CN114854702A - Herbicide tolerance protein, coding gene and application thereof - Google Patents
Herbicide tolerance protein, coding gene and application thereof Download PDFInfo
- Publication number
- CN114854702A CN114854702A CN202210330228.1A CN202210330228A CN114854702A CN 114854702 A CN114854702 A CN 114854702A CN 202210330228 A CN202210330228 A CN 202210330228A CN 114854702 A CN114854702 A CN 114854702A
- Authority
- CN
- China
- Prior art keywords
- plant
- hppd inhibitor
- inhibitor herbicide
- herbicide
- gene
- 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.)
- Granted
Links
- 239000004009 herbicide Substances 0.000 title claims abstract description 267
- 230000002363 herbicidal effect Effects 0.000 title claims abstract description 221
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 208
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 122
- 239000003112 inhibitor Substances 0.000 claims abstract description 122
- 101100339555 Zymoseptoria tritici HPPD gene Proteins 0.000 claims abstract description 49
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 25
- 150000001413 amino acids Chemical class 0.000 claims abstract description 12
- 108010028143 Dioxygenases Proteins 0.000 claims abstract description 8
- 102000016680 Dioxygenases Human genes 0.000 claims abstract description 8
- 241000196324 Embryophyta Species 0.000 claims description 267
- 239000002773 nucleotide Substances 0.000 claims description 80
- 125000003729 nucleotide group Chemical group 0.000 claims description 80
- 230000014509 gene expression Effects 0.000 claims description 62
- 238000000034 method Methods 0.000 claims description 51
- IYMLUHWAJFXAQP-UHFFFAOYSA-N topramezone Chemical group CC1=C(C(=O)C2=C(N(C)N=C2)O)C=CC(S(C)(=O)=O)=C1C1=NOCC1 IYMLUHWAJFXAQP-UHFFFAOYSA-N 0.000 claims description 40
- 230000000694 effects Effects 0.000 claims description 35
- 241000219194 Arabidopsis Species 0.000 claims description 32
- 239000005562 Glyphosate Substances 0.000 claims description 27
- XDDAORKBJWWYJS-UHFFFAOYSA-N glyphosate Chemical compound OC(=O)CNCP(O)(O)=O XDDAORKBJWWYJS-UHFFFAOYSA-N 0.000 claims description 27
- 229940097068 glyphosate Drugs 0.000 claims description 27
- KPUREKXXPHOJQT-UHFFFAOYSA-N mesotrione Chemical compound [O-][N+](=O)C1=CC(S(=O)(=O)C)=CC=C1C(=O)C1C(=O)CCCC1=O KPUREKXXPHOJQT-UHFFFAOYSA-N 0.000 claims description 27
- 239000005578 Mesotrione Substances 0.000 claims description 26
- ZTTKDUXKVPEXCG-UHFFFAOYSA-N 2-cyano-3-cyclopropyl-1-(2-mesyl-4-trifluoromethylphenyl)propan-1,3-dione Chemical compound CS(=O)(=O)C1=CC(C(F)(F)F)=CC=C1C(=O)C(C#N)C(=O)C1CC1 ZTTKDUXKVPEXCG-UHFFFAOYSA-N 0.000 claims description 23
- 244000062793 Sorghum vulgare Species 0.000 claims description 23
- JEXVQSWXXUJEMA-UHFFFAOYSA-N pyrazol-3-one Chemical compound O=C1C=CN=N1 JEXVQSWXXUJEMA-UHFFFAOYSA-N 0.000 claims description 22
- 239000013598 vector Substances 0.000 claims description 22
- 240000008042 Zea mays Species 0.000 claims description 20
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 20
- 230000002401 inhibitory effect Effects 0.000 claims description 20
- 244000068988 Glycine max Species 0.000 claims description 18
- 240000007594 Oryza sativa Species 0.000 claims description 18
- 235000007164 Oryza sativa Nutrition 0.000 claims description 18
- 235000009566 rice Nutrition 0.000 claims description 18
- 235000017060 Arachis glabrata Nutrition 0.000 claims description 17
- 244000105624 Arachis hypogaea Species 0.000 claims description 17
- 235000010777 Arachis hypogaea Nutrition 0.000 claims description 17
- 235000018262 Arachis monticola Nutrition 0.000 claims description 17
- 235000010469 Glycine max Nutrition 0.000 claims description 17
- 235000020232 peanut Nutrition 0.000 claims description 17
- 235000007319 Avena orientalis Nutrition 0.000 claims description 16
- 229920000742 Cotton Polymers 0.000 claims description 16
- 240000008067 Cucumis sativus Species 0.000 claims description 16
- 235000010799 Cucumis sativus var sativus Nutrition 0.000 claims description 16
- 240000005979 Hordeum vulgare Species 0.000 claims description 16
- 235000007340 Hordeum vulgare Nutrition 0.000 claims description 16
- 235000007688 Lycopersicon esculentum Nutrition 0.000 claims description 16
- 240000003768 Solanum lycopersicum Species 0.000 claims description 16
- 235000002595 Solanum tuberosum Nutrition 0.000 claims description 16
- 244000061456 Solanum tuberosum Species 0.000 claims description 16
- 235000011684 Sorghum saccharatum Nutrition 0.000 claims description 16
- 241000209140 Triticum Species 0.000 claims description 16
- 235000021307 Triticum Nutrition 0.000 claims description 16
- CTAPFRYPJLPFDF-UHFFFAOYSA-N isoxazole Chemical compound C=1C=NOC=1 CTAPFRYPJLPFDF-UHFFFAOYSA-N 0.000 claims description 16
- 235000019713 millet Nutrition 0.000 claims description 16
- 241000209510 Liliopsida Species 0.000 claims description 15
- 241000219823 Medicago Species 0.000 claims description 15
- 235000017587 Medicago sativa ssp. sativa Nutrition 0.000 claims description 15
- 235000002637 Nicotiana tabacum Nutrition 0.000 claims description 15
- 241001233957 eudicotyledons Species 0.000 claims description 15
- 244000075850 Avena orientalis Species 0.000 claims description 14
- 235000007558 Avena sp Nutrition 0.000 claims description 14
- 241000219310 Beta vulgaris subsp. vulgaris Species 0.000 claims description 14
- 241000743776 Brachypodium distachyon Species 0.000 claims description 14
- 235000010523 Cicer arietinum Nutrition 0.000 claims description 14
- 244000045195 Cicer arietinum Species 0.000 claims description 14
- 244000020551 Helianthus annuus Species 0.000 claims description 14
- 235000003222 Helianthus annuus Nutrition 0.000 claims description 14
- 235000021536 Sugar beet Nutrition 0.000 claims description 14
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 14
- 235000005822 corn Nutrition 0.000 claims description 14
- 230000001276 controlling effect Effects 0.000 claims description 11
- 230000006378 damage Effects 0.000 claims description 11
- 230000012010 growth Effects 0.000 claims description 9
- IAJOBQBIJHVGMQ-UHFFFAOYSA-N 2-amino-4-[hydroxy(methyl)phosphoryl]butanoic acid Chemical compound CP(O)(=O)CCC(N)C(O)=O IAJOBQBIJHVGMQ-UHFFFAOYSA-N 0.000 claims description 8
- 108010020183 3-phosphoshikimate 1-carboxyvinyltransferase Proteins 0.000 claims description 8
- 239000005561 Glufosinate Substances 0.000 claims description 7
- 230000001131 transforming effect Effects 0.000 claims description 7
- 230000002829 reductive effect Effects 0.000 claims description 6
- 230000001105 regulatory effect Effects 0.000 claims description 6
- 230000002068 genetic effect Effects 0.000 claims description 5
- 238000010362 genome editing Methods 0.000 claims description 5
- 108010000700 Acetolactate synthase Proteins 0.000 claims description 4
- 206010064571 Gene mutation Diseases 0.000 claims description 4
- 108020001991 Protoporphyrinogen Oxidase Proteins 0.000 claims description 4
- 238000012258 culturing Methods 0.000 claims description 4
- 238000003306 harvesting Methods 0.000 claims description 4
- 238000004519 manufacturing process Methods 0.000 claims description 4
- 238000011426 transformation method Methods 0.000 claims description 4
- 102000005135 Protoporphyrinogen oxidase Human genes 0.000 claims description 3
- 230000008654 plant damage Effects 0.000 claims description 3
- 108010016119 Alpha-Ketoglutarate-Dependent Dioxygenase FTO Proteins 0.000 claims description 2
- 102000000383 Alpha-Ketoglutarate-Dependent Dioxygenase FTO Human genes 0.000 claims description 2
- 108030006517 Glyphosate oxidoreductases Proteins 0.000 claims description 2
- 101000951943 Stenotrophomonas maltophilia Dicamba O-demethylase, oxygenase component Proteins 0.000 claims description 2
- 108020002494 acetyltransferase Proteins 0.000 claims description 2
- 102000005421 acetyltransferase Human genes 0.000 claims description 2
- 108010039239 glyphosate N-acetyltransferase Proteins 0.000 claims description 2
- 230000002147 killing effect Effects 0.000 claims description 2
- 244000299507 Gossypium hirsutum Species 0.000 claims 7
- 244000061176 Nicotiana tabacum Species 0.000 claims 7
- 102000018832 Cytochromes Human genes 0.000 claims 1
- 108010052832 Cytochromes Proteins 0.000 claims 1
- 235000018102 proteins Nutrition 0.000 description 101
- 239000013604 expression vector Substances 0.000 description 43
- 210000004027 cell Anatomy 0.000 description 36
- 108020004414 DNA Proteins 0.000 description 35
- 102000040430 polynucleotide Human genes 0.000 description 35
- 108091033319 polynucleotide Proteins 0.000 description 35
- 239000002157 polynucleotide Substances 0.000 description 35
- 238000003259 recombinant expression Methods 0.000 description 34
- 150000007523 nucleic acids Chemical class 0.000 description 29
- 102000039446 nucleic acids Human genes 0.000 description 26
- 108020004707 nucleic acids Proteins 0.000 description 26
- 102000004190 Enzymes Human genes 0.000 description 25
- 108090000790 Enzymes Proteins 0.000 description 25
- 241000219195 Arabidopsis thaliana Species 0.000 description 24
- 230000009466 transformation Effects 0.000 description 20
- IGMNYECMUMZDDF-UHFFFAOYSA-N homogentisic acid Chemical compound OC(=O)CC1=CC(O)=CC=C1O IGMNYECMUMZDDF-UHFFFAOYSA-N 0.000 description 18
- 239000000203 mixture Substances 0.000 description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 16
- 239000000047 product Substances 0.000 description 15
- 239000000243 solution Substances 0.000 description 15
- 238000006243 chemical reaction Methods 0.000 description 14
- 230000009261 transgenic effect Effects 0.000 description 14
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 12
- 235000001014 amino acid Nutrition 0.000 description 12
- 239000003623 enhancer Substances 0.000 description 12
- 239000013612 plasmid Substances 0.000 description 12
- 108090000765 processed proteins & peptides Proteins 0.000 description 12
- 210000001519 tissue Anatomy 0.000 description 12
- 241000701489 Cauliflower mosaic virus Species 0.000 description 11
- 108091028043 Nucleic acid sequence Proteins 0.000 description 10
- 229940024606 amino acid Drugs 0.000 description 10
- 241000219146 Gossypium Species 0.000 description 9
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 9
- 240000006394 Sorghum bicolor Species 0.000 description 9
- 239000002609 medium Substances 0.000 description 9
- 239000000126 substance Substances 0.000 description 9
- 101150046173 HP1 gene Proteins 0.000 description 8
- 241000208125 Nicotiana Species 0.000 description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- 230000006870 function Effects 0.000 description 8
- 238000003752 polymerase chain reaction Methods 0.000 description 8
- 102000004196 processed proteins & peptides Human genes 0.000 description 8
- 229960000268 spectinomycin Drugs 0.000 description 8
- UNFWWIHTNXNPBV-WXKVUWSESA-N spectinomycin Chemical compound O([C@@H]1[C@@H](NC)[C@@H](O)[C@H]([C@@H]([C@H]1O1)O)NC)[C@]2(O)[C@H]1O[C@H](C)CC2=O UNFWWIHTNXNPBV-WXKVUWSESA-N 0.000 description 8
- 238000006467 substitution reaction Methods 0.000 description 8
- 108091026890 Coding region Proteins 0.000 description 7
- 241000588724 Escherichia coli Species 0.000 description 7
- 230000009471 action Effects 0.000 description 7
- 230000015572 biosynthetic process Effects 0.000 description 7
- 239000007788 liquid Substances 0.000 description 7
- 229920001184 polypeptide Polymers 0.000 description 7
- 230000002087 whitening effect Effects 0.000 description 7
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 6
- 241000589158 Agrobacterium Species 0.000 description 6
- 240000004713 Pisum sativum Species 0.000 description 6
- 235000010582 Pisum sativum Nutrition 0.000 description 6
- 108010076504 Protein Sorting Signals Proteins 0.000 description 6
- 235000016383 Zea mays subsp huehuetenangensis Nutrition 0.000 description 6
- 108010050848 glycylleucine Proteins 0.000 description 6
- -1 iron ions Chemical class 0.000 description 6
- 235000009973 maize Nutrition 0.000 description 6
- 230000008488 polyadenylation Effects 0.000 description 6
- 230000006798 recombination Effects 0.000 description 6
- 238000005215 recombination Methods 0.000 description 6
- 238000012163 sequencing technique Methods 0.000 description 6
- 241000894007 species Species 0.000 description 6
- KKADPXVIOXHVKN-UHFFFAOYSA-N 4-hydroxyphenylpyruvic acid Chemical compound OC(=O)C(=O)CC1=CC=C(O)C=C1 KKADPXVIOXHVKN-UHFFFAOYSA-N 0.000 description 5
- 241000701484 Figwort mosaic virus Species 0.000 description 5
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 5
- FKUYMLZIRPABFK-UHFFFAOYSA-N Plastoquinone 9 Natural products CC(C)=CCCC(C)=CCCC(C)=CCCC(C)=CCCC(C)=CCCC(C)=CCCC(C)=CCCC(C)=CCCC(C)=CCC1=CC(=O)C(C)=C(C)C1=O FKUYMLZIRPABFK-UHFFFAOYSA-N 0.000 description 5
- 241000700605 Viruses Species 0.000 description 5
- 206010052428 Wound Diseases 0.000 description 5
- 208000027418 Wounds and injury Diseases 0.000 description 5
- 125000000539 amino acid group Chemical group 0.000 description 5
- 238000012217 deletion Methods 0.000 description 5
- 230000037430 deletion Effects 0.000 description 5
- 238000001976 enzyme digestion Methods 0.000 description 5
- 229930027917 kanamycin Natural products 0.000 description 5
- 229960000318 kanamycin Drugs 0.000 description 5
- 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 5
- 229930182823 kanamycin A Natural products 0.000 description 5
- GQYBCIHRWMPOOF-UHFFFAOYSA-N p-hydroxyphenylpyruvic acid Natural products OC(=O)C(O)=CC1=CC=C(O)C=C1 GQYBCIHRWMPOOF-UHFFFAOYSA-N 0.000 description 5
- 101150113864 pat gene Proteins 0.000 description 5
- FKUYMLZIRPABFK-IQSNHBBHSA-N plastoquinone-9 Chemical compound CC(C)=CCC\C(C)=C\CC\C(C)=C\CC\C(C)=C\CC\C(C)=C\CC\C(C)=C\CC\C(C)=C\CC\C(C)=C\CC\C(C)=C\CC1=CC(=O)C(C)=C(C)C1=O FKUYMLZIRPABFK-IQSNHBBHSA-N 0.000 description 5
- 230000035939 shock Effects 0.000 description 5
- 239000002689 soil Substances 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- 238000011282 treatment Methods 0.000 description 5
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 108020004705 Codon Proteins 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 108091092195 Intron Proteins 0.000 description 4
- 108090000848 Ubiquitin Proteins 0.000 description 4
- 102000044159 Ubiquitin Human genes 0.000 description 4
- 238000007792 addition Methods 0.000 description 4
- 229940041514 candida albicans extract Drugs 0.000 description 4
- 235000021466 carotenoid Nutrition 0.000 description 4
- 150000001747 carotenoids Chemical class 0.000 description 4
- 108010031100 chloroplast transit peptides Proteins 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 238000009396 hybridization Methods 0.000 description 4
- 230000001976 improved effect Effects 0.000 description 4
- 108010058731 nopaline synthase Proteins 0.000 description 4
- 229920000642 polymer Polymers 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 230000002441 reversible effect Effects 0.000 description 4
- JQXXHWHPUNPDRT-WLSIYKJHSA-N rifampicin Chemical compound O([C@](C1=O)(C)O/C=C/[C@@H]([C@H]([C@@H](OC(C)=O)[C@H](C)[C@H](O)[C@H](C)[C@@H](O)[C@@H](C)\C=C\C=C(C)/C(=O)NC=2C(O)=C3C([O-])=C4C)C)OC)C4=C1C3=C(O)C=2\C=N\N1CC[NH+](C)CC1 JQXXHWHPUNPDRT-WLSIYKJHSA-N 0.000 description 4
- 229960001225 rifampicin Drugs 0.000 description 4
- 150000003839 salts Chemical class 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 230000008685 targeting Effects 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 238000013518 transcription Methods 0.000 description 4
- 230000035897 transcription Effects 0.000 description 4
- 239000012137 tryptone Substances 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- 239000012138 yeast extract Substances 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- HHGYNJRJIINWAK-FXQIFTODSA-N Ala-Ala-Arg Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N HHGYNJRJIINWAK-FXQIFTODSA-N 0.000 description 3
- WQVFQXXBNHHPLX-ZKWXMUAHSA-N Ala-Ala-His Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](Cc1cnc[nH]1)C(O)=O WQVFQXXBNHHPLX-ZKWXMUAHSA-N 0.000 description 3
- CVGNCMIULZNYES-WHFBIAKZSA-N Ala-Asn-Gly Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(O)=O CVGNCMIULZNYES-WHFBIAKZSA-N 0.000 description 3
- BTYTYHBSJKQBQA-GCJQMDKQSA-N Ala-Asp-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](C)N)O BTYTYHBSJKQBQA-GCJQMDKQSA-N 0.000 description 3
- MVBWLRJESQOQTM-ACZMJKKPSA-N Ala-Gln-Ser Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(O)=O MVBWLRJESQOQTM-ACZMJKKPSA-N 0.000 description 3
- FBHOPGDGELNWRH-DRZSPHRISA-N Ala-Glu-Phe Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O FBHOPGDGELNWRH-DRZSPHRISA-N 0.000 description 3
- BEMGNWZECGIJOI-WDSKDSINSA-N Ala-Gly-Glu Chemical compound [H]N[C@@H](C)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(O)=O BEMGNWZECGIJOI-WDSKDSINSA-N 0.000 description 3
- OYJCVIGKMXUVKB-GARJFASQSA-N Ala-Leu-Pro Chemical compound C[C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N1CCC[C@@H]1C(=O)O)N OYJCVIGKMXUVKB-GARJFASQSA-N 0.000 description 3
- MEFILNJXAVSUTO-JXUBOQSCSA-N Ala-Leu-Thr Chemical compound C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O MEFILNJXAVSUTO-JXUBOQSCSA-N 0.000 description 3
- XUCHENWTTBFODJ-FXQIFTODSA-N Ala-Met-Ala Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](C)C(O)=O XUCHENWTTBFODJ-FXQIFTODSA-N 0.000 description 3
- IHRGVZXPTIQNIP-NAKRPEOUSA-N Ala-Met-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CCSC)NC(=O)[C@H](C)N IHRGVZXPTIQNIP-NAKRPEOUSA-N 0.000 description 3
- FQNILRVJOJBFFC-FXQIFTODSA-N Ala-Pro-Asp Chemical compound C[C@@H](C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(=O)O)C(=O)O)N FQNILRVJOJBFFC-FXQIFTODSA-N 0.000 description 3
- YNOCMHZSWJMGBB-GCJQMDKQSA-N Ala-Thr-Asp Chemical compound [H]N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(O)=O)C(O)=O YNOCMHZSWJMGBB-GCJQMDKQSA-N 0.000 description 3
- DFCIPNHFKOQAME-FXQIFTODSA-N Arg-Ala-Asn Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(N)=O)C(O)=O DFCIPNHFKOQAME-FXQIFTODSA-N 0.000 description 3
- LLZXKVAAEWBUPB-KKUMJFAQSA-N Arg-Gln-Phe Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O LLZXKVAAEWBUPB-KKUMJFAQSA-N 0.000 description 3
- YNSGXDWWPCGGQS-YUMQZZPRSA-N Arg-Gly-Gln Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CCC(N)=O)C(O)=O YNSGXDWWPCGGQS-YUMQZZPRSA-N 0.000 description 3
- OQCWXQJLCDPRHV-UWVGGRQHSA-N Arg-Gly-Leu Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CC(C)C)C(O)=O OQCWXQJLCDPRHV-UWVGGRQHSA-N 0.000 description 3
- NKNILFJYKKHBKE-WPRPVWTQSA-N Arg-Gly-Val Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](C(C)C)C(O)=O NKNILFJYKKHBKE-WPRPVWTQSA-N 0.000 description 3
- MJINRRBEMOLJAK-DCAQKATOSA-N Arg-Lys-Asp Chemical compound OC(=O)C[C@@H](C(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CCCN=C(N)N MJINRRBEMOLJAK-DCAQKATOSA-N 0.000 description 3
- NGTYEHIRESTSRX-UWVGGRQHSA-N Arg-Lys-Gly Chemical compound NCCCC[C@@H](C(=O)NCC(O)=O)NC(=O)[C@@H](N)CCCN=C(N)N NGTYEHIRESTSRX-UWVGGRQHSA-N 0.000 description 3
- MEFGKQUUYZOLHM-GMOBBJLQSA-N Asn-Arg-Ile Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O MEFGKQUUYZOLHM-GMOBBJLQSA-N 0.000 description 3
- GNKVBRYFXYWXAB-WDSKDSINSA-N Asn-Glu-Gly Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(O)=O GNKVBRYFXYWXAB-WDSKDSINSA-N 0.000 description 3
- UBKOVSLDWIHYSY-ACZMJKKPSA-N Asn-Glu-Ser Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(O)=O UBKOVSLDWIHYSY-ACZMJKKPSA-N 0.000 description 3
- BYLSYQASFJJBCL-DCAQKATOSA-N Asn-Pro-Leu Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(C)C)C(O)=O BYLSYQASFJJBCL-DCAQKATOSA-N 0.000 description 3
- JWQWPRCDYWNVNM-ACZMJKKPSA-N Asn-Ser-Gln Chemical compound C(CC(=O)N)[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(=O)N)N JWQWPRCDYWNVNM-ACZMJKKPSA-N 0.000 description 3
- XLDMSQYOYXINSZ-QXEWZRGKSA-N Asn-Val-Arg Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)NC(=O)[C@H](CC(=O)N)N XLDMSQYOYXINSZ-QXEWZRGKSA-N 0.000 description 3
- IJHUZMGJRGNXIW-CIUDSAMLSA-N Asp-Glu-Arg Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O IJHUZMGJRGNXIW-CIUDSAMLSA-N 0.000 description 3
- VFUXXFVCYZPOQG-WDSKDSINSA-N Asp-Glu-Gly Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(O)=O VFUXXFVCYZPOQG-WDSKDSINSA-N 0.000 description 3
- QNMKWNONJGKJJC-NHCYSSNCSA-N Asp-Leu-Val Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(O)=O QNMKWNONJGKJJC-NHCYSSNCSA-N 0.000 description 3
- KPSHWSWFPUDEGF-FXQIFTODSA-N Asp-Pro-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)CC(O)=O KPSHWSWFPUDEGF-FXQIFTODSA-N 0.000 description 3
