be obtained from plant callus, explants, organs, pollens, embryos or parts thereof. Such regeneration techniques are described generally in Klee et al. (1987) Ann. Rev. of Plant Phys. 38:467-486.
- The nucleic acids of the invention can be used to confer desired traits on essentially any plant. A wide variety of plants and plant cell systems may be engineered for the desired physiological and agronomic characteristics described herein using the nucleic acid constructs of the present invention and the various transformation methods mentioned above. In preferred embodiments, target plants and plant cells for engineering include, but are not limited to, those monocotyledonous and dicotyledonous plants, such as crops including grain crops (e.g., wheat, maize, rice, millet, barley), fruit crops (e.g., tomato, apple, pear, strawberry, orange), forage crops (e.g., alfalfa), root vegetable crops (e.g., carrot, potato, sugar beets, yam), leafy vegetable crops (e.g., lettuce, spinach); flowering plants (e.g., petunia, rose, chrysanthemum), conifers and pine trees (e.g., pine fir, spruce); plants used in phytoremediation (e.g., heavy metal accumulating plants); oil crops (e.g., sunflower, rape seed) and plants used for experimental purposes (e.g., Arabidopsis). Thus, the invention has use over a broad range of plants, including, but not limited to, species from the genera Asparagus, Avena, Brassica, Citrus, Citrullus, Capsicum, Cucurbita, Daucus, Glycine, Hordeum, Lactuca, Lycopersicon, Malus, Manihot, Nicotiana, Oryza, Persea, Pisum, Pyrus, Prunus, Raphanus, Secale, Solanum, Sorghum, Triticum, Vitis, Vigna, and Zea.
- One of skill in the art 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. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed.
- A transformed plant cell, callus, tissue or plant may be identified and isolated by selecting or screening the engineered plant material for traits encoded by the marker genes present on the transforming DNA. For instance, selection may be performed by growing the engineered plant material on media containing an inhibitory amount of the antibiotic or herbicide to which the transforming gene construct confers resistance. Further, transformed plants and plant cells may also be identified by screening for the activities of any visible marker genes (e.g., the β-glucuronidase, luciferase, B or C1 genes) that may be present on the recombinant nucleic acid constructs of the present invention. Such selection and screening methodologies are well known to those skilled in the art.
- Physical and biochemical methods also may be used to identify plant or plant cell transformants containing the gene constructs of the present invention. These methods include but are not limited to: 1) Southern analysis or PCR amplification for detecting and determining the structure of the recombinant DNA insert; 2) Northern blot, S1 RNase protection, primer-extension or reverse transcriptase-PCR amplification for detecting and examining RNA transcripts of the gene constructs; 3) enzymatic assays for detecting enzyme or ribozyme activity, where such gene products are encoded by the gene construct; 4) protein gel electrophoresis, Western blot techniques, immunoprecipitation, or enzyme-linked immunoassays, where the gene construct products are proteins. Additional techniques, such as in situ hybridization, enzyme staining, and immunostaining, also may be used to detect the presence or expression of the recombinant construct in specific plant organs and tissues. The methods for doing all these assays are well known to those skilled in the art.
- Effects of gene manipulation using the methods of this invention can be observed by, for example, northern blots of the RNA (e.g., mRNA) isolated from the tissues of interest. Typically, if the amount of mRNA has increased, it can be assumed that the endogenous dwf7 gene is being expressed at a greater rate than before. Other methods of measuring DWF7 activity can be used. For example, cell length can be measured at specific times. Because dwf7 affects the BR biosynthetic pathway, an assay that measures the amount of BL can also be used. Such assays are known in the art. Different types of enzymatic assays can be used, depending on the substrate used and the method of detecting the increase or decrease of a reaction product or by-product. In addition, the levels of DWF7 protein expressed can be measured immunochemically, i.e., ELISA, RIA, EIA and other antibody based assays well known to those of skill in the art, by electrophoretic detection assays (either with staining or western blotting), and sterol (BL) detection assays.
- The transgene may be selectively expressed in some tissues of the plant or at some developmental stages, or the transgene may be expressed in substantially all plant tissues, substantially along its entire life cycle. However, any combinatorial expression mode is also applicable.
- The present invention also encompasses seeds of the transgenic plants described above wherein the seed has the transgene or gene construct. The present invention further encompasses the progeny, clones, cell lines or cells of the transgenic plants described above wherein said progeny, clone, cell line or cell has the transgene or gene construct.
- Polypeptides
- The present invention also includes DWF7 polypeptides, including such polypeptides as a fusion, or chimeric protein product (comprising the protein, fragment, analog, mutant or derivative joined via a peptide bond to a heterologous protein sequence (of a different protein)). Such a chimeric product can be made by ligating the appropriate nucleic acid sequences encoding the desired amino acid sequences to each other by methods known in the art, in the proper coding frame, and expressing the chimeric product by methods commonly known in the art.
- In addition, DWF7 polypeptides, derivatives (including fragments and chimeric proteins), mutants and analogues can be chemically synthesized. See, e.g., Clark-Lewis et al. (1991)Biochem. 30:3128-3135 and Merrifield (1963) J. Amer. Chem. Soc. 85:2149-2156. For example, DWF7, derivatives, mutants and analogs can be synthesized by solid phase techniques, cleaved from the resin, and purified by preparative high performance liquid chromatography (e.g., see Creighton, 1983, Proteins, Structures and Molecular Principles, W. H. Freeman and Co., N.Y., pp. 50-60). DWF7, derivatives and analog that are proteins can also be synthesized by use of a peptide synthesizer. The composition of the synthetic peptides may be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure; see Creighton, 1983, Proteins, Structures and Molecular Principles, W. H. Freeman and Co., N.Y., pp. 34-49).
- Applications
- The present invention finds use in various applications, for example, including but not limited to those listed above.
- The polynucleotide sequences may additionally be used to isolate mutant dwf7 gene alleles. Such mutant alleles may be isolated from plant species either known or proposed to have a genotype which contributes to altered plant morphology.
- Additionally, such plant dwf7 gene sequences can be used to detect plant dwf7 gene regulatory (e.g., promoter or promotor/enhancer) defects which can affect plant growth.
- The molecules of the present invention can be used to provide plants with increased seed and/fruit production, extended flowering periods and increased branching. The molecules described herein can be used to alter the sterol composition of a plant, thereby increasing or reducing cholesterol content in the plant. A still further utility of the molecules of the present invention is to provide a tool for studying the biosynthesis of brassinosteriods, both in vitro and in vivo.
- The dwf7 gene of the invention also has utility as a transgene encoding a the Δ7 sterol C-5 desaturation protein that mediates one or more steps in brassinosteriod biosynthesis which results in a transgenic plant to alter plant structure or morphology.
- The dwf7 gene also has utility for encoding the DWF7 protein in recombinant vectors which may be inserted into host cells to express the DWF7 protein. Further, the dwf7 polynucleotides of the invention may be utilized (1) as nucleic acid probes to screen nucleic acid libraries to identify other enzymatic genes or mutants; (2) as nucleic acid sequences to be mutated or modified to produce DWF7 protein variants or derivatives; (3) as nucleic acids encoding the Δ7 sterol C-5 desaturases in molecular biology techniques or industrial applications commonly known to those skilled in the art.
- The dwf7 nucleic acid molecules may be used to design antisense molecules, useful, for example, in gene regulation or as antisense primers in amplification reactions of dwf7 gene nucleic acid sequences. With respect to dwf7 gene regulation, such techniques can be used to regulate, for example, plant growth, development or gene expression. Further, such sequences may be used as part of ribozyme and/or triple helix sequences, also useful for dwf7 gene regulation.
- The dwf7 control element (e.g., promoter) of the present invention may be utilized as a plant promoter to express any protein, polypeptide or peptide of interest in a transgenic plant. In particular, the dwf7 promoter may be used to express a protein involved in brassinosteriod biosynthesis.
- The Arabidopsis DWF7 protein of the invention can be used in any biochemical applications (experimental or industrial) where Δ7 sterol C-5 desaturation activity is desired, for example, but not limited to, regulation of BL synthesis, regulation of other sterol synthesis, modification of elongating plant structures, and experimental or industrial biochemical applications known to those skilled in the art.
- III. Experimental
- Below are examples of specific embodiments for carrying out the present invention. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.
- Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.
- Restriction and modifying enzymes, as well as PCR reagents were purchased from commercial sources, and used according to the manufacturers' directions. In the cloning of DNA fragments, except where noted, all DNA manipulations were done according to standard procedures. See, e.g., Sambrook et al., supra. Restriction enzymes, T4 DNA ligase, E. coli, DNA polymerase I, Klenow fragment, and other biological reagents were purchased from commercial suppliers and used according to the manufacturers' directions.
- Materials and Methods
- A. Plant Growth
- For sterile growth of Arabidopsis thaliana plants, seeds of mutants and the wild type were sterilized (50% Clorox and 0.005% Triton X-100) for 8 min, washed three times with sterile distilled water, and dried with 95% ethanol. The seeds were sprinkled on 0.8% agar-solidified media or in liquid media containing 1× Murashige and Skoog (Murashige and Skoog (1962) Physiol. Plant. 15:473-497) salts and 0.5% sucrose (pH 5.8 with KOH). For the plants grown in the dark, the seeds on the plates were illuminated for 3 hr (240 μmol m2 sec1) before being wrapped with two or three layers of aluminum foil. For the mature plants used for morphometric analysis and gas chromatography-selective ion monitoring (GC-SIM) studies, seeds were planted on soil (
Metromix 350; Grace Sierra Co., Milpitas, Calif.) presoaked with distilled water. The flats containing the pots were covered with plastic wrap and cold-treated at 4° C. for 2 days before transfer to a growth chamber (16 hr of light [240 μmol m−2 sec−1]and 8 hr of dark at 22 and 21° C., respectively, and 75 to 90% humidity). The plastic wrap was removed after 2 to 3 days. The pots were subirrigated in distilled water or Hoagland's nutrient solution as required. - B. Morphometric and Physiological Analysis
- At 5 weeks of age, the various morphological traits listed in Table 1 (below) were measured. The number of seeds per silique was determined after the plants were completely dried. Unopened siliques from each plant were selected and crushed, and the number of seeds was counted under a dissecting microscope. To measure the fresh and dry weight, the aerial parts of the plants were cut and immediately weighed to obtain the fresh weight; the plants were then completely dried in a 60° C. oven for 5 days before measuring the dry weight. Flowers were harvested immediately after petal opening. Observations on the structure of flowers were made with flowers at stage 14 (Smyth et al. (1990) Plant Cell 2:755-767), which are right beneath the cluster of developing flowers at the shoot apices. Individual organs of a flower were separated under the dissecting microscope. The length of the organs was measured to a tenth of a millimeter, and the four longest stamens for each flower were measured and the mean value calculated.
- The anatomical studies using a scanning electronic microscope and a light microscope were performed as described by Azpiroz et al. (1998) Plant Cell 10:219-230.