- BRRPVTUFESPTCP-ACZMJKKPSA-N Asp-Ser-Glu Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCC(O)=O BRRPVTUFESPTCP-ACZMJKKPSA-N 0.000 description 3
- XQFLFQWOBXPMHW-NHCYSSNCSA-N Asp-Val-His Chemical compound N[C@@H](CC(=O)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC1=CNC=N1)C(=O)O XQFLFQWOBXPMHW-NHCYSSNCSA-N 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 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 3
- ULXXDWZMMSQBDC-ACZMJKKPSA-N Gln-Asp-Asp Chemical compound C(CC(=O)N)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CC(=O)O)C(=O)O)N ULXXDWZMMSQBDC-ACZMJKKPSA-N 0.000 description 3
- IKFZXRLDMYWNBU-YUMQZZPRSA-N Gln-Gly-Arg Chemical compound NC(=O)CC[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCCN=C(N)N IKFZXRLDMYWNBU-YUMQZZPRSA-N 0.000 description 3
- IWUFOVSLWADEJC-AVGNSLFASA-N Gln-His-Leu Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC(C)C)C(O)=O IWUFOVSLWADEJC-AVGNSLFASA-N 0.000 description 3
- CAXXTYYGFYTBPV-IUCAKERBSA-N Gln-Leu-Gly Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)NCC(O)=O CAXXTYYGFYTBPV-IUCAKERBSA-N 0.000 description 3
- ILKYYKRAULNYMS-JYJNAYRXSA-N Gln-Lys-Phe Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O ILKYYKRAULNYMS-JYJNAYRXSA-N 0.000 description 3
- OSCLNNWLKKIQJM-WDSKDSINSA-N Gln-Ser-Gly Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)NCC(O)=O OSCLNNWLKKIQJM-WDSKDSINSA-N 0.000 description 3
- VEYGCDYMOXHJLS-GVXVVHGQSA-N Gln-Val-Leu Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O VEYGCDYMOXHJLS-GVXVVHGQSA-N 0.000 description 3
- OXEMJGCAJFFREE-FXQIFTODSA-N Glu-Gln-Ala Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C)C(O)=O OXEMJGCAJFFREE-FXQIFTODSA-N 0.000 description 3
- 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 3
- ZGXGVBYEJGVJMV-HJGDQZAQSA-N Glu-Thr-Met Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCSC)C(=O)O)NC(=O)[C@H](CCC(=O)O)N)O ZGXGVBYEJGVJMV-HJGDQZAQSA-N 0.000 description 3
- JVACNFOPSUPDTK-QWRGUYRKSA-N Gly-Asn-Phe Chemical compound NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 JVACNFOPSUPDTK-QWRGUYRKSA-N 0.000 description 3
- FIQQRCFQXGLOSZ-WDSKDSINSA-N Gly-Glu-Asp Chemical compound [H]NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O FIQQRCFQXGLOSZ-WDSKDSINSA-N 0.000 description 3
- YTSVAIMKVLZUDU-YUMQZZPRSA-N Gly-Leu-Asp Chemical compound [H]NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O YTSVAIMKVLZUDU-YUMQZZPRSA-N 0.000 description 3
- LIXWIUAORXJNBH-QWRGUYRKSA-N Gly-Leu-His Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)CN LIXWIUAORXJNBH-QWRGUYRKSA-N 0.000 description 3
- WMGHDYWNHNLGBV-ONGXEEELSA-N Gly-Phe-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)CN)CC1=CC=CC=C1 WMGHDYWNHNLGBV-ONGXEEELSA-N 0.000 description 3
- SSFWXSNOKDZNHY-QXEWZRGKSA-N Gly-Pro-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)CN SSFWXSNOKDZNHY-QXEWZRGKSA-N 0.000 description 3
- HAOUOFNNJJLVNS-BQBZGAKWSA-N Gly-Pro-Ser Chemical compound NCC(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(O)=O HAOUOFNNJJLVNS-BQBZGAKWSA-N 0.000 description 3
- 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 3
- JBCLFWXMTIKCCB-UHFFFAOYSA-N H-Gly-Phe-OH Natural products NCC(=O)NC(C(O)=O)CC1=CC=CC=C1 JBCLFWXMTIKCCB-UHFFFAOYSA-N 0.000 description 3
- 241000238631 Hexapoda Species 0.000 description 3
- HQKADFMLECZIQJ-HVTMNAMFSA-N His-Glu-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CC1=CN=CN1)N HQKADFMLECZIQJ-HVTMNAMFSA-N 0.000 description 3
- DPTBVFUDCPINIP-JURCDPSOSA-N Ile-Ala-Phe Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 DPTBVFUDCPINIP-JURCDPSOSA-N 0.000 description 3
- FVEWRQXNISSYFO-ZPFDUUQYSA-N Ile-Arg-Glu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N FVEWRQXNISSYFO-ZPFDUUQYSA-N 0.000 description 3
- NKRJALPCDNXULF-BYULHYEWSA-N Ile-Asp-Gly Chemical compound [H]N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(O)=O NKRJALPCDNXULF-BYULHYEWSA-N 0.000 description 3
- PHIXPNQDGGILMP-YVNDNENWSA-N Ile-Glu-Glu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N PHIXPNQDGGILMP-YVNDNENWSA-N 0.000 description 3
- MTFVYKQRLXYAQN-LAEOZQHASA-N Ile-Glu-Gly Chemical compound [H]N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(O)=O MTFVYKQRLXYAQN-LAEOZQHASA-N 0.000 description 3
- KFVUBLZRFSVDGO-BYULHYEWSA-N Ile-Gly-Asp Chemical compound CC[C@H](C)[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CC(O)=O KFVUBLZRFSVDGO-BYULHYEWSA-N 0.000 description 3
- RIVKTKFVWXRNSJ-GRLWGSQLSA-N Ile-Ile-Gln Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N RIVKTKFVWXRNSJ-GRLWGSQLSA-N 0.000 description 3
- TWYOYAKMLHWMOJ-ZPFDUUQYSA-N Ile-Leu-Asn Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O TWYOYAKMLHWMOJ-ZPFDUUQYSA-N 0.000 description 3
- VEPIBPGLTLPBDW-URLPEUOOSA-N Ile-Phe-Thr Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)O)C(=O)O)N VEPIBPGLTLPBDW-URLPEUOOSA-N 0.000 description 3
- IVXJIMGDOYRLQU-XUXIUFHCSA-N Ile-Pro-Leu Chemical compound CC[C@H](C)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(C)C)C(O)=O IVXJIMGDOYRLQU-XUXIUFHCSA-N 0.000 description 3
- 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 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
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-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
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 3
- 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 3
- LJHGALIOHLRRQN-DCAQKATOSA-N Leu-Ala-Arg Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N LJHGALIOHLRRQN-DCAQKATOSA-N 0.000 description 3
- YOZCKMXHBYKOMQ-IHRRRGAJSA-N Leu-Arg-Lys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCCCN)C(=O)O)N YOZCKMXHBYKOMQ-IHRRRGAJSA-N 0.000 description 3
- ZDSNOSQHMJBRQN-SRVKXCTJSA-N Leu-Asp-His Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N ZDSNOSQHMJBRQN-SRVKXCTJSA-N 0.000 description 3
- JNDYEOUZBLOVOF-AVGNSLFASA-N Leu-Leu-Gln Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O JNDYEOUZBLOVOF-AVGNSLFASA-N 0.000 description 3
- 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 3
- UCRJTSIIAYHOHE-ULQDDVLXSA-N Leu-Tyr-Arg Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)N UCRJTSIIAYHOHE-ULQDDVLXSA-N 0.000 description 3
- XNKDCYABMBBEKN-IUCAKERBSA-N Lys-Gly-Gln Chemical compound NCCCC[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCC(N)=O XNKDCYABMBBEKN-IUCAKERBSA-N 0.000 description 3
- 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 3
- MDXAULHWGWETHF-SRVKXCTJSA-N Met-Arg-Val Chemical compound CSCC[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](C(C)C)C(O)=O)CCCNC(N)=N MDXAULHWGWETHF-SRVKXCTJSA-N 0.000 description 3
- 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 3
- 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 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
- 108010079364 N-glycylalanine Proteins 0.000 description 3
- 108010002311 N-glycylglutamic acid Proteins 0.000 description 3
- 108010066427 N-valyltryptophan Proteins 0.000 description 3
- 108700026244 Open Reading Frames Proteins 0.000 description 3
- 108010068204 Peptide Elongation Factors Proteins 0.000 description 3
- LSXGADJXBDFXQU-DLOVCJGASA-N Phe-Ala-Asp Chemical compound OC(=O)C[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC1=CC=CC=C1 LSXGADJXBDFXQU-DLOVCJGASA-N 0.000 description 3
- 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 3
- KYYMILWEGJYPQZ-IHRRRGAJSA-N Phe-Glu-Glu Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)CC1=CC=CC=C1 KYYMILWEGJYPQZ-IHRRRGAJSA-N 0.000 description 3
- WPTYDQPGBMDUBI-QWRGUYRKSA-N Phe-Gly-Asn Chemical compound N[C@@H](Cc1ccccc1)C(=O)NCC(=O)N[C@@H](CC(N)=O)C(O)=O WPTYDQPGBMDUBI-QWRGUYRKSA-N 0.000 description 3
- RYQWALWYQWBUKN-FHWLQOOXSA-N Phe-Phe-Glu Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCC(O)=O)C(O)=O RYQWALWYQWBUKN-FHWLQOOXSA-N 0.000 description 3
- HBXAOEBRGLCLIW-AVGNSLFASA-N Phe-Ser-Gln Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N HBXAOEBRGLCLIW-AVGNSLFASA-N 0.000 description 3
- QTDBZORPVYTRJU-KKXDTOCCSA-N Phe-Tyr-Ala Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](C)C(O)=O QTDBZORPVYTRJU-KKXDTOCCSA-N 0.000 description 3
- VIIRRNQMMIHYHQ-XHSDSOJGSA-N Phe-Val-Pro Chemical compound CC(C)[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CC2=CC=CC=C2)N VIIRRNQMMIHYHQ-XHSDSOJGSA-N 0.000 description 3
- GDXZRWYXJSGWIV-GMOBBJLQSA-N Pro-Asp-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@@H]1CCCN1 GDXZRWYXJSGWIV-GMOBBJLQSA-N 0.000 description 3
- WSRWHZRUOCACLJ-UWVGGRQHSA-N Pro-Gly-His Chemical compound C([C@@H](C(=O)O)NC(=O)CNC(=O)[C@H]1NCCC1)C1=CN=CN1 WSRWHZRUOCACLJ-UWVGGRQHSA-N 0.000 description 3
- 240000000111 Saccharum officinarum Species 0.000 description 3
- 235000007201 Saccharum officinarum Nutrition 0.000 description 3
- SRTCFKGBYBZRHA-ACZMJKKPSA-N Ser-Ala-Glu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(O)=O SRTCFKGBYBZRHA-ACZMJKKPSA-N 0.000 description 3
- IDQFQFVEWMWRQQ-DLOVCJGASA-N Ser-Ala-Phe Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O IDQFQFVEWMWRQQ-DLOVCJGASA-N 0.000 description 3
- GHPQVUYZQQGEDA-BIIVOSGPSA-N Ser-Asp-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC(=O)O)NC(=O)[C@H](CO)N)C(=O)O GHPQVUYZQQGEDA-BIIVOSGPSA-N 0.000 description 3
- 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 3
- 108091081024 Start codon Proteins 0.000 description 3
- QQWNRERCGGZOKG-WEDXCCLWSA-N Thr-Gly-Leu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CC(C)C)C(O)=O QQWNRERCGGZOKG-WEDXCCLWSA-N 0.000 description 3
- CRZNCABIJLRFKZ-IUKAMOBKSA-N Thr-Ile-Asp Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)O)NC(=O)[C@H]([C@@H](C)O)N CRZNCABIJLRFKZ-IUKAMOBKSA-N 0.000 description 3
- VUXIQSUQQYNLJP-XAVMHZPKSA-N Thr-Ser-Pro Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CO)C(=O)N1CCC[C@@H]1C(=O)O)N)O VUXIQSUQQYNLJP-XAVMHZPKSA-N 0.000 description 3
- LXXCHJKHJYRMIY-FQPOAREZSA-N Thr-Tyr-Ala Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](C)C(O)=O LXXCHJKHJYRMIY-FQPOAREZSA-N 0.000 description 3
- YOPQYBJJNSIQGZ-JNPHEJMOSA-N Thr-Tyr-Tyr Chemical compound C([C@H](NC(=O)[C@@H](N)[C@H](O)C)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)C1=CC=C(O)C=C1 YOPQYBJJNSIQGZ-JNPHEJMOSA-N 0.000 description 3
- HYNAKPYFEYJMAS-XIRDDKMYSA-N Trp-Arg-Glu Chemical compound [H]N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(O)=O HYNAKPYFEYJMAS-XIRDDKMYSA-N 0.000 description 3
- FKAPNDWDLDWZNF-QEJZJMRPSA-N Trp-Asp-Glu Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N FKAPNDWDLDWZNF-QEJZJMRPSA-N 0.000 description 3
- UIRPULWLRODAEQ-QEJZJMRPSA-N Trp-Ser-Glu Chemical compound C1=CC=C2C(C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(O)=O)=CNC2=C1 UIRPULWLRODAEQ-QEJZJMRPSA-N 0.000 description 3
- GFHYISDTIWZUSU-QWRGUYRKSA-N Tyr-Asn-Gly Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(O)=O GFHYISDTIWZUSU-QWRGUYRKSA-N 0.000 description 3
- ZRPLVTZTKPPSBT-AVGNSLFASA-N Tyr-Glu-Ser Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(O)=O ZRPLVTZTKPPSBT-AVGNSLFASA-N 0.000 description 3
- CDKZJGMPZHPAJC-ULQDDVLXSA-N Tyr-Leu-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 CDKZJGMPZHPAJC-ULQDDVLXSA-N 0.000 description 3
- LUMQYLVYUIRHHU-YJRXYDGGSA-N Tyr-Ser-Thr Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(O)=O LUMQYLVYUIRHHU-YJRXYDGGSA-N 0.000 description 3
- ZXAGTABZUOMUDO-GVXVVHGQSA-N Val-Glu-Lys Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CCCCN)C(=O)O)N ZXAGTABZUOMUDO-GVXVVHGQSA-N 0.000 description 3
- SJRUJQFQVLMZFW-WPRPVWTQSA-N Val-Pro-Gly Chemical compound CC(C)[C@H](N)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O SJRUJQFQVLMZFW-WPRPVWTQSA-N 0.000 description 3
- 230000009418 agronomic effect Effects 0.000 description 3
- 235000004279 alanine Nutrition 0.000 description 3
- 108010068380 arginylarginine Proteins 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 230000003115 biocidal effect Effects 0.000 description 3
- 230000000295 complement effect Effects 0.000 description 3
- 238000005520 cutting process Methods 0.000 description 3
- 229960001484 edetic acid Drugs 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 230000002255 enzymatic effect Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 210000003608 fece Anatomy 0.000 description 3
- 238000009472 formulation Methods 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 3
- VPZXBVLAVMBEQI-UHFFFAOYSA-N glycyl-DL-alpha-alanine Natural products OC(=O)C(C)NC(=O)CN VPZXBVLAVMBEQI-UHFFFAOYSA-N 0.000 description 3
- 108010077435 glycyl-phenylalanyl-glycine Proteins 0.000 description 3
- 108010081551 glycylphenylalanine Proteins 0.000 description 3
- 244000304962 green bristle grass Species 0.000 description 3
- 108010045383 histidyl-glycyl-glutamic acid Proteins 0.000 description 3
- 108010036413 histidylglycine Proteins 0.000 description 3
- 108010025306 histidylleucine Proteins 0.000 description 3
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Substances [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 3
- 108010044374 isoleucyl-tyrosine Proteins 0.000 description 3
- 101150109249 lacI gene Proteins 0.000 description 3
- 108010051673 leucyl-glycyl-phenylalanine Proteins 0.000 description 3
- 108010012058 leucyltyrosine Proteins 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 229930182817 methionine Natural products 0.000 description 3
- 108010068488 methionylphenylalanine Proteins 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 210000000056 organ Anatomy 0.000 description 3
- 108010018625 phenylalanylarginine Proteins 0.000 description 3
- 108010012581 phenylalanylglutamate Proteins 0.000 description 3
- 108010082527 phosphinothricin N-acetyltransferase Proteins 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 239000000523 sample Substances 0.000 description 3
- 238000002864 sequence alignment Methods 0.000 description 3
- 239000007921 spray Substances 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 230000014621 translational initiation Effects 0.000 description 3
- 239000010455 vermiculite Substances 0.000 description 3
- 229910052902 vermiculite Inorganic materials 0.000 description 3
- 235000019354 vermiculite Nutrition 0.000 description 3
- LUBJCRLGQSPQNN-UHFFFAOYSA-N 1-Phenylurea Chemical compound NC(=O)NC1=CC=CC=C1 LUBJCRLGQSPQNN-UHFFFAOYSA-N 0.000 description 2
- YVLPJIGOMTXXLP-UHFFFAOYSA-N 15-cis-phytoene Chemical compound CC(C)=CCCC(C)=CCCC(C)=CCCC(C)=CC=CC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)C YVLPJIGOMTXXLP-UHFFFAOYSA-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
- 102100028626 4-hydroxyphenylpyruvate dioxygenase Human genes 0.000 description 2
- 241000589155 Agrobacterium tumefaciens Species 0.000 description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- 241000209761 Avena Species 0.000 description 2
- 235000000832 Ayote Nutrition 0.000 description 2
- 240000002791 Brassica napus Species 0.000 description 2
- 235000011293 Brassica napus Nutrition 0.000 description 2
- 244000025254 Cannabis sativa Species 0.000 description 2
- 241001515826 Cassava vein mosaic virus Species 0.000 description 2
- 241000233838 Commelina Species 0.000 description 2
- 240000004244 Cucurbita moschata Species 0.000 description 2
- 235000009854 Cucurbita moschata Nutrition 0.000 description 2
- 235000009804 Cucurbita pepo subsp pepo Nutrition 0.000 description 2
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 description 2
- 239000005504 Dicamba Substances 0.000 description 2
- 244000152970 Digitaria sanguinalis Species 0.000 description 2
- 235000010823 Digitaria sanguinalis Nutrition 0.000 description 2
- 244000025670 Eleusine indica Species 0.000 description 2
- 235000014716 Eleusine indica Nutrition 0.000 description 2
- 241000710188 Encephalomyocarditis virus Species 0.000 description 2
- 241001198387 Escherichia coli BL21(DE3) Species 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 208000030036 Hypertelorism-preauricular sinus-punctual pits-deafness syndrome Diseases 0.000 description 2
- 102100034349 Integrase Human genes 0.000 description 2
- FBOZXECLQNJBKD-ZDUSSCGKSA-N L-methotrexate Chemical compound C=1N=C2N=C(N)N=C(N)C2=NC=1CN(C)C1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 FBOZXECLQNJBKD-ZDUSSCGKSA-N 0.000 description 2
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 2
- ZYLJULGXQDNXDK-GUBZILKMSA-N Leu-Gln-Asp Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O ZYLJULGXQDNXDK-GUBZILKMSA-N 0.000 description 2
- FGZVGOAAROXFAB-IXOXFDKPSA-N Leu-Thr-His Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@H](CC(C)C)N)O FGZVGOAAROXFAB-IXOXFDKPSA-N 0.000 description 2
- 241000723994 Maize dwarf mosaic virus Species 0.000 description 2
- 241001457070 Mirabilis mosaic virus Species 0.000 description 2
- 235000007199 Panicum miliaceum Nutrition 0.000 description 2
- 108020005091 Replication Origin Proteins 0.000 description 2
- 241001136275 Sphingobacterium Species 0.000 description 2
- 101150108263 Stk16 gene Proteins 0.000 description 2
- 238000010459 TALEN Methods 0.000 description 2
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 2
- 239000004473 Threonine Substances 0.000 description 2
- 241000723873 Tobacco mosaic virus Species 0.000 description 2
- 108010043645 Transcription Activator-Like Effector Nucleases Proteins 0.000 description 2
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 2
- 102000004243 Tubulin Human genes 0.000 description 2
- 108090000704 Tubulin Proteins 0.000 description 2
- 108010017070 Zinc Finger Nucleases Proteins 0.000 description 2
- 229960000723 ampicillin Drugs 0.000 description 2
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 210000003763 chloroplast Anatomy 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- IWEDIXLBFLAXBO-UHFFFAOYSA-N dicamba Chemical compound COC1=C(Cl)C=CC(Cl)=C1C(O)=O IWEDIXLBFLAXBO-UHFFFAOYSA-N 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 230000005059 dormancy Effects 0.000 description 2
- 238000006911 enzymatic reaction Methods 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 230000035784 germination Effects 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 208000006278 hypochromic anemia Diseases 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 238000003780 insertion Methods 0.000 description 2
- 230000037431 insertion Effects 0.000 description 2
- 230000010354 integration Effects 0.000 description 2
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 2
- OYIKARCXOQLFHF-UHFFFAOYSA-N isoxaflutole Chemical compound CS(=O)(=O)C1=CC(C(F)(F)F)=CC=C1C(=O)C1=C(C2CC2)ON=C1 OYIKARCXOQLFHF-UHFFFAOYSA-N 0.000 description 2
- 150000002545 isoxazoles Chemical class 0.000 description 2
- 239000008101 lactose Substances 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 229960000485 methotrexate Drugs 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 238000001823 molecular biology technique Methods 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 230000003204 osmotic effect Effects 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- SCVFZCLFOSHCOH-UHFFFAOYSA-M potassium acetate Chemical compound [K+].CC([O-])=O SCVFZCLFOSHCOH-UHFFFAOYSA-M 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 235000015136 pumpkin Nutrition 0.000 description 2
- 108020003175 receptors Proteins 0.000 description 2
- 102000005962 receptors Human genes 0.000 description 2
- 230000010076 replication Effects 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 238000002741 site-directed mutagenesis Methods 0.000 description 2
- 239000001509 sodium citrate Substances 0.000 description 2
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 2
- 238000005507 spraying Methods 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- PQTBTIFWAXVEPB-UHFFFAOYSA-N sulcotrione Chemical compound ClC1=CC(S(=O)(=O)C)=CC=C1C(=O)C1C(=O)CCCC1=O PQTBTIFWAXVEPB-UHFFFAOYSA-N 0.000 description 2
- 230000001360 synchronised effect Effects 0.000 description 2
- 230000009885 systemic effect Effects 0.000 description 2
- IUQAXCIUEPFPSF-UHFFFAOYSA-N tembotrione Chemical compound ClC1=C(COCC(F)(F)F)C(S(=O)(=O)C)=CC=C1C(=O)C1C(=O)CCCC1=O IUQAXCIUEPFPSF-UHFFFAOYSA-N 0.000 description 2
- 230000002103 transcriptional effect Effects 0.000 description 2
- 230000001052 transient effect Effects 0.000 description 2
- 238000013519 translation Methods 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- GGKNTGJPGZQNID-UHFFFAOYSA-N (1-$l^{1}-oxidanyl-2,2,6,6-tetramethylpiperidin-4-yl)-trimethylazanium Chemical compound CC1(C)CC([N+](C)(C)C)CC(C)(C)N1[O] GGKNTGJPGZQNID-UHFFFAOYSA-N 0.000 description 1
- 101150072531 10 gene Proteins 0.000 description 1
- YVLPJIGOMTXXLP-UUKUAVTLSA-N 15,15'-cis-Phytoene Natural products C(=C\C=C/C=C(\CC/C=C(\CC/C=C(\CC/C=C(\C)/C)/C)/C)/C)(\CC/C=C(\CC/C=C(\CC/C=C(\C)/C)/C)/C)/C YVLPJIGOMTXXLP-UUKUAVTLSA-N 0.000 description 1