- C. Mapping and Sequencing of the DWARF7 Locus
- The mapping of dwf7 was performed using simple sequence length polymorphism (SSLP) markers (Bell and Ecker (1994) Genomics 19:137-144). Briefly, dwf7-1 mutants (Wassilewskija-2 [Ws-2] background) were crossed to Columbia wild-type plants. Genomic DNA was isolated (Dellaporta et al. 1983) from individual F2 dwarf plants. To locate the mutation to one of the five chromosomes, 20 individual plants were tested with at least two SSLP markers per chromosome. The polymerase chain reaction (PCR) amplified products were analyzed on 4% agarose gels in 1× TAE buffer (40 mM Tris-acetate and 10 mM EDTA). Once the dwf7-1 mutation was shown to be linked to the ngal 62 marker located on chromosome 3 (recombination ratio 11.9%), we tested marker ngal72, which maps at 2.2 centimorgans. No recombination was detected between the dwf7-1 mutation and ngal72 when 86 chromosomes were tested, suggesting that dwf7-1 is linked closely to the ngal72 marker. Linkage between the markers and the dwarf phenotype was determined according to Koornneef and Stam (1992) Genetic analysis. In Methods in Arabidopsis Research, C. Koncz, N.-H. Chua, and J. Schell, eds (Singapore: World Scientific Publishing Co.), pp. 83-99.
- PCR products amplified using primer sets derived from the cDNA sequence of STEROL1 (STE1) were subjected to sequencing. To design sets of primers that do not fall in exon-intron junctions, we predicted possible splice sites by using the RNASPL program available at the internet site of Baylor College of Medicine (Houston, Tex.; http://dot.imgen.bcm.tmc.edu:9331/seq-search/gene-search.html). Primers were designed using the Primer Selection software of DNAstar (DNASTAR Inc., Madison, Wis.).
Oligonucleotide sequences 5′ to 3′ are CAGTGTGAGTAAT T TAGCAT TACTA (S5D_FF), GGAAAGATCATC-AAACAT T TACATGT (S5D_LR), GCGCAATCT TCT T TCGT T T (S5D—1F), TGGACAACAACAACACAAGA (S5D—1R), GATGCACAGAGAGCT-TCATGAC (S5D—2F), CCGGCAAATGGAGAGAGTGTAT (S5D—2R), CACCCATCATATCTACAACAA (S5D—3F), and CATCT T T TGCCG-GCGAATCTAT (S5D—4F) (underlines were added to distinguish forward or reverse primers from the gene acronym S5D). Primers were purchased from Genosys Biotechnologies, Inc. (The Woodlands, Tex.). For template DNA, genomic DNA was isolated from two or three leaves of dwf7-1 and wild-type plants according to the method described by Krysan et al. (1996) Proc. Natl. Acad. Sci. USA 93:8145-8150. Amplification of the DNA fragment spanning the whole coding region was performed with the S5D—4F and S5D—1R primer set with Taq polymerase (Boehringer Mannheim). - Standard PCR reaction mixtures, 1× PCR buffer (10 mM Tris-HCl, 1.5 mM MgCl2, and 50 mM KCl, pH 8.3), 0.2 μM each of forward and reverse primer, 0.2 mM each deoxynucleotide triphosphates, 1 ng of genomic DNA, and 2 units of Taq polymerase were subjected to a PCR program consisting of an initial denaturation at 95° C. for 2 min and then for 35 cycles (95° C. for 30 sec, 56° C. for 30 sec, and 72° C. for 2.5 min), with a final elongation step of 7 min at 72° C. PCR-amplified DNA was size-separated on 0.8% agarose gels in 1× TAE, and the resulting DNA bands were gel-purified using a DNA purification kit (Bio-Rad). The concentration of the extracted DNA was measured by comparing the band intensity with a DNA mass standard (Bethesda Research Laboratories). Sequencing of the DNA was performed at the Arizona Research Laboratory (University of Arizona, Tucson). DNA sequence analysis was conducted using software packages, including one from Genetics Computer Group (Madison, Wis.) and other database search tools available on the Internet.
- The base change in dwf7-1 eliminated the recognition site for a restriction enzyme HaeIII by converting the sequence from GGCC to AGCC. Thus, we utilized this polymorphism to test the co segregation of the dwarf phenotype with the mutation. The 0.8 kb of DNA spanning the mutation was amplified using S5D—3F and S5D—1R primers from 17 different dwarf plants from the mapping lines. Two microliters from each 20 μL of PCR-amplified DNA was digested with the restriction enzyme HaeIII (Boehringer Mannheim). After complete digestion, the samples were resolved on a 2% agarose gel in 1× TAE buffer.
- Genomic DNA sequence flanking the CDNA was identified by sequencing the products obtained from thermal asymmetric interlaced PCR (TAIL PCR) (Liu et al. (1995) Plant J. 8:457-463). Two sets of primers were used to amplify the 5′ and 3′ flanking DNA.
Oligonucleotide sequences 5′ to 3′ are GTAGAAGCACCAGAGGAAACCGGAGATGAAGT (D7-5-1; melting temperature of 69° C.), AAGTATAGTAGGGT TCCGGCGAGG-TA (D7-5-2; melting temperature of 64° C.), ATAGAT TCGCCG-GCAAAAGATGACTC (D7-5-3; melting temperature of 63° C.), TGC-AGGATACCATACGATACACCACACGACAT (D7-3-1; melting temperature of 68° C.), CATACGATACACCACACGACATACAAGCAT-AACTA (D7-3-2; melting temperature of 67° C.), and ATATGGATG-GAT TGGATGT T TGGCTCTC (D7-3-3; melting temperature of 63° C.). The melting temperature of each primer was calculated with the formula 69.3+0.41 (%GC) 650/L (Mazars et al. (1991) Nucleic Acids Res. 19:4783), where L is length of primer. Arbitrary degenerate primers AD1, AD2, and AD3 were synthesized according to the sequence described by Liu et al. (1995) Plant J. 8:457-463. TAIL PCR was performed according to the program originally described by Liu et al. 1995. TAIL PCR-amplified DNA was separated on 1% agarose gels and gel extracted for sequencing. - D. Feeding Experiments
- Biochemical complementation of dwf7-1 plants with different concentrations of brassinolide (BL) was performed in liquid media. BL-supplemented (control, 10−9, 10−8, and 10−7 M) sterile liquid media (1.5 mL) was dispensed into wells of a 24-well plate (Coming Co., Coming, N.Y.). Three seedlings, germinated on agar-solidified media, were transferred into each well. After a week of growth with continuous shaking (230 rpm), the seedlings were lightly stained with toluidine blue, and hypocotyls and roots were measured to the nearest millimeter.
- Feeding experiments using biosynthetic intermediates were performed with 3-week-old mutant plants. The intermediates tested were diluted to the desired concentration with water containing 0.01
% Tween 20. Two microliters of each brassinosteroid (BR) solution was applied daily to the shoot tips of plants by using a micro pipettman. After 1 week of treatment, total growth of inflorescence and pedicels was measured to the nearest millimeter (n=15). - E. Analysis of Endogenous BRs
- Plants were grown for 5 weeks on soil. Two hundred grams of the aerial parts of plants, including stems, flowers, leaves, and siliques, was harvested and subjected to BR extraction. The procedure for extraction and analysis of BR intermediates by using GC-SIM has been described (Fujioka et al. (1997) Plant Cell 9:1951-1962).
- F.13C-Labeled Mevalonic Acid Feeding Experiments
- Before feeding experiments, seedlings were germinated and grown on 0.5× Murashige and Skoog (Murashige and Skoog (1962) Physiol. Plant. 15:473-497) agar medium in the light at 22° C. (25 mL per dish). Eight days after sowing, the seedlings were transferred to a 200-mL flask containing 30 mL of Murashige and Skoog (Murashige and Skoog (1962) Physiol. Plant. 15:473-497) media supplemented with 3% sucrose (Ws-2, five seedlings; dwf7-1, 40 seedlings).
- Compactin (mevastatin; Sigma) was converted to its sodium salt as described previously (Kita et al. (1980) J. Clin. Invest. 66:1094-1100). DL-Mevalonolactone-2-13C (13C-MVA; Isotec, Miamisburg, Ohio.) was dissolved in methanol. Solutions of compactin and 13C-MVA were added aseptically to each 200-mL flask (final concentration, 10 μM compactin and 4.5 mM 13C-MVA) just after the seedlings were transferred, and seedlings were allowed to grow for 11 days at 22° C. in the light on a shaker (110 rpm). After incubation, the seedlings (˜5 g fresh weight of both Ws-2 and dwf7-1 plant materials) were extracted with methanol (250 mL), and the extract was partitioned between CHCl3 and H2O. The CHCl3-soluble fraction was purified with a silica cartridge column (Sep-Pak Vac 12 cc; Waters, Milford, Mass.), which was eluted with 20 mL of CHCl3. The eluate was purified with an octadecylsilane (ODS) cartridge column (Sep-Pak PLUS C18; Waters), which was eluted with 20 mL of methanol. The fraction was subjected to HPLC on an ODS column as follows: column, Senshu Pak ODS 4150-N (150×10 mm); solvent, methanol; flow rate, 2 mL /min; and detection, UV 205 nm. Fractions were collected every 0.5 min (between retention times of 10 to 20 min). Main fractions of each sterol were as follows: 5-dehydroepisterol (retention time of 11.5 to 12 min), episterol (retention time of 12.5 to 13 min), 24-methylenecholesterol (24-MC; retention time of 13 to 13.5 min), 7-dehydrocampestanol (retention time of 14.5 to 15 min), and campesterol (CR; retention time of 15.5 to 16 min).
- Each fraction was converted to a trimethylsilyl derivative and analyzed by gas chromatography-mass spectrometry (GC-MS). GC-MS analyses were performed on a JEOL Automass JMS-
AM 150 mass spectrometer (Tokyo, Japan) connected to a Hewlett-Packard 5890A-II gas chromatograph with a capillary column DB-5 (0.25 mm×15 m; 0.25-μm film thickness). The analytical conditions were the same as previously described (Fujioka et al. 1997). - 5-Dehydroepisterol, episterol, and 7-dehydrocampestanol were chemically synthesized.
- The dwf7-1 mutant originally was identified in a screen of 14,000 T-DNA-transformed lines of Arabidopsis. Genetic complementation tests with other dwf loci indicated that dwf7 belongs to a unique complementation group. dwf7-1 segregated as a monogenic recessive mutation; progeny from a heterozygote segregated 325 (wild-type): 98 (dwf7-1). Although dwf7-1 originated from a T-DNA mutant population, it failed to cosegregate with the kanamycin resistance marker in the T-DNA, suggesting that dwf7-1 was an untagged mutant. Furthermore, mapping the dwf7-1 mutation to the Arabidopsis genome by using simple sequence length polymorphisms (SSLPs; Bell and Ecker (1994) Genomics 19:137-144) confirmed that dwf7 maps to a location different from previously isolated dwarfs. The meiotic recombination ratio between dwf7 and the SSLP marker ngal 72 on
chromosome 3 was scored as 0/86, indicating tight linkage of dwf7 to ngal72. According to a recent recombinant inbred map of Arabidopsis, ngal72 is located 2.2 centimorgans from the top ofchromosome 3. - A second allele of dwf7 was identified among 43 dwarf mutants isolated by screening >50,000 M2 seeds of an EMS mutant population. Similar to dw7-1, the new allele was biochemically complemented by early BR biosynthetic intermediates, including 22 α-hydroxycampesterol (22-OHCR) and cathasterone, and mapped near ngal72. Sequencing revealed a premature stop codon in exon 1 (see below).