- YVLPJIGOMTXXLP-BAHRDPFUSA-N 15Z-phytoene Natural products CC(=CCCC(=CCCC(=CCCC(=CC=C/C=C(C)/CCC=C(/C)CCC=C(/C)CCC=C(C)C)C)C)C)C YVLPJIGOMTXXLP-BAHRDPFUSA-N 0.000 description 1
- FAYURRAZYXBAHG-UHFFFAOYSA-N 2-(dichloromethoxy)benzoic acid Chemical compound OC(=O)C1=CC=CC=C1OC(Cl)Cl FAYURRAZYXBAHG-UHFFFAOYSA-N 0.000 description 1
- 102100027328 2-hydroxyacyl-CoA lyase 2 Human genes 0.000 description 1
- UPMXNNIRAGDFEH-UHFFFAOYSA-N 3,5-dibromo-4-hydroxybenzonitrile Chemical compound OC1=C(Br)C=C(C#N)C=C1Br UPMXNNIRAGDFEH-UHFFFAOYSA-N 0.000 description 1
- CAAMSDWKXXPUJR-UHFFFAOYSA-N 3,5-dihydro-4H-imidazol-4-one Chemical compound O=C1CNC=N1 CAAMSDWKXXPUJR-UHFFFAOYSA-N 0.000 description 1
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- 108010068327 4-hydroxyphenylpyruvate dioxygenase Proteins 0.000 description 1
- 102100039601 ARF GTPase-activating protein GIT1 Human genes 0.000 description 1
- 101710194905 ARF GTPase-activating protein GIT1 Proteins 0.000 description 1
- 241000219144 Abutilon Species 0.000 description 1
- 241000448435 Acalypha australis Species 0.000 description 1
- 101710103719 Acetolactate synthase large subunit Proteins 0.000 description 1
- 101710182467 Acetolactate synthase large subunit IlvB1 Proteins 0.000 description 1
- 101710171176 Acetolactate synthase large subunit IlvG Proteins 0.000 description 1
- 101710176702 Acetolactate synthase small subunit Proteins 0.000 description 1
- 101710147947 Acetolactate synthase small subunit 1, chloroplastic Proteins 0.000 description 1
- 101710095712 Acetolactate synthase, mitochondrial Proteins 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 241000724328 Alfalfa mosaic virus Species 0.000 description 1
- 241001116389 Aloe Species 0.000 description 1
- 241000219318 Amaranthus Species 0.000 description 1
- 108020005544 Antisense RNA Proteins 0.000 description 1
- 240000007087 Apium graveolens Species 0.000 description 1
- 235000015849 Apium graveolens Dulce Group Nutrition 0.000 description 1
- 235000010591 Appio Nutrition 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- 229930192334 Auxin Natural products 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 108091032955 Bacterial small RNA Proteins 0.000 description 1
- 241000218993 Begonia Species 0.000 description 1
- 239000002028 Biomass Substances 0.000 description 1
- 240000008564 Boehmeria nivea Species 0.000 description 1
- 240000007124 Brassica oleracea Species 0.000 description 1
- 235000003899 Brassica oleracea var acephala Nutrition 0.000 description 1
- 235000011299 Brassica oleracea var botrytis Nutrition 0.000 description 1
- 235000011301 Brassica oleracea var capitata Nutrition 0.000 description 1
- 235000001169 Brassica oleracea var oleracea Nutrition 0.000 description 1
- 240000003259 Brassica oleracea var. botrytis Species 0.000 description 1
- 239000005489 Bromoxynil Substances 0.000 description 1
- 235000002566 Capsicum Nutrition 0.000 description 1
- 101001033883 Cenchritis muricatus Protease inhibitor 2 Proteins 0.000 description 1
- 241000747028 Cestrum yellow leaf curling virus Species 0.000 description 1
- 240000006162 Chenopodium quinoa Species 0.000 description 1
- 235000015493 Chenopodium quinoa Nutrition 0.000 description 1
- 240000006740 Cichorium endivia Species 0.000 description 1
- 241001533384 Circovirus Species 0.000 description 1
- 244000241235 Citrullus lanatus Species 0.000 description 1
- 235000012828 Citrullus lanatus var citroides Nutrition 0.000 description 1
- 241000518484 Cotton leaf curl virus Species 0.000 description 1
- 241000219112 Cucumis Species 0.000 description 1
- 235000015510 Cucumis melo subsp melo Nutrition 0.000 description 1
- 108010007218 Cytochromes b6 Proteins 0.000 description 1
- 108010075021 Cytochromes f Proteins 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- 240000004585 Dactylis glomerata Species 0.000 description 1
- 244000000626 Daucus carota Species 0.000 description 1
- 235000002767 Daucus carota Nutrition 0.000 description 1
- 235000009355 Dianthus caryophyllus Nutrition 0.000 description 1
- 240000006497 Dianthus caryophyllus Species 0.000 description 1
- 101100125027 Dictyostelium discoideum mhsp70 gene Proteins 0.000 description 1
- 101150048726 E9 gene Proteins 0.000 description 1
- 244000058871 Echinochloa crus-galli Species 0.000 description 1
- 108091006149 Electron carriers Proteins 0.000 description 1
- 102100038132 Endogenous retrovirus group K member 6 Pro protein Human genes 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 102000004533 Endonucleases Human genes 0.000 description 1
- 241000702371 Enterobacteria phage f1 Species 0.000 description 1
- 101710091045 Envelope protein 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
- 241000620209 Escherichia coli DH5[alpha] Species 0.000 description 1
- 240000006927 Foeniculum vulgare Species 0.000 description 1
- 235000004204 Foeniculum vulgare Nutrition 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
- 101150104463 GOS2 gene Proteins 0.000 description 1
- 235000014820 Galium aparine Nutrition 0.000 description 1
- 206010071602 Genetic polymorphism Diseases 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 101150012639 HPPD gene Proteins 0.000 description 1
- 101150031823 HSP70 gene Proteins 0.000 description 1
- 101710081758 High affinity cationic amino acid transporter 1 Proteins 0.000 description 1
- 101000985215 Homo sapiens 4-hydroxyphenylpyruvate dioxygenase Proteins 0.000 description 1
- 101000899240 Homo sapiens Endoplasmic reticulum chaperone BiP Proteins 0.000 description 1
- ZHLWCBHWYUISFY-UHFFFAOYSA-N Hydroxyphenylpyruvic acid Chemical compound OC(=O)C(=O)C(O)C1=CC=CC=C1 ZHLWCBHWYUISFY-UHFFFAOYSA-N 0.000 description 1
- 108010061833 Integrases Proteins 0.000 description 1
- 244000017020 Ipomoea batatas Species 0.000 description 1
- 235000002678 Ipomoea batatas Nutrition 0.000 description 1
- 108090000769 Isomerases Proteins 0.000 description 1
- 239000005571 Isoxaflutole Substances 0.000 description 1
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 1
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 1
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 1
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 1
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 1
- 239000012880 LB liquid culture medium Substances 0.000 description 1
- 235000003228 Lactuca sativa Nutrition 0.000 description 1
- 240000008415 Lactuca sativa Species 0.000 description 1
- 108091026898 Leader sequence (mRNA) Proteins 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
- WXZOHBVPVKABQN-DCAQKATOSA-N Leu-Met-Asp Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(=O)O)C(=O)O)N WXZOHBVPVKABQN-DCAQKATOSA-N 0.000 description 1
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 1
- 239000006137 Luria-Bertani broth Substances 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- NWBJYWHLCVSVIJ-UHFFFAOYSA-N N-benzyladenine Chemical compound N=1C=NC=2NC=NC=2C=1NCC1=CC=CC=C1 NWBJYWHLCVSVIJ-UHFFFAOYSA-N 0.000 description 1
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 1
- 235000016698 Nigella sativa Nutrition 0.000 description 1
- 244000090896 Nigella sativa Species 0.000 description 1
- 108091092724 Noncoding DNA Proteins 0.000 description 1
- 239000006002 Pepper Substances 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 108091093037 Peptide nucleic acid Proteins 0.000 description 1
- 235000010627 Phaseolus vulgaris Nutrition 0.000 description 1
- 244000046052 Phaseolus vulgaris Species 0.000 description 1
- 231100000674 Phytotoxicity Toxicity 0.000 description 1
- 235000016761 Piper aduncum Nutrition 0.000 description 1
- 240000003889 Piper guineense Species 0.000 description 1
- 235000017804 Piper guineense Nutrition 0.000 description 1
- 235000008184 Piper nigrum Nutrition 0.000 description 1
- 241000205407 Polygonum Species 0.000 description 1
- 244000234609 Portulaca oleracea Species 0.000 description 1
- 235000001855 Portulaca oleracea Nutrition 0.000 description 1
- 241000710078 Potyvirus Species 0.000 description 1
- 101710196435 Probable acetolactate synthase large subunit Proteins 0.000 description 1
- 101710181764 Probable acetolactate synthase small subunit Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 101710188315 Protein X Proteins 0.000 description 1
- 102100029028 Protoporphyrinogen oxidase Human genes 0.000 description 1
- 101710104000 Putative acetolactate synthase small subunit Proteins 0.000 description 1
- 235000002226 Ranunculus ficaria Nutrition 0.000 description 1
- 244000081426 Ranunculus ficaria Species 0.000 description 1
- 244000088415 Raphanus sativus Species 0.000 description 1
- 235000006140 Raphanus sativus var sativus Nutrition 0.000 description 1
- 108700005075 Regulator Genes 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
- 235000008406 SarachaNachtschatten Nutrition 0.000 description 1
- 244000082988 Secale cereale Species 0.000 description 1
- 235000007238 Secale cereale Nutrition 0.000 description 1
- 235000002248 Setaria viridis Nutrition 0.000 description 1
- 235000010086 Setaria viridis var. viridis Nutrition 0.000 description 1
- 108010052160 Site-specific recombinase Proteins 0.000 description 1
- 235000004790 Solanum aculeatissimum Nutrition 0.000 description 1
- 235000008424 Solanum demissum Nutrition 0.000 description 1
- 235000018253 Solanum ferox Nutrition 0.000 description 1
- 235000000208 Solanum incanum Nutrition 0.000 description 1
- 235000013131 Solanum macrocarpon Nutrition 0.000 description 1
- 235000002597 Solanum melongena Nutrition 0.000 description 1
- 244000061458 Solanum melongena Species 0.000 description 1
- 235000009869 Solanum phureja Nutrition 0.000 description 1
- 240000002307 Solanum ptychanthum Species 0.000 description 1
- 235000000341 Solanum ptychanthum Nutrition 0.000 description 1
- 235000017622 Solanum xanthocarpum Nutrition 0.000 description 1
- 235000009337 Spinacia oleracea Nutrition 0.000 description 1
- 244000300264 Spinacia oleracea Species 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 241000187191 Streptomyces viridochromogenes Species 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 108010043934 Sucrose synthase Proteins 0.000 description 1
- 239000005618 Sulcotrione Substances 0.000 description 1
- 229940100389 Sulfonylurea Drugs 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 101710137500 T7 RNA polymerase Proteins 0.000 description 1
- 239000005620 Tembotrione Substances 0.000 description 1
- 108091036066 Three prime untranslated region Proteins 0.000 description 1
- 108700019146 Transgenes Proteins 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- 102000016540 Tyrosine aminotransferases Human genes 0.000 description 1
- 108010042606 Tyrosine transaminase Proteins 0.000 description 1
- 108020004417 Untranslated RNA Proteins 0.000 description 1
- 102000039634 Untranslated RNA Human genes 0.000 description 1
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 1
- 241001506766 Xanthium Species 0.000 description 1
- 241000607479 Yersinia pestis Species 0.000 description 1
- 241001148683 Zostera marina Species 0.000 description 1
- FJJCIZWZNKZHII-UHFFFAOYSA-N [4,6-bis(cyanoamino)-1,3,5-triazin-2-yl]cyanamide Chemical compound N#CNC1=NC(NC#N)=NC(NC#N)=N1 FJJCIZWZNKZHII-UHFFFAOYSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 238000007605 air drying Methods 0.000 description 1
- VFRROHXSMXFLSN-KVTDHHQDSA-N aldehydo-D-mannose 6-phosphate Chemical compound OP(=O)(O)OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)C=O VFRROHXSMXFLSN-KVTDHHQDSA-N 0.000 description 1
- 125000001931 aliphatic group Chemical group 0.000 description 1
- 235000011399 aloe vera Nutrition 0.000 description 1
- 229940051880 analgesics and antipyretics pyrazolones Drugs 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000000692 anti-sense effect Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- MXWJVTOOROXGIU-UHFFFAOYSA-N atrazine Chemical compound CCNC1=NC(Cl)=NC(NC(C)C)=N1 MXWJVTOOROXGIU-UHFFFAOYSA-N 0.000 description 1
- 239000002363 auxin Substances 0.000 description 1
- 108010021384 barley lectin Proteins 0.000 description 1
- 238000003287 bathing Methods 0.000 description 1
- NUPIYIXBLUEVRR-UHFFFAOYSA-N benzenecarbothioic s-acid;pyrimidine Chemical compound C1=CN=CN=C1.OC(=S)C1=CC=CC=C1 NUPIYIXBLUEVRR-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
- 102000023732 binding proteins Human genes 0.000 description 1
- 108091008324 binding proteins Proteins 0.000 description 1
- 230000001851 biosynthetic effect Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 244000022185 broomcorn panic Species 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 229940027138 cambia Drugs 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 235000003733 chicria Nutrition 0.000 description 1
- 229930002875 chlorophyll Natural products 0.000 description 1
- 235000019804 chlorophyll Nutrition 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
- 238000010367 cloning Methods 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 239000003184 complementary RNA Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000009402 cross-breeding Methods 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 108010027183 cytochrome P-450 CYP76B1 (Helianthus tuberosus) Proteins 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- GVJHHUAWPYXKBD-UHFFFAOYSA-N d-alpha-tocopherol Natural products OC1=C(C)C(C)=C2OC(CCCC(C)CCCC(C)CCCC(C)C)(C)CCC2=C1C GVJHHUAWPYXKBD-UHFFFAOYSA-N 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000007123 defense Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 239000007857 degradation product Substances 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 239000005547 deoxyribonucleotide Substances 0.000 description 1
- 125000002637 deoxyribonucleotide group Chemical group 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- KXZOIWWTXOCYKR-UHFFFAOYSA-M diclofenac potassium Chemical compound [K+].[O-]C(=O)CC1=CC=CC=C1NC1=C(Cl)C=CC=C1Cl KXZOIWWTXOCYKR-UHFFFAOYSA-M 0.000 description 1
- 230000003292 diminished effect Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 101150052825 dnaK gene Proteins 0.000 description 1
- 241001493065 dsRNA viruses Species 0.000 description 1
- 210000005069 ears Anatomy 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 210000002257 embryonic structure Anatomy 0.000 description 1
- 210000002472 endoplasmic reticulum Anatomy 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 230000005714 functional activity Effects 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- IAJOBQBIJHVGMQ-BYPYZUCNSA-N glufosinate-P Chemical compound CP(O)(=O)CC[C@H](N)C(O)=O IAJOBQBIJHVGMQ-BYPYZUCNSA-N 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- 230000005484 gravity Effects 0.000 description 1
- 230000009036 growth inhibition Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 101150031530 hpn gene Proteins 0.000 description 1
- 239000010903 husk Substances 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000012994 industrial processing Methods 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 239000000543 intermediate Substances 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 1
- 229940088649 isoxaflutole Drugs 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 239000010871 livestock manure Substances 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 238000007726 management method Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000010198 maturation time Effects 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 230000002438 mitochondrial effect Effects 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 229910001453 nickel ion Inorganic materials 0.000 description 1
- 239000001711 nigella sativa Substances 0.000 description 1
- 231100000989 no adverse effect Toxicity 0.000 description 1
- 108091027963 non-coding RNA Proteins 0.000 description 1
- 102000042567 non-coding RNA Human genes 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 238000007899 nucleic acid hybridization Methods 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 210000003463 organelle Anatomy 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 230000002085 persistent effect Effects 0.000 description 1
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- 230000003711 photoprotective effect Effects 0.000 description 1
- 235000011765 phytoene Nutrition 0.000 description 1
- 108010001545 phytoene dehydrogenase Proteins 0.000 description 1
- 230000008121 plant development Effects 0.000 description 1
- 230000008635 plant growth Effects 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 210000002706 plastid Anatomy 0.000 description 1
- 235000011056 potassium acetate Nutrition 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 230000009465 prokaryotic expression Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 210000001938 protoplast Anatomy 0.000 description 1
- 101150075980 psbA gene Proteins 0.000 description 1
- DWSPRBSLSXQIEJ-UHFFFAOYSA-N pyrasulfotole Chemical compound CC1=NN(C)C(O)=C1C(=O)C1=CC=C(C(F)(F)F)C=C1S(C)(=O)=O DWSPRBSLSXQIEJ-UHFFFAOYSA-N 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- 230000003362 replicative effect Effects 0.000 description 1
- 239000002336 ribonucleotide Substances 0.000 description 1
- 125000002652 ribonucleotide group Chemical group 0.000 description 1
- 101150078341 rop gene Proteins 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 230000001568 sexual effect Effects 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000004550 soluble concentrate Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000009331 sowing Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- YROXIXLRRCOBKF-UHFFFAOYSA-N sulfonylurea Chemical class OC(=N)N=S(=O)=O YROXIXLRRCOBKF-UHFFFAOYSA-N 0.000 description 1
- 229910052717 sulfur Inorganic materials 0.000 description 1
- 239000011593 sulfur Substances 0.000 description 1
- 230000000153 supplemental effect Effects 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 239000000375 suspending agent Substances 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- ZFXYFBGIUFBOJW-UHFFFAOYSA-N theophylline Chemical compound O=C1N(C)C(=O)N(C)C2=C1NC=N2 ZFXYFBGIUFBOJW-UHFFFAOYSA-N 0.000 description 1
- 229960000278 theophylline Drugs 0.000 description 1
- 235000010384 tocopherol Nutrition 0.000 description 1
- 229960001295 tocopherol Drugs 0.000 description 1
- 229930003799 tocopherol Natural products 0.000 description 1
- 239000011732 tocopherol Substances 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- YWBFPKPWMSWWEA-UHFFFAOYSA-O triazolopyrimidine Chemical compound BrC1=CC=CC(C=2N=C3N=CN[N+]3=C(NCC=3C=CN=CC=3)C=2)=C1 YWBFPKPWMSWWEA-UHFFFAOYSA-O 0.000 description 1
- 210000003934 vacuole Anatomy 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 238000004383 yellowing Methods 0.000 description 1
- GVJHHUAWPYXKBD-IEOSBIPESA-N α-tocopherol Chemical compound OC1=C(C)C(C)=C2O[C@@](CCC[C@H](C)CCC[C@H](C)CCCC(C)C)(C)CCC2=C1C GVJHHUAWPYXKBD-IEOSBIPESA-N 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0069—Oxidoreductases (1.) acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
-
- 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)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
- C12N15/8205—Agrobacterium mediated transformation
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8274—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for herbicide resistance
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y113/00—Oxidoreductases acting on single donors with incorporation of molecular oxygen (oxygenases) (1.13)
- C12Y113/11—Oxidoreductases acting on single donors with incorporation of molecular oxygen (oxygenases) (1.13) with incorporation of two atoms of oxygen (1.13.11)
- C12Y113/11027—4-Hydroxyphenylpyruvate dioxygenase (1.13.11.27)
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Biomedical Technology (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Cell Biology (AREA)
- Medicinal Chemistry (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention relates to a herbicide tolerance protein, a coding gene and application thereof, wherein the protein comprises: (a) has an amino acid sequence shown as SEQ ID NO. 6 or SEQ ID NO. 9; (b) a protein derived from (a) having a hydroxyphenylpyruvate dioxygenase, wherein the protein is obtained by substituting and/or deleting and/or adding one or more amino acids in the amino acid sequence of (a). The herbicide tolerance protein can endow plants with tolerance to HPPD inhibitor herbicides, and has wide application prospect in plants.