- dwf7 displays many of the characteristics of other BR dwarfs. The characteristic dwarf phenotype, such as short robust stems, reduced fertility, and dark-green, round, and curled leaves are found in the plants. Compared with 1-month-old wild-type plants, dwf7-1 plants grown for 5 weeks in the light possess short robust inflorescences, dark-green, round leaves, reduced fertility, and short pedicels and siliques. The wild-type generally terminates flowering before 7 weeks of age; however, dwf7-1 continues to produce flowers at this age. At 7 weeks of age, wild-type plants had ceased growing, whereas dwf7-1 plants continued to grow, indicating a prolonged life span.
- Additional morphological defects of 5-week-old light-grown plants are summarized in Table 1. Most noticeably, the height of dwf7-1 plants is strikingly reduced and is only 14% that of wild-type height. The leaf blade width of dwf7-1 mutants is similar to that of wild-type plants; however, the length is greatly reduced (1.8 cm) as compared with that of the wild type (3 cm), resulting in the round shape of dwf7-1 leaves. The overall morphology of dwf7-2 was similar to dwf7-1 except that it was slightly shorter and more sterile.
TABLE 1 Morphometric Analysis of Wild-Type and dwf7-1Plants at 5 Weeks of Age Measurement (n = 15) Wild Type dwf7-1 Inflorescence Height (cm) 31.6 ± 0.9 4.5 ± 0.4 Number of inflorescences 3.9 ± 0.6 4.3 ± 0.5 Reproductive organs Number of reproductive organs 130.2 ± 12.9 89.3 ± 20.9 Length of siliques (mm) 14.8 ± 1.2 3.9 ± 0.8 Number of seedsa 49.7 ± 5.1 12.4 ± 2.4 Leaf Number of resette leaves 9.1 ± 1.2 10.3 ± 1.9 Leaf blade width (cm)b 1.4 ± 0.1 1.4 ± 0.3 Leaf blade length (cm)b 3.0 ± 0.3 1.8 ± 0.3 Weight Fresh weight (g) 1.50 ± 0.19 0.51 ± 0.10 Dry weight (mg) 215 ± 29 53 ± 11 Fresh weight/dry weight 7.0 ± 0.3 9.7 ± 0.6 - Because null mutations in the BR pathway result in a dwarf phenotype, as well as defects in skotomorphogenesis, we compared the dwf7-1 mutant with other BR dwarfs for growth in the dark. Hypocotyl lengths from the longest to the shortest were 18±1.6 (wild-type; units in millimeters ±SE; n=15), 6.3±0.29 (dwf7-1), 4.1±0.03 (det2/dwf6), 1.26±0.09 (dwf4), 1.24±0.08 (cpd/dwf3), and 1.18±0.08 (bril/dwf2). These data indicate that dwf7-1 displays a less severe phenotype (35% that of wild-type hypocotyl length) than do other BR dwarfs (e.g., 7% of wild type in dwf4; Choe et al. (1998) Plant Cell 10:231-243). Furthermore, dwf7-1 frequently displayed closed cotyledons and hooks similar to those of the wild type, whereas severe dwarfs, including bril/dwf2, cpd/dwf3, and dwf4, showed expanded cotyledons and open hooks.
- Unlike severe dwarfs, such as dwf4 and cpd, dwf7-1 mutants are not mechanically sterile. However, the average number of seeds in a silique is reduced in dwf7-1 (n=12) compared with that of the wild-type for reasons yet to be identified (n=49) (Table 1). Scanning electron microscopy demonstrated a relationship between fertility and floral structure. In the wild type, the length of stamens was greater than or similar to that of the gynoecium (quantified in FIG. 2), facilitating dehiscence of pollen on the stigmatic surface. The fertile dwf7-1 flower had a concomitant reduction in the size of the gynoecium and the stamen. Although dwf7-1 flowers (FIG. 2) possess stamens and gynoecia that are shorter than those in the wild type, the fertility of dwf7-1 flowers is possible through the concomitant reduction in the length of both organs. In contrast, only stamen elongation was affected more severely in dwf4-3 flowers (FIG. 2). Because sterile dwf4-3 flowers have shorter filaments than the gynoecium, pollen dehiscence on the stigmatic surface is prevented. The short stamen length in dwf4 is likely to cause dehiscence of pollen on the ovary wall rather than on the stigmatic surface. In fact, when dwf4 pollen is transferred to either wild-type or dwf7-1 stigmas, viable seeds are made.
- The common denominator for the various phenotypes found in dwf7-1 mutants is a reduction in longitudinal growth, which could be due to either a reduced number of cells or a failure in cell elongation. Observations made with other BR dwarf mutants suggest that the number of cells is comparable in the wild type and mutants (Kauschmann et al. (1996) Plant J. 9:701-713; Nomura et al. (1997) Plant Physiol. 113:31-37; Azpiroz et al. (1998) Plant Cell 10:219-230). The length of cells in the epidermis, cortex, and xylem of dwf7-1 was greatly reduced (<30% of wild type). This reduced cell size was converted to the length of the wild type in response to daily application of 10−7 M BL for 1 week. Thus, the reduced organ length in dwf7-1 also is due to a failure of cell elongation.
- The organization of vascular bundles in wild-type and dwf7-1 mutants was also examined. Wild-type inflorescences possessed eight vascular bundles. However, the number of vascular bundles was reduced to six in dwf7-1. Furthermore, the spacing between the vascular bundles in dwf7-1 was irregular. In the wild type, interfascicular parenchyma cells alternated regularly with vascular bundles; however, cross-sections of dwf7-1 showed that two vascular bundles were joined without being separated by parenchyma cells. Within a single vascular bundle, the size and number of xylem cells in dwf7-1 plants generally were reduced, whereas the number of phloem cells was similar to or even greater than that in the wild-type. This characteristic abnormality of vascular bundle organization has been observed consistently in other BR dwarfs (Szekeres et al. (1996) Cell 85:171-182).
- FIG. 3 demonstrates that dwf7-1 seedlings grown in BL-supplemented liquid media were remarkably sensitive to BL. Growth in 1 nM BL induced significant elongation of dwf7-1 hypocotyls (160% increase), whereas the wild-type increase was marginal (5%). Treatment with 10 and 100 nM BL completely rescued dwf7-1 hypocotyls to wild-type length. The strongest response of the wild type to BL was obtained at 100 nM (FIG. 3). Higher concentrations of BL (1 μM) caused a stressed morphology, including inhibition of root growth and swollen, twisted, and fragile hypocotyls in both dwf7-1 and wild-type plants. After BL treatment of dwf7-1, cells in the treated region of the stem were similar in length to wild-type cells.
- The overall morphology of plants is dependent on three factors: cell size, shape, and number (Cosgrove (1997) Plant Cell 9:1031-1041). Various signals modulate these factors. Environmental signals, such as water, temperature, and light, are transduced to invoke internal hormone signals, including auxins, gibberellins, and BRs. These signals then trigger the cell elongation process, including but not limited to cell wall loosening by xyloglucan endotransglycosylases and expansins. Thus, a block in any of the signal transduction cascades from the environmental signals to the cell elongation process could result in dwarfism. Mutants resistant to or deficient in classic hormones, such as auxin (e.g., auxin resistant2[axr2]; Timpte (1992) Genetics 138:1239-1249) and gibberellin ([ga1 to ga5 and gai]; Koornneef and van der Veen (1980) Theor. Appl. Genet. 58:257-263; Koornneef et al. (1985) Physiol. Plant. 65:33-39), often result in dwarfism. Thus, we first tested whether dwf7 is either rescued by or resistant to exogenous application of these hormones. Three-week-old dwf7-1 plants sprayed with 0.1 mM GA3 responded, as did the wild-type (<10% increase of inflorescence height); however, GA3 did not rescue the dwf7-1 phenotype. In addition, dwf7-1 roots grown on indole acetic acid-supplemented agar media (0.1 μM) displayed stunted morphology similar to that of the wild-type, suggesting that dwf7-1 is not resistant to the exogenous application of auxin. The reduction of hypocotyl length in dwf7-1 was rescued by the application of BL (FIG. 3). Both wild-type and dwf7-1 plants responded to BL, but dwf7-1 plants were hypersensitive. The length of dwf7-1 hypocotyls was increased 160% in response to 1 nM BL as compared with the untreated control, whereas the wild-type, responded marginally (5%). In addition, application of BRs to 3-week-old dwf7-1 plants induced the growth of many different organs, including stems, leaves, siliques, petioles, and pedicels, suggesting that the major defect in dwf7-1 is a deficiency of BL.
- Apart from a reduction in cell elongation, a deficiency of endogenous BRs resulted in altered organization of vascular tissue in the inflorescence. Szekeres et al. (1996) Cell 85:171-182 showed that the number of xylem cells in cpd was decreased as compared with the wild-type, whereas the number of phloem cells was increased. The authors reasoned that this could be due to unequal division of cambial cells. Furthermore, previous studies on the effects of BRs on vascular development indicated that BRs play a role in tracheary element formation (Clouse and Zurek (1991) Molecular analysis of brassinolide action in plant growth and development. In Brassinosteroids: Chemistry, Bioactivity and Applications, H. G. Cutler, T. Yokota, and G. Adam, eds (Washington DC: American Chemical Society), pp. 122-140; Iwasaki and Shibaoka (1991) Plant Cell. Physiol. 32:1007-101). Because BRs also have been found in the cambial region of pine, indicative of an important role in this tissue (Kim et al. (1990) Plant Physiol. 94:1709-1713), we hypothesize that the deficiency of BRs in dwarf mutants caused changes in cell fate in vascular cambial cells through yet unknown mechanisms.
- Auxins also are known to be a major factor affecting differentiation of the vascular system (Aloni (1987) Annu. Rev. Plant Physiol. 38:179-204). Lincoln et al. (1990) Plant Cell 2:1071-1080 showed that stem cross-sections of axr1 displayed altered development of the vascular system. The vascular bundles in axr1 mutants are located peripherally and are not as regularly spaced as compared with those in wild-type plants (Lincoln et al. (1990) Plant Cell 2:1071-1080). Furthermore, as opposed to the reduced number of vascular bundles in dwf7-1 (five to seven), axrl plants possess a greater number of bundles (eight to nine) as compared with the wild type (six to eight). Thus, it seems that auxins and BRs play opposing roles in determining the number of vascular bundles. Two other assays in which auxin and BR interactions have been demonstrated are the rice lamina bending assay and hypocotyl hook opening bioassay. Results from these assays include the fact that the degree of effect caused by the combined application of auxin and BR was greater than was the sum of the effect of each, indicative of a synergistic effect of the two hormones (Yopp et al. (1981) Physiol. Plant. 53:445-452; Takeno and Pharis (1982) Plant Cell Physiol. 23:1275-1281 reviewed in Mandava (1988) Annu. Rev. Plant Physiol. Plant Mol. Biol. 39:23-52). However, the details of the mechanisms for interactive and independent action remain to be elucidated.