Description
The application is a divisional application of Chinese patent application 202010194585.0, and the invention name is: herbicide tolerance protein, coding gene and application thereof, application date: year 2020, 3, 19.
Technical Field
The invention relates to a herbicide tolerance protein, a coding gene and application thereof, in particular to a protein with tolerance to an HPPD inhibitor herbicide, a coding gene and application thereof.
Background
Hydroxyphenylpyruvate dioxygenase (HPPD) is an enzyme which is bound to iron ions (Fe) 2+ ) And in the presence of oxygen, catalyzing the reaction of a degradation product of tyrosine, namely, hydroxyphenylpyruvic acid (HPP) to be converted into a precursor of Plastoquinone (PQ), namely homogentisate/Homogentisate (HG), in plants. Tocopherol has the effect of a membrane-associated antioxidant; PQ is not only an electron carrier between PS II and the cytochrome b6/f complex, but also an essential cofactor for phytoene desaturase in carotenoid biosynthesis.
Herbicides that act by inhibiting HPPD are mainly three chemical families of triketones, isoxazoles and pyrazolones. In plants, they block the biosynthesis of PQ from tyrosine by inhibiting HPPD, leading to PQ depletion, carotenoid deficiency. The above mentioned HPPD inhibiting herbicides are plant phloem mobile bleaches which cause the new meristems and leaves exposed to light to appear white, whereas carotenoids are essential for photoprotection, in the absence of carotenoids, uv radiation and reactive oxygen intermediates disrupt chlorophyll synthesis and function, leading to plant growth inhibition, even death.
The method for providing a plant tolerant to HPPD inhibitor herbicides essentially comprises: 1) HPPDs are overexpressed to produce high quantities of HPPD in plants, and despite the presence of the HPPD inhibitor herbicide, the HPPDs are sufficiently reactive with the HPPD inhibitor herbicide to have sufficient functional enzyme available for use. 2) The target HPPD is mutated to a functional HPPD that is less sensitive to herbicides or active metabolites thereof, but which retains the property of being converted to HG. With respect to mutant HPPD classes, while a given mutant HPPD may provide a useful level of tolerance to some HPPD inhibitor herbicides, the same mutant HPPD may not be sufficient to provide a commercial level of tolerance to a different, more desirable HPPD inhibitor herbicide; for example, HPPD inhibitor herbicides can differ in the range of weeds they control, their cost of manufacture, and their environmental friendliness. Thus, there is a need for new methods and/or compositions for conferring HPPD inhibitor herbicide tolerance to different crops and crop varieties.
Disclosure of Invention
The invention aims to provide a novel protein, a coding gene and application thereof, wherein the protein not only has HPPD enzyme activity, but also ensures that a plant transferred with the coding gene of the protein has tolerance to an HPPD inhibitor herbicide.
To achieve the above object, the present invention provides a protein comprising:
(a) has an amino acid sequence shown as SEQ ID NO. 6 or SEQ ID NO. 9;
(b) and (b) a protein derived from (a) and having hydroxyphenylpyruvate dioxygenase activity, wherein the amino acid sequence in (a) is substituted and/or deleted and/or one or more amino acids are added.
To achieve the above object, the present invention provides a gene comprising:
(a) a nucleotide sequence encoding the protein of claim 1; or
(b) A nucleotide sequence which hybridizes with the nucleotide sequence defined in (a) under strict conditions and codes a protein with hydroxyphenylpyruvate dioxygenase activity; or
(c) Has the nucleotide sequence shown in SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 10 or SEQ ID NO 11.
The stringent conditions may be hybridization in a 6 XSSC (sodium citrate), 0.5% SDS (sodium dodecyl sulfate) solution at 65 ℃ and then washing the membrane 1 time each with 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS.
To achieve the above object, the present invention also provides an expression cassette comprising the gene under the control of an operably linked regulatory sequence.
In order to achieve the above object, the present invention also provides a recombinant vector comprising the gene or the expression cassette
To achieve the above object, the present invention also provides a method for extending the herbicide tolerance of a plant, comprising: expressing said protein or the protein encoded by said expression cassette in a plant together with at least one second herbicide tolerance protein different from said protein or the protein encoded by said expression cassette.
Further, the second herbicide tolerance protein is 5-enolpyruvylshikimate-3-phosphate synthase, glyphosate oxidoreductase, glyphosate-N-acetyltransferase, glyphosate decarboxylase, glufosinate acetyltransferase, alpha ketoglutarate-dependent dioxygenase, dicamba monooxygenase, acetolactate synthase, a cytochrome-like protein and/or protoporphyrinogen oxidase.
To achieve the above object, the present invention also provides a method for selecting a transformed plant cell, comprising: transforming a plurality of plant cells with said gene or said expression cassette and culturing said cells at a HPPD inhibitor herbicide concentration that allows growth of transformed cells expressing said gene or said expression cassette, while killing or inhibiting growth of untransformed cells;
preferably, the plant includes monocotyledons and dicotyledons; more preferably, the plant is oat, wheat, barley, millet, corn, sorghum, brachypodium distachyon, rice, tobacco, sunflower, alfalfa, soybean, chickpea, peanut, sugar beet, cucumber, cotton, rape, potato, tomato or arabidopsis;
preferably, the HPPD inhibitor herbicide comprises a pyrazolone HPPD inhibitor herbicide, a triketone HPPD inhibitor herbicide, and/or an isoxazole HPPD inhibitor herbicide; more preferably, the HPPD inhibitor herbicide is topramezone, mesotrione, and/or a diketonitrile.
To achieve the above object, the present invention also provides a method for controlling weeds, comprising: applying an effective dose of an HPPD inhibitor herbicide to a field growing a plant of interest, said plant of interest comprising said gene or said expression cassette or said recombinant vector;
preferably, the plant of interest includes a monocotyledon and a dicotyledon; more preferably, the plant of interest is oat, wheat, barley, millet, corn, sorghum, brachypodium distachyon, rice, tobacco, sunflower, alfalfa, soybean, chickpea, peanut, sugar beet, cucumber, cotton, rape, potato, tomato or arabidopsis; further preferably, the plant of interest is a glyphosate tolerant plant and the weeds are glyphosate resistant weeds;
preferably, the HPPD inhibitor herbicide comprises a pyrazolone HPPD inhibitor herbicide, a triketone HPPD inhibitor herbicide, and/or an isoxazole HPPD inhibitor herbicide; more preferably, the HPPD inhibitor herbicide is topramezone, mesotrione, and/or a diketonitrile.
To achieve the above objects, the present invention also provides a method for protecting a plant from damage caused by or conferring tolerance to a HPPD inhibitor herbicide to a plant, comprising: introducing said gene or said expression cassette or said recombinant vector into a plant such that the introduced plant produces an amount of herbicide tolerance protein sufficient to protect it from HPPD inhibitor herbicide damage;
preferably, the plant includes monocotyledons and dicotyledons; more preferably, the plant is oat, wheat, barley, millet, corn, sorghum, brachypodium distachyon, rice, tobacco, sunflower, alfalfa, soybean, chickpea, peanut, sugar beet, cucumber, cotton, rape, potato, tomato or arabidopsis;
preferably, the HPPD inhibitor herbicide comprises a pyrazolone HPPD inhibitor herbicide, a triketone HPPD inhibitor herbicide, and/or an isoxazole HPPD inhibitor herbicide; more preferably, the HPPD inhibitor herbicide is topramezone, mesotrione, and/or a diketonitrile.
To achieve the above object, the present invention also provides a method for producing a plant tolerant to an HPPD inhibitor herbicide, comprising introducing the gene into the genome of the plant;
preferably, the method of introduction includes a genetic transformation method, a genome editing method, or a gene mutation method;
specifically, the method of producing a plant tolerant to an HPPD inhibitor herbicide comprises: producing an HPPD inhibitor herbicide tolerant plant by selfing a parent plant or crossing with a second plant, said parent plant and/or second plant comprising said gene or said expression cassette, said HPPD inhibitor herbicide tolerant plant inheriting said gene or said expression cassette from said parent plant and/or second plant;
preferably, the plant includes monocotyledons and dicotyledons; more preferably, the plant is oat, wheat, barley, millet, corn, sorghum, brachypodium distachyon, rice, tobacco, sunflower, alfalfa, soybean, chickpea, peanut, sugar beet, cucumber, cotton, rape, potato, tomato or arabidopsis;
preferably, the HPPD inhibitor herbicide comprises a pyrazolone HPPD inhibitor herbicide, a triketone HPPD inhibitor herbicide, and/or an isoxazole HPPD inhibitor herbicide; more preferably, the HPPD inhibitor herbicide is topramezone, mesotrione, and/or a diketonitrile.
To achieve the above objects, the present invention also provides a method of growing a plant tolerant to an HPPD inhibitor herbicide, comprising:
growing at least one plant propagule comprising in its genome the gene or the expression cassette;
growing the plant propagule into a plant;
applying an effective dose of an HPPD inhibitor herbicide to a plant growing environment comprising at least said plant, harvesting plants having reduced plant damage and/or increased plant yield as compared to other plants not having said gene or said expression cassette;
preferably, the plant includes a monocotyledon and a dicotyledon; more preferably, the plant is oat, wheat, barley, millet, corn, sorghum, brachypodium distachyon, rice, tobacco, sunflower, alfalfa, soybean, chickpea, peanut, sugar beet, cucumber, cotton, rape, potato, tomato or arabidopsis;
preferably, the HPPD inhibitor herbicide comprises a pyrazolone HPPD inhibitor herbicide, a triketone HPPD inhibitor herbicide, and/or an isoxazole HPPD inhibitor herbicide; more preferably, the HPPD inhibitor herbicide is topramezone, mesotrione, and/or a diketonitrile.
The present invention also provides a method for obtaining a processed agricultural product comprising treating a harvest of HPPD inhibitor herbicide tolerant plants obtained by said method to obtain a processed agricultural product.
To achieve the above object, the present invention also provides a planting system for controlling weed growth, comprising a HPPD inhibitor herbicide and a plant growing environment in which at least one plant of interest is present, said plant of interest comprising said gene or said expression cassette;
preferably, the plant of interest includes a monocotyledon and a dicotyledon; more preferably, the plant of interest is oat, wheat, barley, millet, corn, sorghum, brachypodium distachyon, rice, tobacco, sunflower, alfalfa, soybean, chickpea, peanut, sugar beet, cucumber, cotton, rape, potato, tomato or arabidopsis; further preferably, the plant of interest is a glyphosate tolerant plant and the weeds are glyphosate resistant weeds;
preferably, the HPPD inhibitor herbicide comprises a pyrazolone HPPD inhibitor herbicide, a triketone HPPD inhibitor herbicide, and/or an isoxazole HPPD inhibitor herbicide; more preferably, the HPPD inhibitor herbicide is topramezone, mesotrione, and/or a diketonitrile.
To achieve the above objects, the present invention also provides the use of the protein for conferring a HPPD inhibitor herbicide on a plant;
preferably, the plant includes monocotyledons and dicotyledons; more preferably, the plant is oat, wheat, barley, millet, corn, sorghum, brachypodium distachyon, rice, tobacco, sunflower, alfalfa, soybean, chickpea, peanut, sugar beet, cucumber, cotton, rape, potato, tomato or arabidopsis;
preferably, the HPPD inhibitor herbicide comprises a pyrazolone HPPD inhibitor herbicide, a triketone HPPD inhibitor herbicide, and/or an isoxazole HPPD inhibitor herbicide; more preferably, the HPPD inhibitor herbicide is topramezone, mesotrione, and/or diketonitrile.
The articles "a" and "an" as used herein mean one or more than one (i.e., at least one). For example, "an element" means one or more elements (components). Furthermore, the terms "comprises" or variations such as "comprising" or "comprising" are to be understood as meaning the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.
In the present invention, the term "hydroxyphenylpyruvate dioxygenase (HPPD)" is synonymous with "4-hydroxyphenylpyruvate dioxygenase (4-HPPD)" and "p-hydroxyphenylpyruvate dioxygenase (p-HPPD)".
The term "HPPD-inhibiting herbicide" is synonymous with "HPPD herbicide" and refers to herbicides that directly or indirectly inhibit HPPD, which herbicides are bleaches, and whose primary site of action is HPPD. The most commercially available HPPD inhibiting herbicides belong to one of three chemical families: (1) triketones, for example sulcotrione (i.e. 2- [ 2-chloro-4- (methylsulfonyl) benzoyl ] -1, 3-cyclohexanedione), mesotrione (mesotrione) (i.e. 2- [4- (methylsulfonyl) -2-nitrobenzoyl ] -1, 3-cyclohexanedione), tembotrione (i.e. 2- [ 2-chloro-4- (methylsulfonyl) -3- [ (2,2, 2-trifluoroethoxy) methyl ] benzoyl ] -1, 3-cyclohexanedione); (2) isoxazoles (in plants, isoxazole HPPD herbicides are rapidly converted to biologically active Diketonitriles (DKN) to exert an inhibitory effect on HPPD, which are thus active forms of isoxazole HPPD inhibiting herbicides), for example isoxaflutole (i.e. 5-cyclopropyl-4-isoxazolyl [2- (methylsulfonyl) -4- (trifluoromethyl) phenyl ] methanone); (3) pyrazolones (pyrazolonates), for example topramezone (i.e. [3- (4, 5-dihydro-3-isoxazolyl) -2-methyl-4- (methylsulfonyl) phenyl ] (5-hydroxy-1-methylpyrazol-4-yl) methanone), pyrasulfopyra-zole (5-hydroxy-1, 3-dimethylpyrazol-4-yl (2-methanesulfonyl-4-trifluoromethylphenyl) methanone).
The topramezone, also called topramezone, in the present invention is [3- (4, 5-dihydro-3-isoxazolyl) -2-methyl-4- (methylsulfonyl) phenyl ] (5-hydroxy-1-methylpyrazol-4-yl) methanone, which is a white crystalline solid. Belongs to a pyrazolone (pyrazolonate) type internal absorption conduction type HPPD herbicide in postemergence treatment, and a common preparation formulation is a 30% suspending agent. Commercial preparations of topramezone, such as fennel, can control grassy and broadleaf weeds, 5.6-6.7g of the preparation per mu can effectively control weeds, including, but not limited to, large crabgrass (cocksfoot grass), barnyard grass, goosegrass, wild millet, green bristlegrass (nigella sativa), chenopodium quinoa, polygonum, ramie, abutilon, amaranthus, purslane, xanthium, and nightshade. The bracteal defense has obvious synergistic effect after adding atrazine, has excellent control effect on the weeds, and also has good control effect on malignant broadleaf weeds such as cephalanoplos segetum (herba Cirsii), endive, acalypha australis and dayflower (herba brassicae), and particularly can effectively control setaria viridis, digitaria sanguinalis, eleusine indica and broomcorn millet with poor control effect on mesotrione.
The effective dose of the topramezone is 25-100g ai/ha, and comprises 50-100g ai/ha, 60-90g ai/ha or 75-85g ai/ha.
In the present invention, the term "resistant" is heritable and allows plants to grow and reproduce under conditions where herbicides are effective for a given plant in general. As recognized by those skilled in the art, even if a given plant is subjected to some degree of herbicide treatment, such as little necrosis, lysis, chlorosis, or other damage, but at least not significantly affected in yield, the plant can still be considered "resistant", i.e., the given plant has an increased ability to withstand herbicide-induced damage of various degrees, while typically causing damage to wild-type plants of the same genotype at the same herbicide dose. The term "resistance" or "tolerance" in the present invention is broader than the term "resistance" and includes "resistance".
The term "conferring" in the context of the present invention means providing a characteristic or trait, such as herbicide tolerance and/or other desirable traits, to a plant.
The term "heterologous" in the context of the present invention means from another source. In the context of DNA, "heterologous" refers to any foreign "non-self DNA, including DNA from another plant of the same species. For example, in the present invention, transgenic methods can be used to express the soybean HPPD gene in soybean plants, which is still considered "heterologous" DNA.
The term "nucleic acid" in the present invention encompasses deoxyribonucleotide or ribonucleotide polymers in either single-or double-stranded form, and unless otherwise limited, encompasses known analogs (e.g., peptide nucleic acids) that have the basic properties of natural nucleotides in that they hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides.
In the present invention, when the term "encoding" or "encoded" is used in the context of a particular nucleic acid, it means that the nucleic acid contains the necessary information to direct the translation of the nucleotide sequence into a particular protein. The information used to encode the protein is specified by the use of codons. A nucleic acid encoding a protein may comprise untranslated sequences (e.g., introns) within translated regions of the nucleic acid, or may lack such intervening untranslated sequences (e.g., in cDNA).
The herbicide tolerance protein of the present invention has HPPD enzymatic activity and confers tolerance in plants to certain classes of HPPD inhibiting herbicides. DNA sequences encoding herbicide tolerance proteins of the invention are useful in providing plants, crops, plant cells and seeds of the invention which provide enhanced tolerance to one or more HPPD herbicides.
The genes encoding the herbicide tolerance proteins of the present invention are useful for producing plants that are tolerant to HPPD inhibiting herbicides. The herbicide tolerance gene is particularly suitable for expression in plants in order to confer herbicide tolerance to the plant.
The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. These terms apply to polymers of amino acid residues in which one or more amino acid residues is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The protein of the invention can be produced from a nucleic acid disclosed herein or by using standard molecular biology techniques. For example, a truncated protein of the invention may be produced by expressing a recombinant nucleic acid of the invention in a suitable host cell, or alternatively by a combination of ex vivo methods such as protease digestion and purification.
The invention also provides nucleic acid molecules comprising a polynucleotide sequence encoding an herbicide tolerance protein that has HPPD enzyme activity and confers tolerance in plants to certain classes of HPPD inhibiting herbicides. In general, the invention includes any polynucleotide sequence encoding any of the herbicide tolerance proteins described herein, as well as any polynucleotide sequence encoding an herbicide tolerance protein having one or more conservative amino acid substitutions relative to the herbicide tolerance protein described herein. Conservative substitutions providing functionally similar amino acids are well known to those skilled in the art, and the following five groups each contain amino acids that are conservative substitutions for one another: aliphatic: glycine (G), alanine (a), valine (V), leucine (L), isoleucine (I); aromatic: phenylalanine (F), tyrosine (Y), tryptophan (W); sulfur-containing: methionine (M), cysteine (C); basic: arginine (I), lysine (K), histidine (H); acidic: aspartic acid (D), glutamic acid (E), asparagine (N), glutamine (Q).
Therefore, sequences having HPPD activity to inhibit herbicide tolerance and hybridizing under stringent conditions to the gene encoding the herbicide tolerance protein of the present invention are included in the present invention. Illustratively, these sequences have at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence homology to the sequences of the invention SEQ ID NO 2, 3, 7, 8, 10, 11.
Any conventional nucleic acid hybridization or amplification method can be used to identify the presence of the herbicide tolerance gene of the present invention. Nucleic acid molecules or fragments thereof are capable of specifically hybridizing to other nucleic acid molecules under certain circumstances. In the present invention, two nucleic acid molecules can be said to be capable of specifically hybridizing to each other if they can form an antiparallel double-stranded nucleic acid structure. Two nucleic acid molecules are said to be "complements" of one another if they exhibit complete complementarity. In the present invention, two nucleic acid molecules are said to exhibit "perfect complementarity" when each nucleotide of the two nucleic acid molecules is complementary to the corresponding nucleotide of the other nucleic acid molecule. Two nucleic acid molecules are said to be "minimally complementary" if they are capable of hybridizing to each other with sufficient stability to allow them to anneal and bind to each other under at least conventional "low stringency" conditions. Similarly, two nucleic acid molecules are said to have "complementarity" if they are capable of hybridizing to each other with sufficient stability to allow them to anneal and bind to each other under conventional "highly stringent" conditions. Deviations from perfect complementarity may be tolerated as long as such deviations do not completely prevent the formation of a double-stranded structure by the two molecules. In order to allow a nucleic acid molecule to act as a primer or probe, it is only necessary to ensure sufficient complementarity in sequence to allow the formation of a stable double-stranded structure in the particular solvent and salt concentrations employed.
In the present invention, a substantially homologous sequence is a nucleic acid molecule that specifically hybridizes under highly stringent conditions to the complementary strand of a compatible nucleic acid molecule. Suitable stringency conditions for promoting DNA hybridization include, for example, treatment with 6.0 XSSC/sodium citrate (SSC) at about 45 ℃ followed by a wash with 2.0 XSSC at 50 ℃, as is well known to those skilled in the art. For example, the salt concentration in the washing step can be selected from the group consisting of about 2.0 XSSC for low stringency conditions, 50 ℃ to about 0.2 XSSC for high stringency conditions, 50 ℃. In addition, the temperature conditions in the washing step can be raised from about 22 ℃ at room temperature for low stringency conditions to about 65 ℃ for high stringency conditions. Both the temperature conditions and the salt concentration may be varied, or one may be held constant while the other is varied. Preferably, the stringent conditions of the present invention may be those in which specific hybridization with the herbicide tolerance gene of the present invention occurs in a 6 XSSC solution of 0.5% SDS at 65 ℃ and then the membranes are washed 1 time each with 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS.
In the present invention, the term "hybridize" or "specifically hybridize" refers to a molecule that can only bind, double-stranded, or hybridize to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA.
Due to the redundancy of the genetic code, a plurality of different DNA sequences may encode the same amino acid sequence. It is well within the skill of the art to generate such alternative DNA sequences encoding the same or substantially the same protein. These different DNA sequences are included in the scope of the present invention. The "substantially identical" sequences refer to sequences having amino acid substitutions, deletions, additions or insertions which do not substantially affect the herbicide tolerance activity, and also include fragments which retain the herbicide tolerance activity.
The term "functional activity" or "activity" as used herein means that the protein/enzyme (alone or in combination with other proteins) for use according to the invention has the ability to degrade or attenuate the activity of a herbicide. The plants producing the proteins of the invention preferably produce an "effective amount" of the protein such that when the plants are treated with the herbicide, the level of protein expression is sufficient to confer full or partial tolerance to the herbicide (if not specifically stated, the amount normally used) to the plant. The herbicide may be used in an amount that normally kills the target plant, in a normal field amount, and at a concentration. Preferably, the plant cells and plants of the invention are protected from growth inhibition or damage caused by herbicide treatment. The transformed plants and plant cells of the invention preferably have tolerance to HPPD inhibiting herbicides, i.e., the transformed plants and plant cells are capable of growing in the presence of an effective amount of a HPPD inhibiting herbicide.
The genes and proteins described in the present invention include not only the specific exemplary sequences, but also portions and/or fragments (including internal and/or terminal deletions compared to the full-length protein), variants, mutants, variant proteins, substitutions (proteins with substituted amino acids), chimeras and fusion proteins that preserve the HPPD herbicide tolerance-inhibiting activity characteristics of the specific exemplary proteins.
The term "variant" of the invention means substantially similar sequences. For polynucleotides, a variant comprises a deletion and/or addition of one or more nucleotides at one or more internal sites within a reference polynucleotide and/or a substitution of one or more nucleotides at one or more sites in an herbicide tolerance gene. The term "reference polynucleotide or polypeptide" in the present invention encompasses a herbicide-tolerant nucleotide sequence or amino acid sequence, respectively. The term "native polynucleotide or polypeptide" in the present invention correspondingly includes naturally occurring nucleotide or amino acid sequences. For polynucleotides, conservative variants include a nucleotide sequence encoding one of these herbicide tolerance proteins of the present invention (due to the degeneracy of the genetic code). Such naturally occurring allelic variants can be identified using well-known molecular biology techniques, for example, using the Polymerase Chain Reaction (PCR) and hybridization techniques outlined below. Variant polynucleotides also include synthetically derived polynucleotides, such as sequences produced by use of site-directed mutagenesis, but which still encode an herbicide tolerance protein of the invention. Typically, variants of a particular polynucleotide of the invention will have at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence homology to the particular polynucleotide as determined by sequence alignment programs and parameters.
By "variant protein" is meant a protein derived from a reference protein by deletion or addition of one or more amino acids at one or more internal sites in said herbicide tolerance protein and/or substitution of one or more amino acids at one or more sites in the herbicide tolerance protein. Variant proteins encompassed by the present invention are biologically active, i.e. they continue to have the desired activity of the herbicide tolerance protein, i.e. still have said HPPD enzyme activity and/or herbicide tolerance. Such variants may arise, for example, from genetic polymorphisms or from manual manipulation. A biologically active variant of an herbicide tolerance protein of the invention will have at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence homology to the entire amino acid sequence of the herbicide tolerance protein as determined by sequence alignment programs and parameters. A biologically active variant of a protein of the invention may differ from a protein in as few as 1-15 amino acid residues, as few as 1-10 (e.g., 6-10), as few as 5 (e.g., 4, 3, 2, or even 1) amino acid residues.
Methods for sequence alignment are well known in the art and can be accomplished using mathematical algorithms, such as the algorithm of Myers and Miller (1988) CABIOS 4: 11-17; smith et al (1981) Adv.appl.Math.2: 482; need the global alignment algorithm of Need eman and Wunsch (1970) J.Mol.biol.48: 443-; and the algorithm of Karlin and Altschul (1990) Proc.Natl.Acad.Sci.USA 872264, as modified in Karlin and Altschul (1993) Proc.Natl.Acad.Sci.USA 90: 5873-. Computer implementations of these mathematical algorithms can be utilized for sequence comparison to determine sequence homology, such implementations including, but not limited to: CLUSTAL (available from Intelligenetics, Mountain View, California) in the PC/Gene program; ALLGN program (version 2.0) and GAP, BESTFIT, BLAST, FASTA and TFASTA in GCG Wisconsin Genetics Software Package version 10 (available from Accelrys Inc.,9685 Scanton Road, San Diego, California, USA).
In certain embodiments, amino acids encoding the herbicide tolerance proteins of the present invention or variants thereof that retain HPPD enzymatic activity can be stacked with any combination of polynucleotide sequences of interest to produce plants with a desired trait. The term "trait" refers to a phenotype derived from a particular sequence or group of sequences. For example, a polynucleotide encoding an amino acid of the herbicide tolerance protein or variant retaining HPPD enzymatic activity may be stacked with any other polynucleotide encoding a polypeptide conferring a desired trait including, but not limited to: resistance to diseases, insects, and herbicides, tolerance to heat and drought, reduced crop maturation time, improved industrial processing (e.g., for converting starch or biomass into fermentable sugars), and improved agronomic quality (e.g., high oil content and high protein content).
The benefits of the combination of two or more modes of action in improving the controlled weed spectrum and/or the naturally more tolerant or resistant weed species can also be extended to the creation of herbicide tolerant chemicals in crops other than HPPD tolerant crops by man (transgenic or non-transgenic), as is well known to those skilled in the art. Indeed, the traits encoding the following resistances may be stacked, alone or in multiple combinations, to provide effective control or prevent the ability of a weed succession to develop resistance to herbicides: glyphosate resistance (e.g., resistant plant or bacteria EPSPS, GOX, GAT), glufosinate resistance (e.g., PAT, Bar), acetolactate synthase (ALS) inhibitory herbicide resistance (e.g., imidazolinone, sulfonylurea, triazolopyrimidine, sulfonanilide, pyrimidine thiobenzoate, and other chemical resistance genes such as AHAS, Csrl, SurA, etc.), phenoxy auxin herbicide resistance (e.g., aryloxyalkanoate dioxygenase-AAD), dicamba herbicide resistance (e.g., dicamba monooxygenase-DMO), bromoxynil resistance (e.g., Bxn), resistance to Phytoene (PDS) inhibitors, resistance to systemic ii inhibitory herbicides (e.g., psbA), resistance to systemic i inhibitory herbicides, resistance to protoporphyrinogen oxidase ix (PPO) inhibitory herbicide desaturase (e.g., PPO-1), resistance to phenylurea (e.g., CYP76B1), Dichloromethoxybenzoic acid degrading enzymes, and the like.
Glyphosate is widely used because it controls a very broad spectrum of broadleaf and grass weed species. However, repeated use of glyphosate in glyphosate-tolerant crop and non-crop applications has (and will continue to) choose to allow weeds to evolve into a more naturally-tolerant species or glyphosate-resistant biotype. Most herbicide resistance management strategies suggest the use of an effective amount of a tank-mix herbicide partner that provides control of the same species but has a different mode of action as a means of delaying the emergence of resistant weeds. The superposition of the herbicide tolerance genes of the present invention with glyphosate tolerance traits (and/or other herbicide tolerance traits) can achieve control of glyphosate resistant weed species (broadleaf weed species controlled by one or more pyrazolone herbicides) in glyphosate tolerant crops by allowing selective use of glyphosate and pyrazolone herbicides (such as topramezone) on the same crop. The application of these herbicides can be simultaneous use in tank mixes containing two or more herbicides of different modes of action, separate use of individual herbicide compositions (with time intervals ranging from 2 hours to 3 months between uses) in successive uses (e.g., pre-planting, pre-emergence, or post-emergence), or alternatively, a combination of any number of herbicides representing the applicable classes of each compound can be used at any time from within 7 months of planting the crop to when the crop is harvested (or for a single herbicide, at the shortest interval).
It is important to have flexibility in controlling broadleaf weeds, i.e., time of use, individual herbicide dosage, and the ability to control persistent or resistant weeds. The application range of glyphosate superposed with glyphosate resistance gene/herbicide tolerance gene in crops can be from 250 to 2500g ae/ha; the pyrazolone herbicide(s) may be present in an amount of from 25 to 500g ai/ha. The optimal combination of times for these applications depends on the specific conditions, species and environment.
Herbicide formulations (such as ester, acid or salt formulations or soluble concentrates, emulsified concentrates or soluble liquids) and tank mix additives (such as adjuvants or compatibilizers) can significantly affect weed control for a given herbicide or combination of one or more herbicides. Any chemical combination of any of the foregoing herbicides is within the scope of the invention.
In addition, the gene encoding the herbicide tolerance protein of the present invention can be added to one or more other input (e.g., insect resistance, fungal resistance, or stress tolerance, etc.) or output (e.g., increased yield, improved oil, increased fiber quality, etc.) traits, either alone or in combination with other herbicide tolerance crop traits. Thus, the present invention can be used to provide a complete agronomic solution with the ability to flexibly and economically control any number of agronomic pests and to improve crop quality.
The combination of these overlays can be produced by any method, including but not limited to: cross-breeding plants or genetic transformation by conventional or top-crossing methods. If these sequences are stacked by genetically transforming these plants, the polynucleotide sequences of interest can be combined at any time and in any order. For example, transgenic plants comprising one or more desired traits may be used as targets for the introduction of additional traits by subsequent transformation. These traits may be introduced in a co-transformation protocol simultaneously with the polynucleotides of interest provided by any combination of expression cassettes. For example, if two sequences are to be introduced, the two sequences may be contained in separate expression cassettes (trans) or in the same expression cassette (cis). Expression of these sequences may be driven by the same promoter or by different promoters. In some cases, it may be desirable to introduce an expression cassette that inhibits expression of the polynucleotide of interest. This can be combined with any combination of other suppression or overexpression cassettes to produce the desired combination of traits in the plant. It is further recognized that polynucleotide sequences can be stacked at a desired genomic location using a site-specific recombination system.
The gene for coding the herbicide tolerance protein has higher tolerance to pyrazolone herbicides, and is the basis of characteristic possibility of important herbicide tolerant crops and selective markers.
The term "expression cassette" in the present invention refers to a nucleic acid molecule capable of directing the expression of a particular nucleotide sequence in an appropriate host cell, including a promoter operably linked to the nucleotide sequence of interest (i.e., a polynucleotide encoding an herbicide tolerance protein or variant retaining HPPD enzyme activity, alone or in combination with one or more additional nucleic acid molecules encoding polypeptides conferring a desired trait), which is operably linked to a termination signal. The coding region typically encodes a protein of interest, but may also encode a functional RNA of interest, such as an antisense RNA or an untranslated RNA in the sense or antisense orientation. The expression cassette comprising the nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components. The expression cassette may also be a naturally occurring expression cassette, but must be obtained in recombinant form useful for heterologous expression. Typically, however, the expression cassette is heterologous to the host, i.e., the particular DNA sequence of the expression cassette does not occur naturally in the host cell and must have been introduced into a new host cell by a transformation event. Expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or an inducible promoter which initiates transcription only when the host cell is exposed to some particular external stimulus. In addition, the promoter is specific for a particular tissue or organ or developmental stage.
The present invention encompasses transforming plants with expression cassettes capable of expressing a polynucleotide of interest (i.e., a polynucleotide encoding an herbicide tolerance protein or a variant thereof that retains HPPD enzyme activity, either alone or in combination with one or more additional nucleic acid molecules encoding polypeptides conferring a desired trait). The expression cassette includes a transcriptional and translational initiation region (i.e., promoter) and a polynucleotide open reading frame in the 5 '-3' direction of transcription. The expression cassette may optionally include transcriptional and translational termination regions (i.e., termination regions) that are functional in plants. In some embodiments, the expression cassette includes a selectable marker gene to allow for selection of stable transformants. The expression constructs of the invention may also comprise leader sequences and/or sequences which allow inducible expression of the polynucleotide of interest.
The regulatory sequences of the expression cassette are operably linked to the polynucleotide of interest. The regulatory sequences of the present invention include, but are not limited to, promoters, transit peptides, terminators, enhancers, leader sequences, introns, and other regulatory sequences operably linked to the herbicide tolerance gene encoding the herbicide tolerance protein.
The promoter is a promoter capable of being expressed in a plant, and the promoter capable of being expressed in the plant is a promoter which ensures that a coding sequence connected with the promoter is expressed in a plant cell. The promoter expressible in plants may be a constitutive promoter. Examples of promoters that direct constitutive expression in plants include, but are not limited to, 35S promoter derived from cauliflower mosaic virus, maize Ubi promoter, promoter of rice GOS2 gene, and the like. Alternatively, the plant expressible promoter may be a tissue specific promoter, i.e. a promoter that directs expression of the coding sequence at a higher level in some tissues of the plant, e.g. in green tissues, than in other tissues of the plant (as can be determined by conventional RNA assays), e.g. the PEP carboxylase promoter. Alternatively, the promoter expressible in a plant may be a wound-inducible promoter. A wound-inducible promoter or a promoter that directs a wound-induced expression pattern means that when a plant is subjected to mechanical or insect feeding induced wounds, the expression of the coding sequence under the control of the promoter is significantly increased compared to under normal growth conditions. Examples of wound-inducible promoters include, but are not limited to, promoters of potato and tomato protease-inhibitory genes (pin I and pin II) and maize protease-inhibitory gene (MPI).
The transit peptide (also known as a secretion signal sequence or targeting sequence) is intended to direct the transgene product to a specific organelle or cellular compartment, and for the receptor protein, the transit peptide may be heterologous, e.g., targeting the chloroplast using a chloroplast transit peptide sequence, or targeting the endoplasmic reticulum using a 'KDEL' retention sequence, or targeting the vacuole using the CTPP of the barley lectin gene.
Such leaders include, but are not limited to, small RNA virus leaders, such as EMCV leaders (encephalomyocarditis virus 5' non-coding region); potyvirus leaders, such as the MDMV (maize dwarf mosaic virus) leader; human immunoglobulin heavy chain binding protein (BiP); untranslated leader sequences of envelope protein mRNA of alfalfa mosaic virus (AMV RNA 4); tobacco Mosaic Virus (TMV) leader sequence.
Such enhancers include, but are not limited to, cauliflower mosaic virus (CaMV) enhancer, Figwort Mosaic Virus (FMV) enhancer, carnation weathering Circovirus (CERV) enhancer, cassava vein mosaic virus (CsVMV) enhancer, Mirabilis Mosaic Virus (MMV) enhancer, midnight fragrant tree yellowing leaf curl virus (CmYLCV) enhancer, multan cotton leaf curl virus (CLCuMV), dayflower yellow mottle virus (CoYMV), and peanut chlorosis streak mosaic virus (PCLSV) enhancer.
For monocot applications, the intron includes, but is not limited to, the maize hsp70 intron, the maize ubiquitin intron, Adh intron 1, the sucrose synthase intron, or the rice Act1 intron. For dicot applications, the introns include, but are not limited to, the CAT-1 intron, the pKANNIBAL intron, the PIV2 intron, and the "superubiquitin" intron.
The terminator may be a suitable polyadenylation signal sequence that functions in plants, including, but not limited to, polyadenylation signal sequence derived from the Agrobacterium tumefaciens nopaline synthase (NOS) gene, polyadenylation signal sequence derived from the protease inhibitor II (PIN II) gene, polyadenylation signal sequence derived from the pea ssRUBISCO E9 gene, and polyadenylation signal sequence derived from the alpha-tubulin (alpha-tubulin) gene.
As used herein, "operably linked" refers to the linkage of nucleic acid sequences such that one provides the functionality required of the linked sequence. In the present invention, the "operative linkage" may be a linkage of a promoter to a sequence of interest such that transcription of the sequence of interest is controlled and regulated by the promoter. "operably linked" when the sequence of interest encodes a protein and expression of the protein is desired indicates that: the promoter is linked to the sequence in such a way that the resulting transcript is translated efficiently. If the linkage of the promoter to the coding sequence is a transcript fusion and expression of the encoded protein is desired, such a linkage is made such that the first translation initiation codon in the resulting transcript is the initiation codon of the coding sequence. Alternatively, if the linkage of the promoter to the coding sequence is a translational fusion and expression of the encoded protein is desired, the linkage is made such that the first translation initiation codon contained in the 5' untranslated sequence is linked to the promoter and is linked in such a way that the resulting translation product is in frame with the translational open reading frame encoding the desired protein. Nucleic acid sequences that may be "operably linked" include, but are not limited to: sequences that provide gene expression functions (i.e., gene expression elements such as promoters, 5 'untranslated regions, introns, protein coding regions, 3' untranslated regions, polyadenylation sites, and/or transcription terminators), sequences that provide DNA transfer and/or integration functions (i.e., T-DNA border sequences, site-specific recombinase recognition sites, integrase recognition sites), sequences that provide selective functions (i.e., antibiotic resistance markers, biosynthetic genes), sequences that provide scorable marker functions, sequences that facilitate sequence manipulation in vitro or in vivo (i.e., polylinker sequences, site-specific recombination sequences), and sequences that provide replication functions (i.e., bacterial origins of replication, autonomously replicating sequences, centromeric sequences).