- It needs to be pointed out that hypocotyl growth in darkness is accomplished through both GA- and BR-dependent cell elongation processes. One piece of evidence for dependence on both GA and BR is that dwf7-1 hypocotyls elongated fivefold in response to darkness as compared with light-grown hypocotyls, although they are still shorter than those of the wild-type. Because BL levels are not detectable in dwf7-1 plants (Table 2), growth of dwf7-1 in the dark could be accomplished mostly by GA-dependent cell elongation processes. Peng and Harberd (1997) Plant Physiol. 113:105 1-1058 and Azpiroz et al. (1998) Plant Cell 10:219-230 found that both gai and dwf4, respectively, partially suppressed the stem elongation phenotype of a light receptor mutant, hy, suggesting that hypocotyl elongation in the absence of light inhibition requires independent growth contributed by both GA and BRs.
- Biochemical complementation of dwf7-1 following application of BL suggested that dwf7-1 is likely to be defective in BR biosynthesis. To pinpoint the defective step in the BR biosynthetic pathway, dwf7-1 mutants were treated with BR biosynthetic intermediates. Due to undetectable bioactivity of some early intermediates (CR to 6-oxocampestanol) in bioassays (Fujioka et al. 1995; Choe et al. (1998) Plant Cell 10:231-243), these were not used. Instead, three biologically active compounds were chosen, 22-OHCR, 6-deoxoCT, and BL, for these feeding tests (see FIG. 1). Because the 22α-hydroxylation reaction is reported to be mediated by DWF4 (Choe et al. (1998) Plant Cell 10:231-243), biochemical complementation of dwf mutants other than dwf4 by 22-OHCR places the defective step upstream of CR.
- Complementing compounds induced growth of internodes and strongly increased pedicel length. The dwf7-1 pedicels treated with 22-OHCR and BL showed growth greater than or equal to that of the wild-type. Measurements of pedicel length shown in FIG. 4 demonstrated that the three compounds tested, 22-OHCR, 6-deoxoCT, and BL, all increased dwf7-1 pedicel length >200% as compared with the control, suggesting that the defective step in BR biosynthesis is located at or before the CR biosynthetic step. Similarly, 3-week-old inflorescences of dwf7-2 were tested with 22-COCR, 6-deoxoCT, teasterone, and BL. All four compounds induced significant elongation of pedicels and internodes, indicating that dwf7-1 and dwf7-2 share the same biosynthetic defect.
- As shown in Table 2, more definitive results indicating a specific defect in BR biosynthesis have been obtained from gas chromatography-selective ion monitoring (GC-SIM) analysis of endogenous BRs and sterols in dwf7-1 plants. The endogenous levels of sterols, such as 24-MC, CR, and campestanol (CN), in wild-type plants, were 3800, 32,900, and 1140 ng/g fresh weight, respectively. However, the levels of all three sterols in dwf7-1 mutants were extremely diminished at 3.1, 1.1, and 1.4% of the wild-type, respectively, suggesting that the biosynthetic block is located before 24-MC. These data are consistent with the results of intermediate feeding studies (FIG. 4).
TABLE 2 Quantification of Endogenous BRs from Wild Type and dwf7-1 by Using GC-SIM BRs Wild Typea dwf7-1 34-MC 3,800 118 CR 32,900 379 CN 1,140 16 6-Deoxoteasterone 0.05 NAb 6-Deoxotyphasterol 2.3 NA 6-Deoxocastasterone 4.0 NDc Typhasterol 0.27 ND CS 0.28 0.13 BL 0.2 ND - Further biochemical feeding studies with13C-labeled mevalonic acid (MVA) and compactin, a MVA biosynthetic inhibitor, were performed to identify the specific sterol biosynthetic step defective in dwf7-1 plants. In a preliminary experiment, the effects of compactin and MVA on the growth of Arabidopsis seedlings in liquid media were investigated. The growth of wild-type Arabidopsis seedlings was almost completely inhibited in the presence of 10 μM compactin. The inhibition, however, was restored to the level of controls by the simultaneous application of 4.5 mM of MVA. Therefore, 4.5 mM 13C-MVA and 10 μM compactin were added to Arabidopsis seedling cultures in the metabolic feeding studies. After 11 days in culture, sterols were extracted and purified by silica and octadecylsilane (ODS) cartridge columns and ODS-HPLC. Purified samples were derivatized and analyzed by gas chromatography-mass spectrometry (GC-MS). As shown in FIG. 5, 13C-MVA was converted to 13C5-episterol and subsequent sterols, such as 13C5-24-MC and 13C5-CR in the wild-type. However, the 13C5-5-dehydroepisterol and downstream compounds were not detected in dwf7-1 mutants, whereas the precursor 13C5-episterol accumulated fourfold as compared with the wild-type. In addition, an uncommon sterol, 13C5-7-dehydrocampestanol (24-epifungisterol), greatly accumulated (FIG. 5). Two lines of evidence-a failure to convert episterol to subsequent sterols, such as 24-MC and CR, and accumulation of 7-dehydrocampestanol in dwf7-1-suggest that the defective step in dwf7-1 is the C-5 desaturation stop.
- A defect either in a biosynthetic enzyme or a factor modulating an enzymatic activity could lead to deficiency of endogenous BRs. To place dwf7 at a specific step in the proposed BR biosynthetic pathway, we first chose to perform feeding studies with BR biosynthetic intermediates. Rescue of dwf7-1 by exogenous application of 22-OHCR suggests that the biosynthetic defect likely resides before the production of CR. Consistent with the results from feeding studies, the endogenous levels of 24-MC, CR, and CN were extremely reduced in dwf7-1 (Table 2). These data indicate that the biosynthetic defect is before 24-MC; dwf7-1 contains only 3% of 24-MC as compared with the wild type. When the phenotypes of dwf7-1 are compared with the downstream biosynthetic mutant dwf4 and the BR-insensitive bril (dwf2) mutant (Clouse et al. (1996) Plant Physiol. 111:671-678), it is obvious that dwf7-1 displays a weaker phenotype despite being a presumptive null mutation. This suggests that there could be an alternative sterol and BR biosynthetic pathway or that there are duplicate genes at individual steps. Providing evidence for the duplicate gene hypothesis, we recently cloned a homolog of the DWF7/STE1 gene (named HOMOLOG OF DWF7, HDF7), shown in FIGS. 10 and 11 (GenBank Accession No. AAF32466). HDF7 is 80% identical in amino acid sequence with STE1. Similarly, Fujioka et al. 1997 reported that the endogenous level of CN in det2, which is defective in a step between CR and CN, is ˜10% that of the wild-type amount. The authors hypothesized that the 10% leakage through the defective step in det2 mutants, even in a null allele, could be associated with a second copy of DET2 that lightly hybridizes in DNA gel blot analyses.
- Placing dwf7 at a single sterol biosynthetic step was accomplished through feeding studies with13C-MVA and compactin. A greater than fourfold accumulation of episterol accompanying the absence of downstream intermediates in dwf7-1 indicates that the Δ7 sterol C-5 desaturase step is blocked in dwf7. In addition, the feeding studies identified an accumulation of 7-dehydrocampestanol, which is an uncommon sterol in plants (FIG. 5). Accumulation of this compound only in dwf7-1 suggests that sterol biosynthesis in dwf7-1 could proceed to a C-24 reduction step, skipping C-5 desaturation as well as the next immediate C-7 reduction. The C-24 reductase seems to convert episterol independently of the immediate upstream enzyme. The absence of a detectable amount of C-7-reduced compounds in dwf7-1 suggests that the enzym atic step is highly dependent on the C-5 desaturation reaction. This confirms the sequence of reactions originally proposed by Taton and Rahier (1991) Biochem. Biophys. R.es. Commun. 181:465-473, Taton and Rahier (1996) Arch. Biochem. Biophys. 325:279-288.
- An EMS-induced mutant (ste1-1) of STE1 encoding a Δ7 sterol C-5 desaturase did not possess a dwarf phenotype (Gachotte et al. (1995) Plant J. 8:407-416). However, because it is likely that ste1-1 is a leaky allele, it was hypothesized that dwf7-1 might be a strong or null allele. The genomic DNA of the STE1 gene was sequenced and two introns and three exons identified by comparing them with the published STE1 cDNA sequence. The organization of the STE1 gene is represented schematically in FIG. 6. Sequencing the STE1 locus in the dwf7 alleles revealed mutations. The mutations found in dwf7-1 and dwf7-2 were located in the third and the first exons, respectively. Both of the dwf7 alleles contained a base change from a guanine to an adenine, converting tryptophan (TGG) to a stop codon (TAG in dwf7-1 and TGA in dwf7-2).
- In addition to creating a stop codon, the mutation in dwf7-1 eliminated a HaellI restriction enzyme recognition site (GGCC to AGCC). Taking advantage of this restriction enzyme site change, we tested the linkage of this mutation to the dwf7-1 phenotype. DNAs isolated from 17 different dwarf plants from a segregating F2 population were subjected to polymerase chain reaction (PCR) analysis by using S5D—3F and S5D—1R primers (underlines were used to distinguish forward or reverse primers from the gene acronym S5D), and the PCR products were digested with HaeIII. Agarose gel electrophoresis definitively showed that none of the PCR products, from 17 mutant templates was restricted, whereas products from wild-type templates were all restricted at the HaeIII site. These data suggest that the creation of the premature stop codon in
exon 3 is the cause of the dwF7-1-conferred phenotype. - To better understand the importance of these nonsense mutations, we analyzed the sequence of STE1 in relation to other C-5 desaturase proteins isolated from fungi. The STE1 protein is composed of 281 predicted amino acids with a theoretical pI of 6.39 and molecular mass of 33 kD. Whereas yeast ERG3 (38% identical; Arthington et al. (1991) Gene 107:173-174; GenBank accession number M62623) is predicted to contain four transmembrane domains, STE1 possesses three putative transmembrane domains. The overall amino acid sequence identities of STE1 with C-5 desaturases from fission yeast (GenBank accession number AB004539) and Candida glabrata (Geber et al. (1995) Antimicrob. Agents Chemother. 39:2708-2717; GenBank accession number L40390) were 37 and 33%, respectively (gap creation weight of 4; gap extension weight of 1). In addition, multiple sequence alignment of STE1 with the three yeast sequences, shown in FIG. 7, revealed that the transmembrane domains and histidine clusters, which were first reported by Gachotte et al. (1996) Plant J. 9:391-398, are well conserved between the proteins. The three characteristic histidine boxes flank the last transmlembrane domain. The nonsense mutations are located in the first exon (dwf7-2) and the third exon, immediately before the third histidine box (dwf7-1), indicating that at least one histidine domain is deleted in each of the dwf7 mutants as a result of the premature stop codons.