The genome of a plant, plant tissue or plant cell as defined in the present invention refers to any genetic material within a plant, plant tissue or plant cell and includes the nuclear and plastid and mitochondrial genomes.
In the present invention, the term "plant part" or "plant tissue" includes plant cells, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant clumps, and plant cells that are intact in plants or parts of plants such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, nuclei, ears, cobs, husks, stems, roots, root tips, anthers, and the like.
The herbicide tolerance proteins of the present invention can be applied to a variety of plants, including but not limited to alfalfa, beans, cauliflower, cabbage, carrot, celery, cotton, cucumber, eggplant, lettuce, melon, pea, pepper, pumpkin, radish, rape, spinach, soybean, pumpkin, tomato, arabidopsis, peanut, or watermelon; preferably, the dicotyledonous plant is cucumber, soybean, arabidopsis thaliana, tobacco, cotton, peanut or rape. The monocotyledonous plant includes, but is not limited to, maize, rice, sorghum, wheat, barley, rye, millet, sugarcane, oats, or turf grass; preferably, the monocotyledonous plant is maize, rice, sorghum, wheat, barley, millet, sugar cane or oats.
The term "plant transformation" in the context of the present invention refers to the cloning of a herbicide resistant or tolerant polynucleotide into an expression system, alone or in combination with one or more additional nucleic acid molecules encoding polypeptides conferring a desired trait, into a plant cell. The receptor and target expression cassette of the present invention can be introduced into a plant cell in a variety of well-known methods. In the context of a polynucleotide, the term "introduced" (e.g., a nucleotide construct of interest) is intended to mean that the polynucleotide is provided to the plant in such a way that the polynucleotide gains access to or is achieved by the interior of a plant cell. Where more than one polynucleotide is to be introduced, these polynucleotides may be assembled as part of a single nucleotide construct, or as separate nucleotide constructs, and may be located on the same or different transformation vectors. Thus, or as part of a breeding scheme, the polynucleotides may be introduced into a host cell of interest, e.g., in a single transformation event in a plant, in separate transformation events. The methods of the invention do not depend on a particular method for introducing one or more polynucleotides into a plant, but merely obtain access or achievement of the polynucleotide(s) to the interior of at least one cell of a plant. Methods known in the art for introducing one or more polynucleotides into a plant include, but are not limited to, transient transformation methods, stable transformation methods, virus-mediated methods, or genome editing techniques.
The term "stable transformation" refers to the introduction of an exogenous gene into the genome of a plant and stable integration into the genome of the plant and any successive generation thereof, resulting in stable inheritance of the exogenous gene.
The term "transient transformation" refers to the introduction of a nucleic acid molecule or protein into a plant cell that performs a function but does not integrate into the plant genome, resulting in the inability of a foreign gene to be stably inherited.
The term "genome editing technology" refers to a genome modification technology that can perform precise operations on genome sequences to achieve operations such as site-directed mutagenesis, insertion, deletion, and the like of genes. Currently, the genome editing technologies mainly include HE (home endonucleases), ZFN (Zinc finger nucleases), TALEN (transcription activator-like effector nucleases), CRISPR (Clustered regularly interspaced short palindromic repeats).
Numerous transformation vectors available for plant transformation are known to those skilled in the art, and the genes associated with the present invention can be used in combination with any of the above vectors. The choice of vector will depend on the preferred transformation technique and the species of interest for transformation. For certain target species, different antibiotic or herbicide selection markers may be preferred. Selection markers conventionally used in transformation include the nptll gene conferring resistance to kanamycin and related antibiotics or related herbicides (this gene was published by Bevan et al in 1983 at page 184-187 of Nature Science 304), the pat and bar genes conferring resistance to the herbicide glufosinate (also known as glufosinate; see White et al in 1990 at page 1062 of Nucl. AcidsRes 18, Spencer et al in 1990 at page 79 of the door. Appl. Genet. 631 and US patents 5561236 and 5276268), the hpn gene conferring resistance to the antibiotic hygromycin (Blochinger & Diggelmann, mol. biol.4:2929-2931) and the Boufr gene conferring resistance to methotrexate (Glyphosate et al in 1983 at page 2 of EMBO J9), the Boufr gene conferring resistance to glyphosate (see also WO 1104-519) and the glyphosate gene conferring resistance to methotrexate (Glyphosate gene) and the EP 881 gene conferring resistance to glyphosate (see WO 3, WO 2-WO 2), the glyphosate gene conferring resistance to glyphosate and the SPS-2004 at page 8811) 1154 pages; us published patents 20070004912, 20050246798 and 20050060767) and the 6-phosphomannose isomerase gene that provides metabolic mannose (us patents 5767378 and 5994629).
Methods for regenerating plants are also well known in the art. For example, Ti plasmid vectors have been utilized for delivery of foreign DNA, as well as direct DNA uptake, liposomes, electroporation, microinjection, and microprojectiles.
The planting system in the present invention refers to a combination of plants, any one of which exhibits herbicide tolerance and/or herbicide treatments available at different stages of plant development, resulting in plants with high yield and/or reduced damage.
In the present invention, the weeds mean plants that compete with cultivated target plants in the environment where the plants grow.
The terms "control" and/or "control" herein mean the application of at least an effective dose of a pyrazolone herbicide directly (e.g., by spraying) into the plant growing environment to minimize weed development and/or stop growth. At the same time, the cultivated plant of interest should be morphologically normal and can be cultured under conventional methods for consumption and/or production of the product; preferably, there is reduced plant damage and/or increased plant yield as compared to a non-transgenic wild type plant. The plant with reduced damage, including but not limited to improved stalk resistance, and/or increased kernel weight, etc. The "controlling" and/or "controlling" action of the herbicide tolerance protein on weeds can be present independently and not diminished and/or eliminated by the presence of other materials that can "control" and/or "control" weeds. In particular, any tissue of the transgenic plant (containing the gene encoding the herbicide tolerance protein) which is present and/or produced simultaneously and/or asynchronously, said herbicide tolerance protein and/or another substance which controls weeds, the presence of said another substance neither affecting the "controlling" and/or "controlling" action of said herbicide tolerance protein on weeds nor causing said "controlling" and/or "controlling" action to be effected wholly and/or partially by said another substance, independently of said herbicide tolerance protein.
"plant propagules" as used herein include, but are not limited to, vegetative propagules and vegetative propagules. The plant sexual propagules include, but are not limited to, plant seeds; the vegetative propagation body of the plant refers to a vegetative organ or a special tissue of the plant body, and can generate a new plant under the condition of in vitro; the vegetative organ or a specific tissue includes, but is not limited to, roots, stems and leaves, such as: plants with roots as vegetative propagules include strawberry, sweet potato, and the like; plants with stems as vegetative propagules include sugarcane and potato (tubers); the plant with leaves as asexual propagules includes aloe, begonia, etc.
The present invention can impart new herbicide resistance traits to plants, and no adverse effect on phenotype including yield was observed. The plants of the invention are tolerant to, for example, at least one herbicide subject to 2 x, 3 x, or 4 x normal use levels. These increased levels of resistance are within the scope of the present invention. For example, various techniques known in the art can be predictably optimized and further developed to increase expression of a given gene.
The invention provides a herbicide tolerance protein, a coding gene and application thereof, and has the following advantages:
1. the herbicide tolerance protein can endow plants with tolerance to HPPD inhibitor herbicide, and can tolerate the topramezone with field concentration of at least 1 time.
2. The herbicide tolerance protein has wide application prospect in plants.
The technical solution of the present invention is further described in detail by the accompanying drawings and embodiments.
Drawings
FIG. 1 is a schematic structural diagram of a prokaryotic recombinant expression vector DBN11765 containing the HP1 nucleotide sequence;
FIG. 2 is a graph showing the effect of enzyme activity of the herbicide tolerance protein HP1 under conditions of different concentration gradients of HPPD inhibitor herbicides (topramezone, mesotrione or diketonitrile);
FIG. 3 is a graph showing the effect of enzyme activity of the herbicide tolerance protein HP1-1 under conditions of different concentration gradients of HPPD inhibitor herbicides (topramezone, mesotrione or diketonitrile);
FIG. 4 is a graph showing the effect of enzyme activity of the herbicide tolerance protein HP1-2 under conditions of different concentration gradients of HPPD inhibitor herbicides (topramezone, mesotrione or diketonitrile);
FIG. 5 is a schematic structural diagram of an Arabidopsis thaliana recombinant expression vector DBN11770 containing the HP1M nucleotide sequence;
FIG. 6 is a schematic structural diagram of a control recombinant expression vector DBN11770N of the present invention.
Detailed Description
The technical schemes of the herbicide tolerance protein, the coding gene and the application thereof are further illustrated by the following specific examples.
First example, mutation and screening of HP1 Gene
1. Synthesis of HP1 Gene
The nucleotide sequence of the HP1 gene is synthesized and is shown as SEQ ID NO. 2 in the sequence table, and the HP1 protein is coded and is shown as SEQ ID NO. 1 in the sequence table. The nucleotide sequence of HP1M which codes the amino acid sequence corresponding to the HP1 is obtained according to the preference codon of Arabidopsis thaliana, and is shown as SEQ ID NO. 3 in the sequence table.
2. Construction of HP1 Gene mutation library
The synthesized HP1 gene was PCR-amplified, cloned into pUC118 vector according to the procedure described in the pUC118 vector (Takara, CAT: 3318) available from Takara, and the ligated product was introduced into E.coli DH5 α as a template, and error-prone PCR was performed using a forward primer and a reverse primer, so that the HP1 gene was mutated by random base mismatching.
The primers and error-prone PCR reaction system are as follows:
a forward primer: 5' -cccaagcttgatggccgccgattccgaaaatc-3' as shown in SEQ ID NO:4 in the sequence Listing (HindIII restriction sites are underlined);
reverse primer: 5' -cgcggatcctcaggcatcgaccgtcacaacg-3', as shown in SEQ ID NO:5 in the sequence Listing (underlined is BamHI enzyme cutting site);
the error-prone PCR reaction system (total volume 50. mu.L) was:
the concentration of the plasmid DNA template is 1-10 ng/. mu.L, the concentration of the forward primer is 10. mu.M, and the concentration of the reverse primer is 10. mu.M.
The error-prone PCR reaction conditions were:
and (2) carrying out agarose gel electrophoresis on the error-prone PCR product by 7g/L, cutting the gel and recovering, carrying out double enzyme digestion on the error-prone PCR product subjected to gel cutting recovery and a pUC118 vector by utilizing HindIII and BamHI, purifying the enzyme digestion product, carrying out enzyme ligation by T4-DNA ligase, and then transforming the enzyme digestion product into escherichia coli DH5 alpha sensitive to topramezone to construct an HP1 gene random mutation library.
3. Screening of HP1 Gene mutation library
The transformants in the library were inoculated into 48-well plates containing 300. mu.M of topramezone in 2mL of LB liquid medium (ampicillin 100mg/L, tyrosine 800mg/L) and shake-cultured in a shaker at a constant temperature of 180rpm and 37 ℃. Observing the growth condition of the strain, adding 1mM IPTG into the LB liquid culture medium when the strain grows to OD 0.4-0.6, continuing to culture, and observing the color change of the culture medium.
As tyrosine aminotransferase exists in Escherichia coli, tyrosine can be catalyzed to generate p-hydroxyphenyl pyruvic acid (HPP), the HPP can be used as a substrate of the HPPD, a product HG after the catalytic reaction of the HPPD can be spontaneously oxidized and polymerized to generate brownish red puama, but the HPPD inhibitor can inhibit the activity of the HPPD enzyme and further inhibit the color generation. Thus, the mutant library can be screened at high throughput based on whether it produces a red-brown color in LB liquid medium, and E.coli DH 5. alpha. that still produces a red-brown color on the medium containing 300. mu.M topramezone can be isolated to obtain a resistance gene.
4. Obtaining a mutated resistance gene
Sequencing results show that two HP1 mutant resistance genes are obtained and are respectively named as HP1-1 and HP1-2, the 475 th site of the HP1-1 nucleotide sequence is mutated from the original A to G, and the 159 th site of the amino acid sequence is mutated from the original threonine to alanine; the 772 th and 773 th positions of the HP1-2 nucleotide sequence are mutated from original CA to AT, so that the 258 th position of the amino acid sequence is mutated from original glutamine to methionine.
The amino acid sequence of the herbicide tolerance protein HP1-1 is shown as SEQ ID NO. 6 in the sequence table, and the nucleotide sequence of HP1-1 which codes the amino acid sequence corresponding to the herbicide tolerance protein HP1-1 is shown as SEQ ID NO. 7 in the sequence table; an HP1-1M nucleotide sequence encoding an amino acid sequence corresponding to the herbicide tolerance protein HP1-1 is obtained according to the Arabidopsis thaliana preference codon, and is shown as SEQ ID NO:8 in the sequence table.
The amino acid sequence of the herbicide tolerance protein HP1-2 is shown as SEQ ID NO. 9 in the sequence table, and the nucleotide sequence of HP1-2 which codes the amino acid sequence corresponding to the herbicide tolerance protein HP1-2 is shown as SEQ ID NO. 10 in the sequence table; an HP1-2M nucleotide sequence encoding an amino acid sequence corresponding to the herbicide tolerance protein HP1-2 was obtained according to Arabidopsis thaliana bias codons, as shown in SEQ ID NO:11 of the sequence Listing.
Second example, testing of the tolerance Effect of the herbicide tolerance proteins HP1-1 and HP1-2 on HPPD inhibitor herbicides
1. Synthesis of nucleotide sequences for HP1, HP1-1, and HP1-2
The 5 'and 3' ends of the HP1 nucleotide sequence (SEQ ID NO:2), the HP-1 nucleotide sequence (SEQ ID NO:7) and the HP1-2 nucleotide sequence (SEQ ID NO:10) were respectively connected to a universal adapter primer 1:
5' end universal adaptor primer 1: 5'-taagaaggagatatacatatg-3', as shown in SEQ ID NO:12 of the sequence Listing;
3' end universal adaptor primer 1: 5'-gtggtggtggtggtgctcgag-3', as shown in SEQ ID NO 13 of the sequence list.
2. Construction of prokaryotic recombinant expression vector and obtaining of recombinant strain
The prokaryotic expression vector DBNBC-01 is linearized by utilizing restriction enzymes Nde I and Xho I, the digestion product is purified to obtain a linearized DBNBC-01 expression vector skeleton (vector skeleton: pET-29a (+)), the HP1 nucleotide sequence connected with the universal joint primer 1 and the linearized DBNBC-01 expression vector skeleton are subjected to recombination reaction, the operation steps are carried out according to the instruction of an In-Fusion seamless connection product kit (Clontech, CA, USA, CAT: 121416) of Takara company, a recombinant expression vector DBN11765 is constructed, the vector structure is shown as figure 1 (F1 Ori: the replication origin of bacteriophage F1; Kan: kanamycin resistance gene; Ori: the replication origin; Rop: Rop gene, encoding ROP protein; T7 promoter: T7 RNA promoter; HP 1: HP1 nucleotide sequence (SEQ ID NO: 2); affinity tag 6xHis, encoding 6 consecutive His as a tag for purification of the target protein; t7 terminator: a terminator of T7 RNA polymerase; LacI operator: a manipulation region of the lactose operon; lacI promoter: a lacI promoter; lacI: lactose operon regulatory gene).
The recombinant expression vector DBN11765 is used for transforming escherichia coli BL21(DE3) competent cells by a heat shock method, wherein the heat shock conditions are as follows: 50 μ L of E.coli BL21(DE3) competent cells, 10 μ L of plasmid DNA (recombinant expression vector DBN11765), water bath at 42 ℃ for 30 s; shaking at 37 deg.C for 1h (shaking table at 100 rpm); then, the mixture was cultured on the LB solid plate containing 50mg/L of Kanamycin (Kanamycin) at 37 ℃ for 12 hours, and white colonies were picked up and cultured overnight at 37 ℃ in LB liquid medium (tryptone 10g/L, yeast extract 5g/L, NaCl 10g/L, Kanamycin 50mg/L, pH adjusted to 7.5 with NaOH). The plasmid is extracted by an alkaline method. Sequencing and identifying the extracted plasmid, wherein the result shows that the nucleotide sequences of the recombinant expression vector DBN11765 between Nde I sites and Xho I sites are respectively the nucleotide sequences shown by SEQ ID NO. 2 in the sequence table, and the recombinant strain BL21(HP1) is preserved for later use.
According to the method for constructing the recombinant expression vector DBN11765, the HP1-1 nucleotide sequence and the HP1-2 nucleotide sequence connected with the universal joint primer 1 are respectively subjected to recombination reaction with the linearized DBNBC-01 expression vector skeleton, and the recombinant expression vectors DBN11774 and DBN11775 are sequentially obtained. The recombinant expression vectors DBN11774 and DBN11775 are respectively transformed into competent cells of escherichia coli BL21(DE3) by a heat shock method, plasmids of the competent cells are extracted by an alkaline method, and sequencing verification is carried out on the extracted plasmids, so that the nucleotide sequences in the recombinant expression vectors DBN11774 and DBN11775 respectively contain the nucleotide sequence shown by SEQ ID NO. 7 and the nucleotide sequence shown by SEQ ID NO. 10 in a sequence table, namely the nucleotide sequence of HP1-1 and the nucleotide sequence of HP1-2 are correctly inserted. The resulting recombinant strains BL21(HP1-1) and BL21(HP1-2) were stored for future use.
3. Expression and purification of herbicide tolerance protein in escherichia coli
The recombinant strains BL21(HP1), BL21(HP1-1) and BL21(HP1-2) are respectivelyThe single clone was inoculated into 100mL of LB medium (tryptone 10g/L, yeast extract 5g/L, NaCl 10g/L, ampicillin 100mg/L, pH adjusted to 7.5 with NaOH) and cultured to OD 600nm 0.6-0.8, IPTG was added at a concentration of 0.4mM and induced at 16 ℃ for 12 h. The cells were collected, resuspended in 15mL of PBS buffer (50mM, pH 7.4), sonicated (X0-900D ultrasonic processor ultrasonic processor, 30% intensity) for 10min, centrifuged, and the supernatant collected, and the obtained herbicide-resistant proteins were purified by a nickel ion affinity column, and the size of the band was determined to be identical to the theoretically predicted band size by SDS-PAGE protein electrophoresis.
4. Determination of the tolerance of HP1, HP1-1 and HP1-2 to HPPD inhibitor herbicides
Enzyme activity reaction system (1 mL): contains 25. mu.g of a reaction enzyme (the herbicide-tolerant protein HP1, HP1-1 or HP1-2 obtained by the above purification), 0.2mM HPP substrate, 1mM Fe 2+ 1mM ascorbic acid, a gradient of topramezone (mesotrione or diketonitrile) at different concentrations (0. mu.M, 5. mu.M, 10. mu.M, 20. mu.M, 30. mu.M, 40. mu.M and 50. mu.M, respectively), in a buffer system of 50mM PBS buffer (pH 7.4), and reacted in a water bath at 30 ℃ for 20min, each reaction being started with the addition of the reaction enzyme and stopped in a boiling water bath for 5 min.
The enzyme reaction system thus terminated was centrifuged and filtered, and 20. mu.L of the filtrate was subjected to High Performance Liquid Chromatography (HPLC) analysis under the following conditions: the mobile phase was acetonitrile/water (30:70, V/V), 0.1% trifluoroacetic acid was added to the water. C18 reversed phase chromatographic column (5 μm, 250mm × 4.6mm), column temperature 40 deg.C, VWD-3100 single wavelength ultraviolet detector, detection wavelength 292nm, sample injection amount 20 μ L, and flow rate 1.0 mL/min. By HPLC analysis, the peak time of the enzymatic reaction product is consistent with that of the homogentisate standard sample, and no homogentisate is generated in the reaction system without adding enzyme. The resistance conditions of HP1, HP1-1 and HP1-2 are detected according to different concentrations of HPPD inhibitor herbicides (topramezone, mesotrione or diketonitrile) added into the system, and specifically are as follows: calculating relative enzyme activity when HPPD inhibitor herbicide is added by taking enzyme activity when HPPD inhibitor herbicide is not added as 100%, and measuring by HPLCDetermination of the amount of homogentisate produced to calculate the IC of HPPD inhibitor herbicides on HP1, HP1-1 and HP1-2 50 (concentration of HPPD inhibitor herbicide at which the enzyme activity catalyzes 50% inhibition). One unit of enzyme activity is defined as: the amount of enzyme required to produce 1. mu. mol of homogentisate within 1min at 30 ℃ and pH 7.4. The results of the experiments are shown in FIGS. 2-4 (graphs of the enzyme activity effects of the herbicide tolerance proteins HP1, HP1-1 and HP1-2 under different concentration gradients of HPPD inhibitor herbicides (topramezone, mesotrione or diketonitrile)), and IC of topramezone, mesotrione and diketonitrile for HP1, HP1-1 and HP1-2 were calculated by non-linear fitting using Prism 6.0 software 50 The results are shown in Table 1.