- The Δ7 sterol C-5 desaturase-mediated reaction is common to both photosynthetic and nonphotosynthetic organisms. Many genes encoding a C-5 desaturase have been cloned from fungi. First, Arthington et al. (1991) Gene 107:173-174 cloned the ERG3 gene from Saccharomyces cerevisiae. The authors found that viable erg3 mutants, which normally accumulate Δ7 sterols, were restored to wild-type phenotype when transformed with a wild-type genomic clone of the Δ7 sterol C-5 desaturase gene. Taguchi et al. (1994) Microbiology 140:353-359 showed that the yeast mutant syrl displays dual phenotypes, resistance to the phytotoxin syringomycin and susceptibility to higher concentrations of Ca2+, presumably due to altered membranes. Sequencing the ERG3 locus in the syr1 mutant revealed that syr1 is an allele of ERG3. Furthermore, Geber et al. (1995) Antimicrob. Agents Chemother. 39:2708-2717 cloned both ERG3 and ERG11 (14α-sterol-demethylase) from C. glabrata. The authors found that lethal erg11 mutations can be suppressed by an additional mutation in erg3. They reasoned that formation of toxic 3β, 6α-diol sterols in erg11 mutants is prevented due to the defect in C-5 desaturation in erg11 erg3 double mutants.
- In plants, Gachotte et al. (1995) Plant J. 8:407-416 found that the Arabidopsis ste1-1 mutant, which is deficient in C-5 desaturated sterols, can be partially complemented by the yeast ERG3 gene. Accordingly, the authors hypothesized that ste1-1 possesses a mutation in the sterol C-5 desaturase gene. They isolated the Arabidopsis C-5 desaturase gene through heterologous complementation of a yeast erg3 null mutant with an Arabidopsis cDNA library (Gachotte et al. (1996) Plant J. 9:391-398). Finally, the partial human cDNA for the C-5 desaturase has been identified by Matsushima et al. (1996) Cell Genet. 74:252-254. Alignment of the sequences of these enzymes revealed that C-5 desaturases from different organisms; are highly conserved in overall sequence as well as in specific domains. The overall amino acid sequence identity and similarity among STE1 and ERG3 and the human ortholog is 38% (50%) and 35% (47%), respectively (similarity within parentheses). As indicated in FIG. 6 and FIG. 7, key domains including the transmembrane domains and the histidine clusters are well conserved between all the C-5 desaturases.
- Closely spaced histidine residues, HX3H in helices, serve as typical metal binding motifs in many proteins (Regan (1993) Annu. Rev. Biophys. Biomol. Struct. 22:257-281). Shanklin et al. (1994) Biochemistry 33:12787-12794 showed that three membrane-associated bacterial enzymes, fatty acid desaturase, alkane hydroxylase, and xylene monooxygenase, possess eight histidine residues that are conserved in three regions dispersed in these enzymes, HX(3-4)H, HX(2-3)HH, and HX(2-3)HH (where X stands for any amino acid). DNA constructs containing site-directed mutations at any of these eight histidine residues of the rat Δ9 desaturase failed to complement the yeast mutant ole1, which is defective in the same enzymatic step, suggesting that the individual histidine residues are essential for the function of the enzyme. On the basis of these observations, Shanklin et al. (1994) Biochemistry 33:12787-12794 hypothesized that the histidine clusters conserved in these enzymes constitute new structural domains of diiron binding centers (Shanklin et al. (1994) Biochemistry 33:12787-12794). Gachotte et al. (1996) Plant J. 9:391-398 first recognized the conserved histidine clusters in STE1 and yeast proteins. We confirmed that the motifs are highly conserved in STE1 and the yeast ERG3 enzymes with the same context of HX3H, HX2HH, and HX2HH (FIG. 7), revealing the presence of a putative iron binding motif in Δ7 stero1C-5 desaturases.
- More direct evidence of metal ion involvement in Δ7 sterol C-5 desaturase function was obtained by Taton and Rahier (1996) Arch. Biochem. Biophys. 325:279-288. These authors discovered that the enzyme prepared from maize microsomes is inhibited by cyanide, whereas it is insensitive to carbon monoxide, indicative of the involvement of a metal ion, presumably an iron, for the proper function of the enzyme. Furthermore, we noticed that the typical histidine moiety also was conserved in a different group of oxidases such as RANP-1 (Uwabe et al. (1997) Neuroscience 80:501-509), C-4 methyl sterol oxidase (Li and Kaplan (1996) J. Biol. Chem. 271:16927-16933), and aldehyde decarbonylase (Aarts et al. (1995) Plant Cell 7:2115-2127). Occurrence of these histidine boxes in a wide variety of oxidases indicates that this domain plays a common and essential role in the function of membrane oxidases. Therefore, it is likely that the mutations in dwf7-1 and dwf7-2 would be deleterious to protein function. The premature stop codon in dwf7-2 would eliminate all important known domains, whereas the third histidine box and several amino acid residues that are 100% conserved in the C terminus of the protein are eliminated in dwf7-1. Intriguingly, the location of the mutations in dwf7-1 and dwf7-2 seems to be related to the phenotypic severity of the mutant alleles. dwf7-2, which contains an earlier stop codon, was shorter in height and less fertile than dwf7-1. A more precise comparison between the two alleles is not possible because the EMS allele, dwf7-2, has not been outcrossed to remove any background mutations that might have increased the severity of the phenotype of dwf7-2. Despite the differences in severity, both dwf7 alleles are likely complete loss-of-function alleles. The resulting nonfunctional enzyme causes a block in sterol biosynthesis. This shortage of substrate sterols in dwf7-1 and dwf7-2 leads to a deficiency of endogenous BRs and causes the characteristic dwarfism in dwf7 plants.
- Thus, novel dwf7 mutants, as well as methods of using the same, are disclosed. Although preferred embodiments of the subject invention have been described in some detail, it is understood that obvious variations can be made without departing from the spirit and the scope of the invention as defined by the appended claims.
-
0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 25 <210> SEQ ID NO 1 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Primer S5D_FF <400> SEQUENCE: 1 cagtgtgagt aatttagcat tacta 25 <210> SEQ ID NO 2 <211> LENGTH: 26 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Primer S5D_LR <400> SEQUENCE: 2 ggaaagatca tcaaacattt acatgt 26 <210> SEQ ID NO 3 <211> LENGTH: 19 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Primer S5D_1F <400> SEQUENCE: 3 gcgcaatctt ctttcgttt 19 <210> SEQ ID NO 4 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Primer S5D_1R <400> SEQUENCE: 4 tggacaacaa caacacaaga 20 <210> SEQ ID NO 5 <211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Primer S5D_2F <400> SEQUENCE: 5 gatgcacaga gagcttcatg ac 22 <210> SEQ ID NO 6 <211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Primer S5D_2R <400> SEQUENCE: 6 ccggcaaatg gagagagtgt at 22 <210> SEQ ID NO 7 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Primer S5D_3F <400> SEQUENCE: 7 cacccatcat atctacaaca a 21 <210> SEQ ID NO 8 <211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Primer S5D_4F <400> SEQUENCE: 8 catcttttgc cggcgaatct at 22 <210> SEQ ID NO 9 <211> LENGTH: 32 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Primer D7-5-1 <400> SEQUENCE: 9 gtagaagcac cagaggaaac cggagatgaa gt 32 <210> SEQ ID NO 10 <211> LENGTH: 26 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Primer D7-5-2 <400> SEQUENCE: 10 aagtatagta gggttccggc gaggta 26 <210> SEQ ID NO 11 <211> LENGTH: 26 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Primer D7-5-3 <400> SEQUENCE: 11 atagattcgc cggcaaaaga tgactc 26 <210> SEQ ID NO 12 <211> LENGTH: 32 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Primer D7-3-1 <400> SEQUENCE: 12 tgcaggatac catacgatac accacacgac at 32 <210> SEQ ID NO 13 <211> LENGTH: 35 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Primer D7-3-2 <400> SEQUENCE: 13 catacgatac accacacgac