TABLE 1 half inhibitory concentrations of HPPD inhibitor herbicides topramezone, mesotrione and diketonitrile for HP1, HP1-1 and HP1-2
The results in table 1 show that: the herbicide tolerance proteins HP1, HP1-1 and HP1-2 all have varying degrees of tolerance to HPPD inhibitor herbicides (topramezone, mesotrione and diketonitrile); the purified herbicide tolerance proteins HP1-1 and HP1-2 were more resistant to HPPD inhibitor herbicides than the herbicide tolerance protein HP 1.
Third example, obtaining and validation of transgenic Arabidopsis plants
1. Respectively constructing arabidopsis thaliana recombinant expression vectors containing HP1, HP1-1 or HP1-2 genes
The 5 'and 3' ends of the HP1M nucleotide sequence (SEQ ID NO:3) described in the above first example 1, the HP1-1M nucleotide sequence (SEQ ID NO:8) described in the above first example 4, and the HP1-2M nucleotide sequence (SEQ ID NO:11) were ligated to universal adapter primer 2:
5' end universal adaptor primer 2: 5'-agtttttctgattaacagactagt-3', as shown in SEQ ID NO:14 of the sequence Listing;
3' end universal adaptor primer 2: 5'-caaatgtttgaacgatcggcgcgcc-3', as shown in SEQ ID NO. 15 of the sequence list.
Carrying out double enzyme digestion reaction on a plant expression vector DBNBC-02 by using restriction enzymes Spe I and Asc I, thereby linearizing the plant expression vector, purifying enzyme digestion products to obtain a linearized DBNBC-02 expression vector skeleton (the vector skeleton: pCAMBIA2301 (can be provided by CAMBIA organization)), carrying out recombination reaction on the HP1M nucleotide sequence connected with the universal joint primer 2 and the linearized DBNBC-02 expression vector skeleton, and constructing a recombinant expression vector DBN11770 according to the instruction of Takara In-Fusion seamless connection product kit (Clontech, CA, USA, CAT: 121416), wherein the structural schematic diagram of the recombinant expression vector DBN11770 is shown In figure 5 (Spec: spectinomycin gene; right border; 34S enhancer of the mosaic figwort virus (TsCBV: 16); prCBP: promoter of rape eukaryotic elongation factor gene 1 alpha (chloroplast f1) (AtCTP: 17), sp 2: Arabidopsis peptide (SEQ ID NO: 18); EPSPS: 5-enolpyruvylshikimate-3-phosphate synthase gene (SEQ ID NO: 19); tPSE 9: the terminator of the pea RbcS gene (SEQ ID NO: 20); prAtUbi 10: the promoter of the Arabidopsis Ubiquitin (Ubiquitin)10 gene (SEQ ID NO: 21); spAtCTP 2: arabidopsis chloroplast transit peptide (SEQ ID NO: 18); HP 1M: HP1M nucleotide sequence (SEQ ID NO: 3); tNos: a terminator of the nopaline synthase gene (SEQ ID NO: 22); pr 35S: the cauliflower mosaic virus 35S promoter (SEQ ID NO: 23); PAT: phosphinothricin N-acetyltransferase gene (SEQ ID NO: 24); t 35S: cauliflower mosaic virus 35S terminator (SEQ ID NO: 25); LB: left border).
Transforming the recombinant expression vector DBN11770 into Escherichia coli T by a heat shock method 1 Competent cells under heat shock conditions: 50 μ L of E.coli T 1 Competent cells, 10. mu.L of plasmid DNA (recombinant expression vector DBN11770), water bath at 42 ℃ for 30 s; shaking at 37 deg.C for 1h (shaking table at 100 rpm); then, the mixture was cultured on the LB solid plate (tryptone 10g/L, yeast extract 5g/L, NaCl 10g/L, agar 15g/L, pH adjusted to 7.5 with NaOH) containing 50mg/L Spectinomycin (Spectinomycin) at 37 ℃ for 12 hours, white colonies were picked up, and the mixture was cultured on LB liquid medium (tryptone 10g/L, yeast extract 5g/L, NaCl 10g/L, Spectinomycin 50mg/L, N)aOH to pH 7.5) at 37 ℃ overnight. Extracting the plasmid by an alkaline method: centrifuging the bacterial solution at 12000rpm for 1min, removing supernatant, and suspending the precipitated bacterial solution with 100 μ l ice-precooled solution I (25mM Tris-HCl, 10mM EDTA (ethylene diamine tetraacetic acid), 50mM glucose, pH 8.0); add 200. mu.L of freshly prepared solution II (0.2M NaOH, 1% SDS (sodium dodecyl sulfate)), invert the tube 4 times, mix, and place on ice for 3-5 min; adding 150 μ L ice-cold solution III (3M potassium acetate, 5M acetic acid), mixing well immediately, and standing on ice for 5-10 min; centrifuging at 4 deg.C and 12000rpm for 5min, adding 2 times volume of anhydrous ethanol into the supernatant, mixing, and standing at room temperature for 5 min; centrifuging at 4 deg.C and 12000rpm for 5min, removing supernatant, washing precipitate with 70% ethanol (V/V), and air drying; adding 30. mu.L of TE (10mM Tris-HCl, 1mM EDTA, pH8.0) containing RNase (20. mu.g/mL) to dissolve the precipitate; bathing in water at 37 deg.C for 30min to digest RNA; storing at-20 deg.C for use. The extracted plasmid is subjected to sequencing identification, and the result shows that the nucleotide sequence of the recombinant expression vector DBN11770 between Spe I and Asc I sites is the nucleotide sequence shown by SEQ ID NO. 3 in the sequence table, namely the HP1M nucleotide sequence.
According to the method for constructing the recombinant expression vector DBN11770, the HP1-1M nucleotide sequence and the HP1-2M nucleotide sequence which are respectively connected with the universal joint primer 2 are respectively subjected to recombination reaction with the linearized DBNBC-02 expression vector skeleton, and the recombinant expression vectors DBN11777 and DBN11778 are sequentially obtained. The nucleotide sequences in the recombinant expression vectors DBN11777 and DBN11778 respectively contain the nucleotide sequence shown by SEQ ID NO. 8 and the nucleotide sequence shown by SEQ ID NO. 11 in the sequence table through sequencing verification, namely the HP1-1M nucleotide sequence and the HP1-2M nucleotide sequence are correctly inserted.
According to the above-mentioned method for constructing recombinant expression vector DBN11770, a control recombinant expression vector DBN11770N was constructed, and its vector structure was shown in FIG. 6 (Spec: spectinomycin gene; RB: right border; eFMV: enhancer of 34S of figwort mosaic virus (SEQ ID NO:16), prBrCBP: promoter of rape eukaryotic elongation factor gene 1 α (Tsf1) (SEQ ID NO:17), spAtCTP 2: Arabidopsis chloroplast transit peptide (SEQ ID NO:18), EPSPS: 5-enolpyruvylshikimate-3-phosphate synthase gene (SEQ ID NO:19), tPSE 9: terminator of pea RbcS gene (SEQ ID NO:20), pr 35S: cauliflower mosaic virus 35S promoter (SEQ ID NO:23), PAT: phosphinothricin N-acetyltransferase gene (SEQ ID NO:24), t 35S: cauliflower mosaic virus terminator (SEQ ID NO:25), LB: left border).
2. Agrobacterium transformed by arabidopsis recombinant expression vector
The correctly constructed recombinant expression vectors DBN11770, DBN11777, DBN11778 and the control recombinant expression vector DBN11770N are transformed into agrobacterium GV3101 by a liquid nitrogen method respectively, wherein the transformation conditions are as follows: 100. mu.L of Agrobacterium GV3101, 3. mu.L of plasmid DNA (recombinant expression vector); placing in liquid nitrogen for 10min, and heating in 37 deg.C water bath for 10 min; inoculating the transformed agrobacterium GV3101 into an LB test tube, culturing for 2h at the temperature of 28 ℃ and the rotation speed of 200rpm, smearing the agrobacterium GV3101 on an LB solid plate containing 50mg/L Rifampicin (Rifampicin) and 50mg/L spectinomycin until positive monoclonals grow out, picking the monoclonals, culturing and extracting plasmids of the monoclonals, and sequencing and identifying the extracted plasmids, wherein the results show that the structures of the recombinant expression vectors DBN11770, DBN11777, DBN11778 and DBN11770N are completely correct.
3. Obtaining transgenic Arabidopsis plants
Wild type Arabidopsis seeds were suspended in 0.1% (w/v) agarose solution. The suspended seeds were stored at 4 ℃ for 2 days to complete the need for dormancy to ensure synchronous germination of the seeds. Horse dung soil was mixed with vermiculite and irrigated with water underground until moist, and the soil mixture was drained for 24 h. The pretreated seeds were planted on the soil mixture and covered with a moisture-retaining cover for 7 days. Germinating the seeds and keeping the light intensity at a constant temperature (22 ℃) and a constant humidity (40-50%) of 120-150 mu mol/m 2 s -1 Under long-day conditions (16h light/8 h dark) plants were grown in a greenhouse. Plants were initially irrigated with Hoagland nutrient solution followed by deionized water to keep the soil moist but not drenched.
Arabidopsis thaliana was transformed using the floral dip method. One or more 15-30mL precultures of LB broth containing spectinomycin (50mg/L) and rifampicin (10mg/L) were inoculated with selected Agrobacterium colonies. The preculture was incubated overnight at a temperature of 28 ℃ with constant shaking at 220 rpm. Each preculture was used to inoculate two 500ml cultures of the YEP broth containing spectinomycin (50mg/L) and rifampicin (10mg/L) and the cultures were incubated overnight at a temperature of 28 ℃ with continuous shaking. The cells were pelleted by centrifugation at about 4000rpm for 20min at room temperature and the resulting supernatant was discarded. The cell pellet was gently resuspended in 500mL of osmotic medium containing 1/2 XMS salt/B5 vitamins, 10% (w/v) sucrose, 0.044. mu.M benzylaminopurine (10. mu.L/L (stock in 1mg/mL DMSO)), and 300. mu.L/LSilvet L-77. About 1 month old arabidopsis plants were soaked for 5min in the osmotic medium containing resuspended cells to ensure that the latest inflorescences were submerged. Then, the Arabidopsis thaliana plants were placed on their sides and covered, and were kept moist for 24 hours in a dark environment, and then were normally cultured at a temperature of 22 ℃ under 16 hours of light/8 hours of dark photoperiod. The seeds were harvested after about 4 weeks.
Freshly harvested (HP1M nucleotide sequence, HP1-1M nucleotide sequence, HP1-2M nucleotide sequence, and control vector DBN11770N) T 1 The seeds were dried at room temperature for 7 days. Seeds were planted in 26.5cm by 51cm germinating discs, each receiving 200mg of T 1 Seeds (approximately 10000 seeds) which had been previously suspended in distilled water and stored at a temperature of 4 ℃ for 2 days to complete the need for dormancy to ensure synchronous germination of the seeds.
Mixing the horse dung with vermiculite, irrigating with water until the mixture is wet, and draining water by gravity. The pretreated seeds were evenly seeded on the soil mixture with a pipette and covered with a moisture hood for 4-5 days. The hood was removed 1 day prior to initial transformant selection using post-emergence glufosinate-ammonium spray (selection for co-transformed PAT gene). After 7 planting Days (DAP) and again at 11DAP, using a DeVilbiss compressed air nozzle, T is sprayed with a 0.2% solution of Liberty herbicide (200g ai/L glufosinate) at a spray volume of 10 mL/dish (703L/ha) 1 Plants (cotyledonary stage and 2-4 leaf stage, respectively) to provide an effective amount of glufosinate of 280g ai/ha per application. Surviving plants (actively growing plants) were identified 4-7 days after the final spray and transplanted into 7cm x 7cm square pots (3-5 per dish) made with horse manure and vermiculite, respectively. Covering the transplanted plants with a moisture-retaining cover for 3-4 days, and placing in a culture room at 22 deg.C orDirectly transferred into a greenhouse. The caps were then removed and the plants were planted in a greenhouse (temperature 22 + -5 deg.C, 50 + -30% RH, 14h light: 10h dark, minimum 500. mu.E/M) for at least 1 day before testing the ability of the HP1M gene, HP1-1M gene, and HP1-2M gene to provide tolerance to pyrazoxolone herbicides 2 s -1 Natural + supplemental light).
4. Detection of herbicide tolerance effects in transgenic Arabidopsis plants
First T was selected from a background of untransformed seeds using a glufosinate selection protocol 1 A transformant. The transgenic recombinant expression vector DBN11770 is an Arabidopsis plant (HP1) transferred with an HP1M nucleotide sequence, the transgenic recombinant expression vector DBN11777 is an Arabidopsis plant (HP1-1) transferred with an HP1-1M nucleotide sequence, the transgenic recombinant expression vector DBN11778 is an Arabidopsis plant (HP1-2) transferred with an HP1-2M nucleotide sequence, and the transgenic recombinant expression vector DBN11770N is an Arabidopsis plant (control vector) transferred with a control recombinant expression vector. About 20000 HP 1T were selected 1 Seeds and 213T strains were identified 1 Passage-positive transformants (PAT gene), a transformation efficiency of about 1.0%; about 20000 HP 1-1T were screened 1 Seed and identified 195T strains 1 Generation-positive transformants (PAT gene), transformation efficiency of about 1.0%; about 20000 HP 1-2T were screened 1 Seed and identified 195T strains 1 Generation-positive transformants (PAT gene), transformation efficiency of about 1.0%; about 20000 control vectors were screened for T 1 Seeds, and 172T strains were identified 1 Passage-positive transformants (PAT gene), about 0.86% transformation efficiency.
HP1 Arabidopsis thaliana T 1 Plant, HP1-1 Arabidopsis thaliana T 1 Plant, HP1-2 Arabidopsis thaliana T 1 Arabidopsis thaliana T of plant and control vector 1 Plants and wild type arabidopsis thaliana plants (CK) (18 days after sowing) were sprayed with 3 concentrations of topramezone to test arabidopsis thaliana for herbicide tolerance, namely 25gai/ha (1-fold field strength, 1 ×), 100g ai/ha (4-fold field strength, 4 ×) and 0g ai/ha (water, 0 ×). After 7 days of spraying (7DAT), the herbicide-receiving status of each plant was counted according to the leaf whitening area ratio (leaf whitening area ratio ═ leaf whitening area/total leaf area × 100%)Degree of damage: the substantially non-whitening phenotype is grade 0, the leaf whitening area proportion is less than 50% and grade 1, the leaf whitening area proportion is more than 50% and grade 2, and the leaf whitening area proportion is 100% and grade 3. According to the formula X ═ Σ (NxS)/(TxM)]X 100 the resistance performance of the transformation event for each recombinant expression vector was scored. (X-phytotoxicity score, N-same-stage damaged plant number, S-phytotoxicity grade number, T-total plant number and M-highest phytotoxicity grade), and carrying out resistance evaluation according to the score: high resistant plants (0-15 points), medium resistant plants (16-33 points), low resistant plants (34-67 points), and non-resistant plants (68-100 points). The results of the experiment are shown in table 2.
TABLE 2 transgenic Arabidopsis T 1 Tolerance experiment result of plant topramezone
The results in table 2 show that: compared with Arabidopsis thaliana T transferred into control vector DBN11770N 1 Plants and wild type Arabidopsis thaliana plants, Arabidopsis thaliana T with HP1M nucleotide sequence transferred 1 Plant, Arabidopsis thaliana T transferred with HP1-1M nucleotide sequence 1 Plant and Arabidopsis thaliana T transferred with HP1-2M nucleotide sequence 1 The plants can generate tolerance to topramezone to different degrees; compared with Arabidopsis thaliana T with HP1M nucleotide sequence 1 Plant, Arabidopsis thaliana T transformed with HP1-1M nucleotide sequence 1 Plant and Arabidopsis thaliana T transferred with HP1-2M nucleotide sequence 1 The plant has better resistance to topramezone.
In conclusion, when the amino acid sequence of the herbicide tolerance protein HP1 is mutated from threonine to alanine at the 159 th position or from glutamine to methionine at the 258 th position (the herbicide tolerance protein HP1-1 or HP1-2) the protein can show higher tolerance to HPPD inhibitor herbicides, and the herbicide tolerance protein can tolerate the topramezone with 1-fold field concentration, so the protein has wide application prospect on plants.