atacaagcat aacta 35 <210> SEQ ID NO 14 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Primer D7-3-3 <400> SEQUENCE: 14 atatggatgg attggatgtt tggctctc 28 <210> SEQ ID NO 15 <211> LENGTH: 364 <212> TYPE: PRT <213> ORGANISM: delta-7 sterol C-5 desaturase (Candida glabrata) <400> SEQUENCE: 15 Met Asp Leu Val Leu Glu Thr Leu Asp His Tyr Ile Phe Asp Asp Val 1 5 10 15 Tyr Ala Lys Ile Ala Pro Val Glu Leu Gln Arg Gly Ile Asp Asp Ser 20 25 30 Leu Val Asn Ala Leu Ser Leu Asn Lys Ile Val Ser Asn Ser Thr Leu 35 40 45 Leu His Glu Thr Leu Ser Ile Thr Asn Ser Leu Lys Arg Val Asn Lys 50 55 60 Asp Val Tyr Gly Leu Thr Pro Phe Leu Phe Asp Phe Thr Glu Lys Thr 65 70 75 80 Tyr Ala Ser Leu Leu Pro Arg Asn Asn Leu Ile Arg Glu Phe Phe Ser 85 90 95 Leu Trp Ala Val Val Thr Val Phe Gly Leu Leu Leu Tyr Leu Ile Thr 100 105 110 Ala Ser Leu Ser Tyr Val Phe Val Phe Asp Arg Thr Ile Phe Asn His 115 120 125 Pro Lys Tyr Leu Lys Asn Gln Met Tyr Leu Glu Ile Lys Leu Ala Val 130 135 140 Ser Ala Ile Pro Thr Met Ser Leu Leu Thr Val Pro Trp Phe Met Leu 145 150 155 160 Glu Leu Asn Gly Tyr Ser Lys Leu Tyr Tyr Asp Val Asp Trp Glu His 165 170 175 His Gly Leu Arg Lys Leu Leu Ile Glu Tyr Ala Thr Phe Ile Phe Phe 180 185 190 Thr Asp Cys Gly Ile Tyr Leu Ala His Arg Trp Leu His Trp Pro Arg 195 200 205 Val Tyr Lys Ala Leu His Lys Pro His His Lys Trp Leu Val Cys Thr 210 215 220 Pro Phe Ala Ser His Ala Phe His Pro Val Asp Gly Tyr Phe Gln Ser 225 230 235 240 Leu Ser Tyr His Ile Tyr Pro Met Ile Leu Pro Leu His Lys Ile Ser 245 250 255 Tyr Leu Ile Leu Phe Thr Phe Val Asn Phe Trp Ser Val Met Ile His 260 265 270 Asp Gly Gln His Met Ser Asn Asn Pro Val Val Asn Gly Thr Ala Cys 275 280 285 His Thr Val His His Leu Tyr Phe Asn Tyr Asn Tyr Gly Gln Phe Thr 290 295 300 Thr Leu Trp Asp Arg Leu Gly Gly Ser Tyr Arg Arg Pro Glu Asp Ser 305 310 315 320 Leu Phe Asp Pro Lys Leu Lys Met Asp Lys Lys Val Leu Glu Lys Gln 325 330 335 Ala Arg Glu Thr Ala Ala Tyr Ile Gln Glu Val Glu Gly Asp Asp Thr 340 345 350 Asp Arg Val Tyr Asn Thr Asp Lys Lys Lys Thr Asn 355 360 <210> SEQ ID NO 16 <211> LENGTH: 365 <212> TYPE: PRT <213> ORGANISM: delta-7 sterol C-5 desaturase (Saccharomyces cerevisiae) <400> SEQUENCE: 16 Met Asp Leu Val Leu Glu Val Ala Asp His Tyr Val Leu Asp Asp Leu 1 5 10 15 Tyr Ala Lys Val Leu Pro Ala Ser Leu Ala Ala Asn Ile Pro Val Lys 20 25 30 Trp Gln Lys Leu Leu Gly Leu Asn Ser Gly Phe Ser Asn Ser Thr Ile 35 40 45 Leu Gln Glu Thr Leu Asn Ser Lys Asn Ala Val Lys Glu Cys Arg Arg 50 55 60 Phe Tyr Gly Gln Val Pro Phe Leu Phe Asp Met Ser Thr Thr Ser Phe 65 70 75 80 Ala Ser Leu Leu Pro Arg Ser Ser Ile Leu Arg Glu Phe Leu Ser Leu 85 90 95 Trp Val Ile Val Thr Ile Phe Gly Leu Leu Leu Tyr Leu Phe Thr Ala 100 105 110 Ser Leu Ser Tyr Val Phe Val Phe Asp Lys Ser Ile Phe Asn His Pro 115 120 125 Arg Tyr Leu Lys Asn Gln Met Ala Met Glu Ile Lys Leu Ala Val Ser 130 135 140 Ala Ile Pro Trp Met Ser Met Leu Thr Val Pro Trp Phe Val Met Glu 145 150 155 160 Leu Asn Gly His Ser Lys Leu Tyr Met Lys Ile Asp Tyr Glu Asn His 165 170 175 Gly Val Arg Lys Leu Ile Ile Glu Tyr Phe Thr Phe Ile Phe Phe Thr 180 185 190 Asp Cys Gly Val Tyr Leu Ala His Arg Trp Leu His Trp Pro Arg Val 195 200 205 Tyr Arg Ala Leu His Lys Pro His His Lys Trp Leu Val Cys Thr Pro 210 215 220 Phe Ala Ser His Ser Phe His Pro Val Asp Gly Phe Leu Gln Ser Ile 225 230 235 240 Ser Tyr His Ile Tyr Pro Leu Ile Leu Pro Leu His Lys Val Ser Tyr 245 250 255 Leu Ile Leu Phe Thr Phe Val Asn Phe Trp Thr Val Met Ile His Asp 260 265 270 Gly Gln Tyr Leu Ser Asn Asn Pro Ala Val Asn Gly Thr Ala Cys His 275 280 285 Thr Val His His Leu Tyr Phe Asn Tyr Asn Tyr Gly Gln Phe Thr Thr 290 295 300 Leu Trp Asp Arg Leu Gly Gly Ser Tyr Arg Arg Pro Asp Asp Ser Leu 305 310 315 320 Phe Asp Pro Lys Leu Arg Asp Ala Lys Glu Thr Trp Asp Ala Gln Val 325 330 335 Lys Glu Val Glu His Phe Ile Lys Glu Val Glu Gly Asp Asp Asn Asp 340 345 350 Arg Ile Tyr Glu Asn Asp Pro Asn Thr Lys Lys Asn Asn 355 360 365 <210> SEQ ID NO 17 <211> LENGTH: 329 <212> TYPE: PRT <213> ORGANISM: delta-7 sterol C-5 desaturase (Schizosaccharomyces pombe) <400> SEQUENCE: 17 Met Asp Val Val Leu Gln Tyr Ala Asp Lys Tyr Val Phe Asp Thr Phe 1 5 10 15 Tyr Gly Lys Ile Ala Glu Ser Phe Asp Ser Ser Ser Ser Phe Ala Asn 20 25 30 Thr Ala Val Asn Ser Thr Thr Leu Gly Leu Ala Glu Lys Val Asn Phe 35 40 45 Ala Ile Thr Ser Gly Leu Leu Asp Arg Asn Asn Val Trp Arg Gln Phe 50 55 60 Thr Ser Leu Phe Leu Ile Thr Trp Ile Met Gly Thr Leu Ser Tyr Phe 65 70 75 80 Leu Ser Ala Ser Phe Ala Tyr Tyr Val Tyr Phe Asp Arg Glu Glu Ala 85 90 95 Arg Arg His Pro Lys Phe Leu Lys Asn Gln Glu His Leu Glu Leu Met 100 105 110 Val Ala Leu Lys Asn Leu Pro Gly Met Ala Ile Leu Thr Ala Pro Trp 115 120 125 Phe Leu Ala Glu Ile Arg Gly Tyr Gly Tyr Val Tyr Asp Lys Leu Asp 130 135 140 Glu Tyr Gly Tyr Phe Tyr Leu Phe Phe Ser Ile Ala Leu Phe Leu Leu 145 150 155 160 Phe Ser Asp Phe Leu Ile Tyr Trp Ile His Arg Ala Leu His His Arg 165 170 175 Trp Leu Tyr Ala Pro Leu His Lys Leu His His Lys Trp Ile Val Pro 180 185 190 Thr Pro Tyr Ser Ser His Ala Phe His Tyr Leu Asp Gly Tyr Ser Gln 195 200 205 Ser Leu Pro Tyr His Met Phe Pro Phe Phe Phe Pro Leu Asn Lys Tyr 210 215 220 Val Tyr Leu Leu Leu Phe Gly Ser Val Asn Tyr Trp Thr Val Leu Ile 225 230 235 240 His Asp Gly Lys Tyr Phe Ser Asn Asn Ala Val Val Asn Gly Ala Ala 245 250 255 His His Ala Ala His His Met Tyr Phe Asn Tyr Asn Tyr Gly Gln Phe 260 265 270 Phe Thr Leu Phe Asp Arg Leu Cys Ser Ser Tyr Arg Gln Pro Asp Gln 275 280 285 Glu Leu Phe Asp Ala Glu Leu Arg Asn Glu Lys Leu Gln Glu Gln Arg 290 295 300 Ile Arg Phe Met Glu Thr Val Gln Tyr Thr Val Glu Gly Lys Asp Asp 305 310 315 320 Arg Thr Tyr Ala Ser Lys Lys Asp Asn 325 <210> SEQ ID NO 18 <211> LENGTH: 281 <212> TYPE: PRT <213> ORGANISM: DWF7/STE1 (Arabidopsis) <400> SEQUENCE: 18 Met Ala Ala Asp Asn Ala Tyr Leu Met Gln Phe Val Asp Glu Thr Ser 1 5 10 15 Phe Tyr Asn Arg Ile Val Leu Ser His Leu Leu Pro Ala Asn Leu Trp 20 25 30 Glu Pro Leu Pro His Phe Leu Gln Thr Trp Leu Arg Asn Tyr Leu Ala 35 40 45 Gly Thr Leu Leu Tyr Phe Ile Ser Gly Phe Leu Trp Cys Phe Tyr Ile 50 55 60 Tyr Tyr Leu Lys Ile Asn Val Tyr Leu Pro Lys Asp Ala Ile Pro Thr 65 70 75 80 Ile Lys Ala Met Arg Leu Gln Met Phe Val Ala Met Lys Ala Met Pro 85 90 95 Trp Tyr Thr Leu Leu Pro Thr Val Ser Glu Ser Met Ile Glu Arg Gly 100 105 110 Trp Thr Lys Cys Phe Ala Ser Ile Asp Glu Phe Gly Trp Ile Leu Tyr 115 120 125 Phe Val Tyr Ile Ala Ile Tyr Leu Val Phe Val Glu Phe Gly Ile Tyr 130 135 140 Trp Met His Arg Glu Leu His Asp Ile Lys Pro Leu Tyr Lys Tyr Leu 145 150 155 160 His Ala Thr His His Ile Tyr Asn Lys Gln Asn Thr Leu Ser Pro Phe 165 170 175 Ala Gly Leu Ala Phe His Pro Val Asp Gly Ile Leu Gln Ala Val Pro 180 185 190 His Val Ile Ala Leu Phe Ile Val Pro Ile His Phe Thr Thr His Ile 195 200 205 Gly Leu Leu Phe Met Glu Ala Ile Trp Thr Ala Asn Ile His Asp Cys 210 215 220 Ile His Gly Asn Ile Trp Pro Val Met Gly Ala Gly Tyr His Thr Ile 225 230 235 240 His His Thr Thr Tyr Lys His Asn Tyr Gly His Tyr Thr Ile Trp Met 245 250 255 Asp Trp Met Phe Gly Ser Leu Arg Asp Pro Leu Leu Glu Glu Asp Asp 260 265 270 Asn Lys Asp Ser Phe Lys Lys Ala Glu 275 280 <210> SEQ ID NO 19 <211> LENGTH: 291 <212> TYPE: PRT <213> ORGANISM: delta-7 sterol C-5 desaturase concensus <400> SEQUENCE: 19 Met Asp Leu Val Leu Glu Ala Asp His Tyr Val Phe Asp Asp Tyr Ala 1 5 10 15 Lys Ile Pro Leu Ala Ile Asp Ser Leu Leu Asn Val Ser Asn Ser Thr 20 25 30 Leu Glu Thr Leu Asn Lys Val Asn Tyr Gly Pro Phe Leu Phe Asp Phe 35 40 45 Thr Glu Thr Ser Phe Ser Leu Leu Pro Arg Asn Asn Leu Trp Arg Glu 50 55 60 Phe Leu Ser Leu Trp Leu Ile Val Thr Ile Phe Gly Leu Leu Tyr Ile 65 70 75 80 Ala Ser Leu Ser Tyr Phe Phe Asp Ile Phe Asn His Pro Lys Tyr Leu 85 90 95 Lys Asn Gln Met Leu Glu Ile Lys Ala Val Ala Ile Pro Trp Met Ser 100 105 110 Leu Leu Thr Val Pro Trp Phe Met Glu Leu Gly Tyr Ser Lys Leu Tyr 115 120 125 Lys Ile Asp Glu His Gly Arg Lys Leu Phe Ile Glu Ala Thr Phe Phe 130 135 140 Phe Thr Asp Gly Ile Tyr Ala His Arg Trp Leu His Trp Pro Tyr Lys 145 150 155 160 Ala Leu His Lys Pro His His Lys Trp Leu Val Cys Thr Pro Phe Ala 165 170 175 Ser His Ala Phe His Pro Val Asp Gly Tyr Leu Gln Ser Leu Tyr His 180 185 190 Ile Tyr Pro Leu Leu Pro Leu His Lys Ser Tyr Leu Leu Phe Thr Phe 195 200 205 Val Asn Phe Trp Thr Val Met Ile His Asp Gly Gln Ser Asn Asn Pro 210 215 220 Val Val Asn Gly Ala Cys His Thr Val His His Leu Tyr Phe Asn Tyr 225 230 235 240 Asn Tyr Gly Gln Phe Thr Thr Leu Trp Asp Arg Leu Gly Gly Ser Tyr 245 250 255 Arg Arg Pro Asp Ser Leu Phe Asp Pro Lys Leu Arg Asp Lys Lys Glu 260 265 270 Gln Arg Glu Thr Tyr Ile Glu Val Glu Gly Asp Asp Asp Arg Tyr Asp 275 280 285 Lys Lys Asn 290 <210> SEQ ID NO 20 <211> LENGTH: 1889 <212> TYPE: DNA <213> ORGANISM: Genomic dwf7 (Arabidopsis) <400> SEQUENCE: 20 gaagatcgat caatcaatca tcaaactctc tgtgtgccac atgcattact actgttgact 60 tgttcaataa aggtaaagta agatcaatcc ggcgaatctt ctttcgtttt ccggcaccga 120 tctcggtgga tctccgattc acatggcggc ggataatgct tatctgatgc agtttgttga 180 cgaaacctct ttttacaacc gaatcgttct gagtcatctt ttgccggcga atctatggga 240 acccttacct cattttctcc agacatggct ccgaaattac ctcgccggaa ccctactata 300 cttcatctcc ggtttcctct ggtgcttcta catctattac cttaaaatca acgtttacct 360 tcccaaaggt ctcgactttc acttttgtat tcactattgc ttaatcgctt tctatgttat 420 cgatttttca atttaaggaa gagggtttct tcgtgtactg tacagtaatt tggatttgat 480 gtggatagtt catgtttgca tttattgatt atttgtgcat attctccatc taagggattg 540 aacagttagt ggcttatata agtttttgtg caaccaatga gaagtcgtac atctttgaag 600 ttgaattttc tacttgccat ttaagtccac ttaaattgtt tgttgaagtg attgtctact 660 ttcagacaca ttctttttct gcttctctga gactctgtct tagtttgaaa tcttttttgg 720 tctgttttgc ttcagatgca attcctacaa taaaggctat gcgtttgcaa atgtttgtgg 780 caatgaaggc tatgccatgg tacactcttc ttccaactgt ctccgagagt atgattgaac 840 gtggttggac caaatgtttt gctagcatag acgaattcgg ctggattctg tattttgttt 900 acatcgccat ctatcttgtt ttcgttgagt ttggtattta ttggatgcac agagagcttc 960 atgacattaa gcctctctat aagtatctcc atgccaccca tcatatctac aacaagcaga 1020 atacactctc tccatttgcc ggtaagtgtt ttcagtttgt tcttctttag ttcttgtaaa 1080 agattggtag catttagttt cttaccagaa aagactttgt cagcagctgc ttgtactcca 1140 aatcacattt tgcattcctt atccataaag taaccagaaa ggctagaatt atataaatgt 1200 cagctgcatt acttcacata tgtcagagag acttctgact taaccagagt ttagatcttt 1260 gtgtttctct tctggtctcg gactgattgg aaatgacgag aagttctttt atctacttcc 1320 ctggagtgta tcttggttaa tccaaggatg tgacatctaa tattacttgt aacttcctta 1380 cgtttttgtt tacagggctt gcatttcacc cagtagacgg gatacttcag gctgtaccgc 1440 atgtgatagc gctgtttata gtgccaattc atttcacaac tcatataggt cttttgttca 1500 tggaagcgat atggacggcg aacatccatg actgcatcca tggcaacatc tggccagtaa 1560 tgggtgcagg ataccatacg atacaccaca cgacatacaa gcataactat ggtcattata 1620 ccatatggat ggattggatg tttggctctc ttagggatcc tctcttagaa gaagatgaca 1680 acaaagacag cttcaagaaa gcagagtgag aatgcccact tgggttttgt tcttctgttt 1740 tgtcttgtgt tgttgttgtt caaagtttca gcctttcttg ttctttttct tcttcttctt 1800 attcatgtgt ctctctcaac ctttccaatt atattgttac aaacatttgc tgtctagttt 1860 aaaacatgta aatgtttgat gatctttgc 1889 <210> SEQ ID NO 21 <211> LENGTH: 281 <212> TYPE: PRT <213> ORGANISM: DWF7 <400> SEQUENCE: 21 Met Ala Ala Asp Asn Ala Tyr Leu Met Gln Phe Val Asp Glu Thr Ser 1 5 10 15 Phe Tyr Asn Arg Ile Val Leu Ser His Leu Leu Pro Ala Asn Leu Trp 20 25 30 Glu Pro Leu Pro His Phe Leu Gln Thr Trp Leu Arg Asn Tyr Leu Ala 35 40 45 Gly Thr Leu Leu Tyr Phe Ile Ser Gly Phe Leu Trp Cys Phe Tyr Ile 50 55 60 Tyr Tyr Leu Lys Ile Asn Val Tyr Leu Pro Lys Asp Ala Ile Pro Thr 65 70 75 80 Ile Lys Ala Met Arg Leu Gln Met Phe Val Ala Met Lys Ala Met Pro 85 90 95 Trp Tyr Thr Leu Leu Pro Thr Val Ser Glu Ser Met Ile Glu Arg Gly 100 105 110 Trp Thr Lys Cys Phe Ala Ser Ile Asp Glu Phe Gly Trp Ile Leu Tyr 115 120 125 Phe Val Tyr Ile Ala Ile Tyr Leu Val Phe Val Glu Phe Gly Ile Tyr 130 135 140 Trp Met His Arg Glu Leu His Asp Ile Lys Pro Leu Tyr Lys Tyr Leu 145 150 155 160 His Ala Thr His His Ile Tyr Asn Lys Gln Asn Thr Leu Ser Pro Phe 165 170 175 Ala Gly Leu Ala Phe His Pro Val Asp Gly Ile Leu Gln Ala Val Pro 180 185 190 His Val Ile Ala Leu Phe Ile Val Pro Ile His Phe Thr Thr His Ile 195 200 205 Gly Leu Leu Phe Met Glu Ala Ile Trp Thr Ala Asn Ile His Asp Cys 210 215 220 Ile His Gly Asn Ile Trp Pro Val Met Gly Ala Gly Tyr His Thr Ile 225 230 235 240 His His Thr Thr Tyr Lys His Asn Tyr Gly His Tyr Thr Ile Trp Met 245 250 255 Asp Trp Met Phe Gly Ser Leu Arg Asp Pro Leu Leu Glu Glu Asp Asp 260 265 270 Asn Lys Asp Ser Phe Lys Lys Ala Glu 275 280 <210> SEQ ID NO 22 <211> LENGTH: 2925 <212> TYPE: DNA <213> ORGANISM: Genomic HDF7 <400> SEQUENCE: 22 gtttggtatt tattggatgc acagagagct tcatgacatt aagcctctct ataagtatct 60 ccatgccacc catcatatct acaacaagca gaatacactc tctccatttg ccggtaagtg 120 ttttcagttt gttcttcttt agttcttgta aaagattggt agcatttagt ttcttaccag 180 aaaagacttt gtcagcagct gcttgtactc caaatcacat tttgcattcc ttatccataa 240 agtaaccaga aaggctagaa ttatataaat gtcagctgca ttacttcaca tatgtcagag 300 agacttctga cttaaccaga gtttagatct ttgtgtttct cttctggtct cggactgatt 360 ggaaatgacg agaagttctt ttatctactt ccctggagtg tatcttggtt aatccaagga 420 tgtgacatct aaatattact tgtaacttcc ttacgttttt gtttacaggg cttgcattca 480 cccagtagac gggatactta aggctgtacc gcatgtgata gcgctgttat agtgccaatt 540 catttcacaa ctcatatagg tcttttgttc atggaagcga tatggacggc gaacatccat 600 gactgcatcc atggcaacat ctggccagta atgggtgcag gataccatac gatacaccac 660 acgacataca agcataacta tggtcattat accatatgga tggattggat gtttggctct 720 cttagggatc ctctcttaga agaagatgac aacaaagaca gcttcaagaa agcagagtga 780 gaatgcccac ttgggttttg ttcttctgtt ttgtcttgtg ttgttgttgt tcaaagtttc 840 agcctttctt gttctttttc ttcttcttct tattcatgtg tctctctcaa cctttccaat 900 tatattgtta caaacatttg ctgtctagtt taaaacatgt aaatgtttga tgatctttgc 960 aagactccat ttttgtttaa ggtaaacctt gaatctcata gattgtcgat tgttggtatt 1020 tccattttca ggtacggttc tgtagactgt agtcttgctg accagtccgg cttaaccacc 1080 ccaaatttca aagatctcac caatcaaaat gctggctggc cccaatatat agatgggcca 1140 gttaatccgt ctagctttac tctttagacc taccttagac agttagacac ctgctaatta 1200 atgagtttcc tttttcttgt tcagcaagtt acctgtgtta cttgagagtt gagttaatgg 1260 tagtaaacgc aatttaaccc ttataagttt aatcgtattc aacgaatgac ccagagactt 1320 taaataaatc catcgtaacc ctccacttca aaattctttt taaaaagtag caaatcattt 1380 aaatattgta agtttgcttc attcgaaatt gtagctacag atctcaaagc tcctcctgtt 1440 ggccatatct ctctctaaca aacgcatagt aacacttgac cacagtttga cttctcggcg 1500 gtttcatggc ggcgactatg gcagattata atgatcagat cgtcaatgag acctcttttt 1560 acaaccgaat ggttctgagt caccttttgc cggtgaatct atgggaacct ttaccacatt 1620 tcctccagac atggctccgg aactacctcg ccggaaacat actctacttc atctccggct 1680 tcctctggtg cttctacatc tattacctta aactcaacgt ttacgtcccc aaaggttact 1740 tttttcaatt tcgatgttct gttttgaaac ctttcttttg ttgattcctt cgattgtatc 1800 gcctgataga ttgtgttata cgttaacctt tttttcttac tgttactttc agttcttgtc 1860 ttctacttct catttaatta gttttaaagt ttaatatttt tggctaatcc acatttttta 1920 agttgaatct tccatgaaat ttgagctcaa aatataccat gaaattgaaa tttgtggttc 1980 ttagttctat ttcttgcttg gtttcttcta tttttgtggt tagaatccat tcctacgaga 2040 aaggcaatgc ttttgcaaat atacgtggca atgaaggcta tgccttggta cactcttctt 2100 ccagctgtct ctgagtatat gatcgagcat ggttggacca aatgttactc tacacttgac 2160 catttcaact ggttcctctg tttcctctac atagctctct atcttgtttt agttgagttt 2220 atgatttatt gggttcacaa agagcttcat gacattaaat ttctctataa gcatctccat 2280 gctacccatc atatgtacaa caagcaaaac acactctctc catttgccgg tatgtcaaag 2340 ctatatgttc tcaatctaaa ttcaagagct tgtatcaatg gtgacttctt tacttgatgt 2400 ttttcgggtt ttcagggctc gcattccatc cgctggacgg gatacttcag gctataccgc 2460 acgtgatagc gctgtttata gtgccgattc atctcataac acatctgagt cttttgtttt 2520 tggaagggat atggacagca agcatccatg attgcataca tggtaacatc tggcctataa 2580 tgggtgcagg ataccatacc atacaccata caacatacaa gcataactat ggtcattata 2640 ccatatggat ggactggatg tttggctctc ttatggttcc tttagcagaa aaagacagtt 2700 tcaaggagaa agaaaagtga gaatgttcaa tgctcacatg tattcttcat atgttgctct 2760 tctcgtgact cttattaaaa cctttctaat cactttggtg gaattaaaaa catgactgca 2820 taatttgatg caaagtttca gacttttatt gctaaaaatc tctgatgatt attaacctca 2880 attatataat tgctggatga agagttcaaa tttggactaa atctg 2925 <210> SEQ ID NO 23 <211> LENGTH: 279 <212> TYPE: PRT <213> ORGANISM: HDF7 <400> SEQUENCE: 23 Met Ala Ala Thr Met Ala Asp Tyr Asn Asp Gln Ile Val Asn Glu Thr 1 5 10 15 Ser Phe Tyr Asn Arg Met Val Leu Ser His Leu Leu Pro Val Asn Leu 20 25 30 Trp Glu Pro Leu Pro His Phe Leu Gln Thr Trp Leu Arg Asn Tyr Leu 35 40 45 Ala Gly Asn Ile Leu Tyr Phe Ile Ser Gly Phe Leu Trp Cys Phe Tyr 50 55 60 Ile Tyr Tyr Leu Lys Leu Asn Val Tyr Val Pro Lys Glu Ser Ile Pro 65 70 75 80 Thr Arg Lys Ala Met Leu Leu Gln Ile Tyr Val Ala Met Lys Ala Met 85 90 95 Pro Trp Tyr Thr Leu Leu Pro Ala Val Ser Glu Tyr Met Ile Glu His 100 105 110 Gly Trp Thr Lys Cys Tyr Ser Thr Leu Asp His Phe Asn Trp Phe Leu 115 120 125 Cys Phe Leu Tyr Ile Ala Leu Tyr Leu Val Leu Val Glu Phe Met Ile 130 135 140 Tyr Trp Val His Lys Glu Leu His Asp Ile Lys Phe Leu Tyr Lys His 145 150 155 160 Leu His Ala Thr His His Met Tyr Asn Lys Gln Asn Thr Leu Ser Pro 165 170 175 Phe Ala Gly Leu Ala Phe His Pro Leu Asp Gly Ile Leu Gln Ala Ile 180 185 190 Pro His Val Ile Ala Leu Phe Ile Val Pro Ile His Leu Ile Thr His 195 200 205 Leu Ser Leu Leu Phe Leu Glu Gly Ile Trp Thr Ala Ser Ile His Asp 210 215 220 Cys Ile His Gly Asn Ile Trp Pro Ile Met Gly Ala Gly Tyr His Thr 225 230 235 240 Ile His His Thr Thr Tyr Lys His Asn Tyr Gly His Tyr Thr Ile Trp 245 250 255 Met Asp Trp Met Phe Gly Ser Leu Met Val Pro Leu Ala Glu Lys Asp 260 265 270 Ser Phe Lys Glu Lys Glu Lys 275 <210> SEQ ID NO 24 <211> LENGTH: 60 <212> TYPE: PRT <213> ORGANISM: dwf7-2 <400> SEQUENCE: 24 Met Ala Ala Asp Asn Ala Tyr Leu Met Gln Phe Val Asp Glu Thr Ser 1 5 10 15 Phe Tyr Asn Arg Ile Val Leu Ser His Leu Leu Pro Ala Asn Leu Trp 20 25 30 Glu Pro Leu Pro His Phe Leu Gln Thr Trp Leu Arg Asn Tyr Leu Ala 35 40 45 Gly Thr Leu Leu Tyr Phe Ile Ser Gly Phe Leu Trp 50 55 60 <210> SEQ ID NO 25 <211> LENGTH: 230 <212> TYPE: PRT <213> ORGANISM: dwf7-1 <400> SEQUENCE: 25 Met Ala Ala Asp Asn Ala Tyr Leu Met Gln Phe Val Asp Glu Thr Ser 1 5 10 15 Phe Tyr Asn Arg Ile Val Leu Ser His Leu Leu Pro Ala Asn Leu Trp 20 25 30 Glu Pro Leu Pro His Phe Leu Gln Thr Trp Leu Arg Asn Tyr Leu Ala 35 40 45 Gly Thr Leu Leu Tyr Phe Ile Ser Gly Phe Leu Trp Cys Phe Tyr Ile 50 55 60 Tyr Tyr Leu Lys Ile Asn Val Tyr Leu Pro Lys Asp Ala Ile Pro Thr 65 70 75 80 Ile Lys Ala Met Arg Leu Gln Met Phe Val Ala Met Lys Ala Met Pro 85 90 95 Trp Tyr Thr Leu Leu Pro Thr Val Ser Glu Ser Met Ile Glu Arg Gly 100 105 110 Trp Thr Lys Cys Phe Ala Ser Ile Asp Glu Phe Gly Trp Ile Leu Tyr 115 120 125 Phe Val Tyr Ile Ala Ile Tyr Leu Val Phe Val Glu Phe Gly Ile Tyr 130 135 140 Trp Met His Arg Glu Leu His Asp Ile Lys Pro Leu Tyr Lys Tyr Leu 145 150 155 160 His Ala Thr His His Ile Tyr Asn Lys Gln Asn Thr Leu Ser Pro Phe 165 170 175 Ala Gly Leu Ala Phe His Pro Val Asp Gly Ile Leu Gln Ala Val Pro 180 185 190 His Val Ile Ala Leu Phe Ile Val Pro Ile His Phe Thr Thr His Ile 195 200 205 Gly Leu Leu Phe Met Glu Ala Ile Trp Thr Ala Asn Ile His Asp Cys 210 215 220 Ile His Gly Asn Ile Trp 225 230
Claims (35)
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US09/775,879 US20020068822A1 (en) | 2000-02-02 | 2001-02-02 | Dwf7 mutants |
US10/736,318 US7183459B2 (en) | 2000-02-02 | 2003-12-15 | Dwf7 mutants |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US17990100P | 2000-02-02 | 2000-02-02 | |
US09/775,879 US20020068822A1 (en) | 2000-02-02 | 2001-02-02 | Dwf7 mutants |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US10/736,318 Division US7183459B2 (en) | 2000-02-02 | 2003-12-15 | Dwf7 mutants |
Publications (1)
Publication Number | Publication Date |
---|---|
US20020068822A1 true US20020068822A1 (en) | 2002-06-06 |
Family
ID=26875796
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US09/775,879 Abandoned US20020068822A1 (en) | 2000-02-02 | 2001-02-02 | Dwf7 mutants |
US10/736,318 Expired - Fee Related US7183459B2 (en) | 2000-02-02 | 2003-12-15 | Dwf7 mutants |
Family Applications After (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US10/736,318 Expired - Fee Related US7183459B2 (en) | 2000-02-02 | 2003-12-15 | Dwf7 mutants |
Country Status (1)
Country | Link |
---|---|
US (2) | US20020068822A1 (en) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1669443A1 (en) * | 2003-09-12 | 2006-06-14 | Riken | Plant dwarfing gene |
CN113201551A (en) * | 2021-05-26 | 2021-08-03 | 云南中烟工业有限责任公司 | Tobacco delta 7-sterol C5(6) -desaturase gene and application thereof |
CN117568290A (en) * | 2023-04-28 | 2024-02-20 | 江西农业大学 | Dp7-DR protein related to dioscin synthesis, gene, application and method |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8326977B2 (en) * | 2008-07-16 | 2012-12-04 | Fujitsu Limited | Recording medium storing system analyzing program, system analyzing apparatus, and system analyzing method |
CN106093303B (en) * | 2016-06-08 | 2018-05-08 | 中国农业科学院烟草研究所 | The screening technique of tabacum sterol mutant |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB9109063D0 (en) | 1991-04-26 | 1991-06-12 | Ici Plc | Modification of lignin synthesis in plants |
-
2001
- 2001-02-02 US US09/775,879 patent/US20020068822A1/en not_active Abandoned
-
2003
- 2003-12-15 US US10/736,318 patent/US7183459B2/en not_active Expired - Fee Related
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1669443A1 (en) * | 2003-09-12 | 2006-06-14 | Riken | Plant dwarfing gene |
EP1669443A4 (en) * | 2003-09-12 | 2007-08-22 | Riken | Plant dwarfing gene |
CN113201551A (en) * | 2021-05-26 | 2021-08-03 | 云南中烟工业有限责任公司 | Tobacco delta 7-sterol C5(6) -desaturase gene and application thereof |
CN117568290A (en) * | 2023-04-28 | 2024-02-20 | 江西农业大学 | Dp7-DR protein related to dioscin synthesis, gene, application and method |
Also Published As
Publication number | Publication date |
---|---|
US20040133948A1 (en) | 2004-07-08 |
US7183459B2 (en) | 2007-02-27 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Choe et al. | The Arabidopsis dwf7/ste1 mutant is defective in the Δ7 sterol C-5 desaturation step leading to brassinosteroid biosynthesis | |
Hong et al. | The rice brassinosteroid-deficient dwarf2 mutant, defective in the rice homolog of Arabidopsis DIMINUTO/DWARF1, is rescued by the endogenously accumulated alternative bioactive brassinosteroid, dolichosterone | |
AU726846B2 (en) | Nucleic acid molecules encoding cytochrome p450-type proteins involved in the brassinosteroid synthesis in plants | |
US7935532B2 (en) | DWF4 polynucleotides, polypeptides and uses thereof | |
US20070101457A1 (en) | Methods of controlling reproduction in plants | |
JP3183407B2 (en) | Plants with a modified response to ethylene | |
US20020120111A1 (en) | Dwf5 mutants | |
EP1398382A1 (en) | Sd1 gene involved in plant semidwarfing and uses thereof | |
US7183459B2 (en) | Dwf7 mutants | |
US20080222754A1 (en) | Methods of Modulating Glucosinolate Production in Plants | |
WO2000047715A9 (en) | Dwf4 polynucleotides, polypeptides and uses thereof | |
US20100281580A1 (en) | Use of a gene encoding a histidine protein kinase to create drought resistant plants | |
US20090025100A1 (en) | DWF12 and Mutants Thereof | |
AU720590B2 (en) | Novel plant steroid 5alpha reductase, DET2 | |
US20020170093A1 (en) | Nucleic acid molecules associated with plant cell proliferation and growth and uses thereof | |
WO1997030581A1 (en) | Male-sterile plants | |
WO2000016609A1 (en) | Novel method of regulating seed development in plants and genetic sequences therefor | |
US20070130640A1 (en) | Methods of modulating auxin production in plants | |
Choe | Genetics and biology of Arabidopsis brassinosteroid dwarf mutants |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AS | Assignment |
Owner name: ARIZONA BOARD OF REGENTS ON BEHALF OF THE UNIVERSI Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:CHOE, SUNGHWA;FELDMANN, KENNETH A.;REEL/FRAME:012338/0664;SIGNING DATES FROM 20010809 TO 20010813 |
|
STCB | Information on status: application discontinuation |
Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION |
|
AS | Assignment |
Owner name: NATIONAL SCIENCE FOUNDATION, VIRGINIA Free format text: CONFIRMATORY LICENSE;ASSIGNOR:UNIVERSITY OF ARIZONA;REEL/FRAME:054488/0543 Effective date: 20200304 |
|
AS | Assignment |
Owner name: NATIONAL SCIENCE FOUNDATION, VIRGINIA Free format text: CONFIRMATORY LICENSE;ASSIGNOR:UNIVERSITY OF ARIZONA;REEL/FRAME:063601/0099 Effective date: 20200304 |