Finally, it should be noted that the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Sequence listing
<110> Beijing Dabei agricultural Biotechnology Co., Ltd
Nanjing Agricultural University
<120> herbicide tolerance protein, encoding gene and use thereof
<130> DBNBC152
<160> 25
<170> SIPOSequenceListing 1.0
<210> 1
<211> 350
<212> PRT
<213> amino acid sequence of HP1 (Sphingobacterium)
<400> 1
Met Ala Ala Asp Ser Glu Asn Pro Leu Gly Leu His Gly Phe Ala Phe
1 5 10 15
Ala Glu Phe Thr Ser Pro Asp Pro Ala Ala Met Ala Arg Gln Phe Glu
20 25 30
Gln Leu Gly Phe Val Pro Ala Ala Arg Arg Lys Asp Arg Gly Leu Thr
35 40 45
Leu Tyr Arg Gln Gly Arg Ile Ala Phe Ile Leu Asn Ala Gly Glu Gly
50 55 60
Glu Gln Ala Ser Ala Phe Arg Ala Ala His Gly Pro Ser Ala Asn Gly
65 70 75 80
Met Ala Phe Asn Val Ala Asp Ala Lys Ala Ala His Arg His Ala Ile
85 90 95
Gln Ser Gly Ala Thr Asp Ala Asp Thr Ala Gln Ser Ala Leu Pro Gly
100 105 110
Thr Tyr Ala Ile Glu Gly Ile Gly Asp Ser Leu Leu Tyr Leu Val Asp
115 120 125
Ser Asp Pro Phe Ala Asp Trp Asp Glu Val Pro Gly Trp Arg Glu Ala
130 135 140
Ser Ala Glu Arg Gly Val Gly Leu Asp Leu Leu Asp His Leu Thr His
145 150 155 160
Asn Val Arg Arg Gly Gln Met Arg Val Trp Ser Glu Phe Tyr Ala Thr
165 170 175
Leu Phe Gly Phe Glu Glu Gln Lys Phe Phe Asp Ile Lys Gly Gln Ala
180 185 190
Thr Gly Leu Phe Ser Gln Ala Met Ile Ala Pro Asp His Glu Ile Arg
195 200 205
Ile Pro Leu Asn Glu Ser Gln Asp Asp Asn Ser Gln Ile Glu Glu Phe
210 215 220
Ile Arg Glu Tyr Asn Gly Glu Gly Ile Gln His Leu Ala Leu Thr Thr
225 230 235 240
Pro Asp Ile Tyr Ser Thr Val Glu Lys Leu Arg Ala Asn Gly Val Arg
245 250 255
Leu Gln Asp Thr Ile Asp Thr Tyr Tyr Asp Leu Val Asp Glu Arg Val
260 265 270
Pro Gly His Gly Glu Asp Leu Ala Arg Leu Arg Lys Asn Arg Ile Leu
275 280 285
Ile Asp Gly Asp Val His Asp Glu Gly Leu Leu Leu Gln Ile Phe Thr
290 295 300
Glu Thr Met Phe Gly Pro Ile Phe Phe Glu Ile Ile Gln Arg Lys Gly
305 310 315 320
Asn Glu Gly Phe Gly Asn Gly Asn Phe Gln Val Leu Tyr Glu Ser Ile
325 330 335
Glu Leu Asp Gln Ile Arg Arg Gly Val Val Thr Val Asp Ala
340 345 350
<210> 2
<211> 1053
<212> DNA
<213> nucleotide sequence of HP1 (Sphingobacterium)
<400> 2
atggccgccg attccgaaaa tccgttgggc ctgcatggct ttgcgtttgc tgaatttacc 60
tcacccgatc cggccgcgat ggcgcggcag ttcgagcagc ttggatttgt tccggcggcg 120
cgccggaagg acagggggct gacgctttac cggcaggggc gcatcgcgtt cattctgaat 180
gccggggagg gcgaacaggc cagcgcgttt cgcgccgccc atggcccatc cgccaacggc 240
atggcgttta acgtcgccga cgcgaaagca gcgcaccggc acgccattca aagtggggcg 300
acagacgctg ataccgcgca gagcgctctg ccggggactt atgccattga gggcataggc 360
gactcgctgc tctatctggt ggacagcgac ccctttgcgg actgggatga ggtgcccggt 420
tggcgagagg cgtcggcgga gcggggcgtc ggcctcgacc tgctcgacca cctgacccat 480
aatgtccgcc gggggcagat gcgcgtatgg tccgaattct acgcgacgct gttcggtttc 540
gaggaacaga agttcttcga catcaaaggg caggcgaccg gtctgttcag ccaggcgatg 600
atcgccccgg atcatgaaat ccgcatccct ctgaatgaaa gccaggatga taacagccag 660
atcgaggagt ttatccgcga atataatggc gagggtatcc agcatctcgc cctgacgacg 720
ccggatatct attccacggt ggagaagcta cgcgccaatg gcgtgcggct gcaggatacg 780
atcgacacct attatgatct ggtcgacgag cgggtgccgg ggcatggcga ggatctggcg 840
cgattgcgca aaaaccgcat cctgatcgat ggcgatgtcc atgatgaggg gctgctgctg 900
cagatattca ccgagactat gttcggcccg atcttcttcg agatcatcca gcgcaagggc 960
aatgagggtt tcgggaacgg caatttccag gtactctacg aatccataga attggaccag 1020
atccggcgcg gcgttgtgac ggtcgatgcc tga 1053
<210> 3
<211> 1053
<212> DNA
<213> nucleotide Sequence of HP1M (Artificial Sequence)
<400> 3
atggctgcag attctgagaa cccattgggt cttcatggat ttgctttcgc agagtttact 60
tctccagatc cagctgcaat ggctagacaa tttgagcagt tgggtttcgt tcctgctgca 120
agaaggaagg ataggggttt gactctttat agacaaggaa ggattgcttt tattcttaat 180
gcaggagagg gtgaacaggc ttctgcattc agagctgcac acggtccatc agctaacgga 240
atggctttta acgtggctga tgcaaaggct gcacataggc acgctattca atctggtgct 300
actgatgcag atacagctca gtcagcattg cctggaacat atgctattga gggaattgga 360
gattctcttt tgtaccttgt tgattcagat ccattcgcag attgggatga agtgcctgga 420
tggagagagg cttctgcaga aaggggagtt ggtttggatc ttttggatca tcttacccac 480
aacgttagaa ggggtcaaat gagagtgtgg tcagagttct acgctacttt gtttggattc 540
gaggaacaga agtttttcga tattaagggt caagcaacag gattgttttc tcaggctatg 600
attgcaccag atcatgagat taggattcca cttaacgaat ctcaagatga taactcacag 660
atcgaggagt ttattaggga gtataacgga gaaggtattc aacacttggc tcttactaca 720
ccagatatct attcaacagt tgaaaagttg agagcaaatg gtgtgaggct tcaagataca 780
atcgatacct attacgattt ggttgatgag agagtgcctg gacatggtga agatttggct 840
agacttagga agaacaggat tcttattgat ggagatgttc acgatgaggg acttttgctt 900
caaattttta ccgaaactat gttcggtcca attttctttg agattattca gaggaaggga 960
aacgagggat ttggtaacgg aaacttccaa gttttgtacg agtctatcga acttgatcag 1020
attagaaggg gagttgtgac cgtggatgct tga 1053
<210> 4
<211> 32
<212> DNA
<213> Forward primer (Artificial Sequence)
<400> 4
cccaagcttg atggccgccg attccgaaaa tc 32
<210> 5
<211> 31
<212> DNA
<213> reverse primer (Artificial Sequence)
<400> 5
cgcggatcct caggcatcga ccgtcacaac g 31
<210> 6
<211> 350
<212> PRT
<213> amino acid Sequence of HP1-1 (Artificial Sequence)
<400> 6
Met Ala Ala Asp Ser Glu Asn Pro Leu Gly Leu His Gly Phe Ala Phe
1 5 10 15
Ala Glu Phe Thr Ser Pro Asp Pro Ala Ala Met Ala Arg Gln Phe Glu
20 25 30
Gln Leu Gly Phe Val Pro Ala Ala Arg Arg Lys Asp Arg Gly Leu Thr
35 40 45
Leu Tyr Arg Gln Gly Arg Ile Ala Phe Ile Leu Asn Ala Gly Glu Gly
50 55 60
Glu Gln Ala Ser Ala Phe Arg Ala Ala His Gly Pro Ser Ala Asn Gly
65 70 75 80
Met Ala Phe Asn Val Ala Asp Ala Lys Ala Ala His Arg His Ala Ile
85 90 95
Gln Ser Gly Ala Thr Asp Ala Asp Thr Ala Gln Ser Ala Leu Pro Gly
100 105 110
Thr Tyr Ala Ile Glu Gly Ile Gly Asp Ser Leu Leu Tyr Leu Val Asp
115 120 125
Ser Asp Pro Phe Ala Asp Trp Asp Glu Val Pro Gly Trp Arg Glu Ala
130 135 140
Ser Ala Glu Arg Gly Val Gly Leu Asp Leu Leu Asp His Leu Ala His
145 150 155 160
Asn Val Arg Arg Gly Gln Met Arg Val Trp Ser Glu Phe Tyr Ala Thr
165 170 175
Leu Phe Gly Phe Glu Glu Gln Lys Phe Phe Asp Ile Lys Gly Gln Ala
180 185 190
Thr Gly Leu Phe Ser Gln Ala Met Ile Ala Pro Asp His Glu Ile Arg
195 200 205
Ile Pro Leu Asn Glu Ser Gln Asp Asp Asn Ser Gln Ile Glu Glu Phe
210 215 220
Ile Arg Glu Tyr Asn Gly Glu Gly Ile Gln His Leu Ala Leu Thr Thr
225 230 235 240
Pro Asp Ile Tyr Ser Thr Val Glu Lys Leu Arg Ala Asn Gly Val Arg
245 250 255
Leu Gln Asp Thr Ile Asp Thr Tyr Tyr Asp Leu Val Asp Glu Arg Val
260 265 270
Pro Gly His Gly Glu Asp Leu Ala Arg Leu Arg Lys Asn Arg Ile Leu
275 280 285
Ile Asp Gly Asp Val His Asp Glu Gly Leu Leu Leu Gln Ile Phe Thr
290 295 300
Glu Thr Met Phe Gly Pro Ile Phe Phe Glu Ile Ile Gln Arg Lys Gly
305 310 315 320
Asn Glu Gly Phe Gly Asn Gly Asn Phe Gln Val Leu Tyr Glu Ser Ile
325 330 335
Glu Leu Asp Gln Ile Arg Arg Gly Val Val Thr Val Asp Ala
340 345 350
<210> 7
<211> 1053
<212> DNA
<213> HP1-1 nucleotide Sequence (Artificial Sequence)
<400> 7
atggccgccg attccgaaaa tccgttgggc ctgcatggct ttgcgtttgc tgaatttacc 60
tcacccgatc cggccgcgat ggcgcggcag ttcgagcagc ttggatttgt tccggcggcg 120
cgccggaagg acagggggct gacgctttac cggcaggggc gcatcgcgtt cattctgaat 180
gccggggagg gcgaacaggc cagcgcgttt cgcgccgccc atggcccatc cgccaacggc 240
atggcgttta acgtcgccga cgcgaaagca gcgcaccggc acgccattca aagtggggcg 300
acagacgctg ataccgcgca gagcgctctg ccggggactt atgccattga gggcataggc 360
gactcgctgc tctatctggt ggacagcgac ccctttgcgg actgggatga ggtgcccggt 420
tggcgagagg cgtcggcgga gcggggcgtc ggcctcgacc tgctcgacca cctggcccat 480
aatgtccgcc gggggcagat gcgcgtatgg tccgaattct acgcgacgct gttcggtttc 540
gaggaacaga agttcttcga catcaaaggg caggcgaccg gtctgttcag ccaggcgatg 600
atcgccccgg atcatgaaat ccgcatccct ctgaatgaaa gccaggatga taacagccag 660
atcgaggagt ttatccgcga atataatggc gagggtatcc agcatctcgc cctgacgacg 720
ccggatatct attccacggt ggagaagcta cgcgccaatg gcgtgcggct gcaggatacg 780
atcgacacct attatgatct ggtcgacgag cgggtgccgg ggcatggcga ggatctggcg 840
cgattgcgca aaaaccgcat cctgatcgat ggcgatgtcc atgatgaggg gctgctgctg 900
cagatattca ccgagactat gttcggcccg atcttcttcg agatcatcca gcgcaagggc 960
aatgagggtt tcgggaacgg caatttccag gtactctacg aatccataga attggaccag 1020
atccggcgcg gcgttgtgac ggtcgatgcc tga 1053
<210> 8
<211> 1053
<212> DNA
<213> HP1-1M nucleotide Sequence (Artificial Sequence)
<400> 8
atggctgcag attctgagaa cccattgggt cttcatggat ttgctttcgc agagtttact 60
tctccagatc cagctgcaat ggctagacaa tttgagcagt tgggtttcgt tcctgctgca 120
agaaggaagg ataggggttt gactctttat agacaaggaa ggattgcttt tattcttaat 180
gcaggagagg gtgaacaggc ttctgcattc agagctgcac acggtccatc agctaacgga 240
atggctttta acgtggctga tgcaaaggct gcacataggc acgctattca atctggtgct 300
actgatgcag atacagctca gtcagcattg cctggaacat atgctattga gggaattgga 360
gattctcttt tgtaccttgt tgattcagat ccattcgcag attgggatga agtgcctgga 420
tggagagagg cttctgcaga aaggggagtt ggtttggatc ttttggatca tcttgcccac 480
aacgttagaa ggggtcaaat gagagtgtgg tcagagttct acgctacttt gtttggattc 540
gaggaacaga agtttttcga tattaagggt caagcaacag gattgttttc tcaggctatg 600
attgcaccag atcatgagat taggattcca cttaacgaat ctcaagatga taactcacag 660
atcgaggagt ttattaggga gtataacgga gaaggtattc aacacttggc tcttactaca 720
ccagatatct attcaacagt tgaaaagttg agagcaaatg gtgtgaggct tcaagataca 780
atcgatacct attacgattt ggttgatgag agagtgcctg gacatggtga agatttggct 840
agacttagga agaacaggat tcttattgat ggagatgttc acgatgaggg acttttgctt 900
caaattttta ccgaaactat gttcggtcca attttctttg agattattca gaggaaggga 960
aacgagggat ttggtaacgg aaacttccaa gttttgtacg agtctatcga acttgatcag 1020
attagaaggg gagttgtgac cgtggatgct tga 1053
<210> 9
<211> 350
<212> PRT
<213> amino acid Sequence of HP1-2 (Artificial Sequence)
<400> 9
Met Ala Ala Asp Ser Glu Asn Pro Leu Gly Leu His Gly Phe Ala Phe
1 5 10 15
Ala Glu Phe Thr Ser Pro Asp Pro Ala Ala Met Ala Arg Gln Phe Glu
20 25 30
Gln Leu Gly Phe Val Pro Ala Ala Arg Arg Lys Asp Arg Gly Leu Thr
35 40 45
Leu Tyr Arg Gln Gly Arg Ile Ala Phe Ile Leu Asn Ala Gly Glu Gly
50 55 60
Glu Gln Ala Ser Ala Phe Arg Ala Ala His Gly Pro Ser Ala Asn Gly
65 70 75 80
Met Ala Phe Asn Val Ala Asp Ala Lys Ala Ala His Arg His Ala Ile
85 90 95
Gln Ser Gly Ala Thr Asp Ala Asp Thr Ala Gln Ser Ala Leu Pro Gly
100 105 110
Thr Tyr Ala Ile Glu Gly Ile Gly Asp Ser Leu Leu Tyr Leu Val Asp
115 120 125
Ser Asp Pro Phe Ala Asp Trp Asp Glu Val Pro Gly Trp Arg Glu Ala
130 135 140
Ser Ala Glu Arg Gly Val Gly Leu Asp Leu Leu Asp His Leu Thr His
145 150 155 160
Asn Val Arg Arg Gly Gln Met Arg Val Trp Ser Glu Phe Tyr Ala Thr
165 170 175
Leu Phe Gly Phe Glu Glu Gln Lys Phe Phe Asp Ile Lys Gly Gln Ala
180 185 190
Thr Gly Leu Phe Ser Gln Ala Met Ile Ala Pro Asp His Glu Ile Arg
195 200 205
Ile Pro Leu Asn Glu Ser Gln Asp Asp Asn Ser Gln Ile Glu Glu Phe
210 215 220
Ile Arg Glu Tyr Asn Gly Glu Gly Ile Gln His Leu Ala Leu Thr Thr
225 230 235 240
Pro Asp Ile Tyr Ser Thr Val Glu Lys Leu Arg Ala Asn Gly Val Arg
245 250 255
Leu Met Asp Thr Ile Asp Thr Tyr Tyr Asp Leu Val Asp Glu Arg Val
260 265 270
Pro Gly His Gly Glu Asp Leu Ala Arg Leu Arg Lys Asn Arg Ile Leu
275 280 285
Ile Asp Gly Asp Val His Asp Glu Gly Leu Leu Leu Gln Ile Phe Thr
290 295 300
Glu Thr Met Phe Gly Pro Ile Phe Phe Glu Ile Ile Gln Arg Lys Gly
305 310 315 320
Asn Glu Gly Phe Gly Asn Gly Asn Phe Gln Val Leu Tyr Glu Ser Ile
325 330 335
Glu Leu Asp Gln Ile Arg Arg Gly Val Val Thr Val Asp Ala
340 345 350
<210> 10
<211> 1053
<212> DNA
<213> HP1-2 nucleotide Sequence (Artificial Sequence)
<400> 10
atggccgccg attccgaaaa tccgttgggc ctgcatggct ttgcgtttgc tgaatttacc 60
tcacccgatc cggccgcgat ggcgcggcag ttcgagcagc ttggatttgt tccggcggcg 120
cgccggaagg acagggggct gacgctttac cggcaggggc gcatcgcgtt cattctgaat 180
gccggggagg gcgaacaggc cagcgcgttt cgcgccgccc atggcccatc cgccaacggc 240
atggcgttta acgtcgccga cgcgaaagca gcgcaccggc acgccattca aagtggggcg 300
acagacgctg ataccgcgca gagcgctctg ccggggactt atgccattga gggcataggc 360
gactcgctgc tctatctggt ggacagcgac ccctttgcgg actgggatga ggtgcccggt 420
tggcgagagg cgtcggcgga gcggggcgtc ggcctcgacc tgctcgacca cctgacccat 480
aatgtccgcc gggggcagat gcgcgtatgg tccgaattct acgcgacgct gttcggtttc 540
gaggaacaga agttcttcga catcaaaggg caggcgaccg gtctgttcag ccaggcgatg 600
atcgccccgg atcatgaaat ccgcatccct ctgaatgaaa gccaggatga taacagccag 660
atcgaggagt ttatccgcga atataatggc gagggtatcc agcatctcgc cctgacgacg 720
ccggatatct attccacggt ggagaagcta cgcgccaatg gcgtgcggct gatggatacg 780
atcgacacct attatgatct ggtcgacgag cgggtgccgg ggcatggcga ggatctggcg 840
cgattgcgca aaaaccgcat cctgatcgat ggcgatgtcc atgatgaggg gctgctgctg 900
cagatattca ccgagactat gttcggcccg atcttcttcg agatcatcca gcgcaagggc 960
aatgagggtt tcgggaacgg caatttccag gtactctacg aatccataga attggaccag 1020
atccggcgcg gcgttgtgac ggtcgatgcc tga 1053
<210> 11
<211> 1053
<212> DNA
<213> HP1-2M nucleotide Sequence (Artificial Sequence)
<400> 11
atggctgcag attctgagaa cccattgggt cttcatggat ttgctttcgc agagtttact 60
tctccagatc cagctgcaat ggctagacaa tttgagcagt tgggtttcgt tcctgctgca 120
agaaggaagg ataggggttt gactctttat agacaaggaa ggattgcttt tattcttaat 180
gcaggagagg gtgaacaggc ttctgcattc agagctgcac acggtccatc agctaacgga 240
atggctttta acgtggctga tgcaaaggct gcacataggc acgctattca atctggtgct 300
actgatgcag atacagctca gtcagcattg cctggaacat atgctattga gggaattgga 360
gattctcttt tgtaccttgt tgattcagat ccattcgcag attgggatga agtgcctgga 420
tggagagagg cttctgcaga aaggggagtt ggtttggatc ttttggatca tcttacccac 480
aacgttagaa ggggtcaaat gagagtgtgg tcagagttct acgctacttt gtttggattc 540
gaggaacaga agtttttcga tattaagggt caagcaacag gattgttttc tcaggctatg 600
attgcaccag atcatgagat taggattcca cttaacgaat ctcaagatga taactcacag 660
atcgaggagt ttattaggga gtataacgga gaaggtattc aacacttggc tcttactaca 720
ccagatatct attcaacagt tgaaaagttg agagcaaatg gtgtgaggct tatggataca 780
atcgatacct attacgattt ggttgatgag agagtgcctg gacatggtga agatttggct 840
agacttagga agaacaggat tcttattgat ggagatgttc acgatgaggg acttttgctt 900
caaattttta ccgaaactat gttcggtcca attttctttg agattattca gaggaaggga 960
aacgagggat ttggtaacgg aaacttccaa gttttgtacg agtctatcga acttgatcag 1020
attagaaggg gagttgtgac cgtggatgct tga 1053
<210> 12
<211> 21
<212> DNA
<213> 5' Universal adaptor primer 1(Artificial Sequence)
<400> 12
taagaaggag atatacatat g 21
<210> 13
<211> 21
<212> DNA
<213> 3' terminal Universal adaptor primer 1(Artificial Sequence)
<400> 13
gtggtggtgg tggtgctcga g 21
<210> 14
<211> 24
<212> DNA
<213> 5' Universal adaptor primer 2(Artificial Sequence)
<400> 14
agtttttctg attaacagac tagt 24
<210> 15
<211> 25
<212> DNA
<213> 3' terminal Universal adaptor primer 2(Artificial Sequence)
<400> 15
caaatgtttg aacgatcggc gcgcc 25
<210> 16
<211> 542
<212> DNA
<213> enhancer of 34S of Figwort mosaic virus (Figwort mosaic virus)
<400> 16
aattctcagt ccaaagcctc aacaaggtca gggtacagag tctccaaacc attagccaaa 60
agctacagga gatcaatgaa gaatcttcaa tcaaagtaaa ctactgttcc agcacatgca 120
tcatggtcag taagtttcag aaaaagacat ccaccgaaga cttaaagtta gtgggcatct 180
ttgaaagtaa tcttgtcaac atcgagcagc tggcttgtgg ggaccagaca aaaaaggaat 240
ggtgcagaat tgttaggcgc acctaccaaa agcatctttg cctttattgc aaagataaag 300
cagattcctc tagtacaagt ggggaacaaa ataacgtgga aaagagctgt cctgacagcc 360
cactcactaa tgcgtatgac gaacgcagtg acgaccacaa aagaattagc ttgagctcag 420
gatttagcag cattccagat tgggttcaat caacaaggta cgagccatat cactttattc 480
aaattggtat cgccaaaacc aagaaggaac tcccatcctc aaaggtttgt aaggaagaat 540
tc 542
<210> 17
<211> 1534
<212> DNA
<213> Brassica napus eukaryotic elongation factor Gene 1. alpha. (promoter Brassica napus of Tsf1)
<400> 17
gattatgaca ttgctcgtgg aatgggacag ttatggtatt tttttgtaat aaattgtttc 60
cattgtcatg agattttgag gttaatctat gagacattga atcacttagc attagggatt 120
aagtagtcac aaatcgcatt caagaagctg aagaacacgt tatggtctaa tggttgtgtc 180
tctttattag aaaatgttgg tcagtagcta tatgcactgt ttctgtaaaa ccatgttggt 240
gttgtgttta tttcaagaca catgttgagt ccgttgattc agagcttttg tcttcgaaca 300
caatctagag agcaaatttg ggttcaattt ggatatcaat atgggttcga ttcagataga 360
acaataccct ttgatgtcgg gtttcgattt ggttgagatt catttttatc gggtttggtt 420
cgattttcga attcggttta ttcgccccct catagcatct acattctgca gattaatgta 480
caagttatgg aaaaaaaaat gtggttttcg aattcggttt agtagctaaa cgttgcttgc 540
agtgtagtta tgggaattat gaaacacgac cgaaggtatc aattagaaga acgggtcaac 600
gggtaagtat tgagaaatta ccggagggta aaaataaaca gtattctttt tttttcttaa 660
cgaccgacca aggttaaaaa aagaaaggag gacgagatac aggggcatga ctgtaattgt 720
acataagatc tgatctttaa accctaggtt tccttcgcat cagcaactat aaataattct 780
gagtgccact cttcttcatt cctagatctt tcgccttatc gctttagctg aggtaagcct 840
ttctatacgc atagacgctc tcttttctct tctctcgatc ttcgttgaaa cggtcctcga 900
tacgcatagg atcggttaga atcgttaatc tatcgtctta gatcttcttg attgttgaat 960
tgagcttcta ggatgtattg tatcatgtga tggatagttg attggatctc tttgagtgaa 1020
ctagctagct ttcgatgcgt gtgatttcag tataacagga tccgatgaat tatagctcgc 1080
ttacaattaa tctctgcaga tttattgttt aatcttggat ttgatgctcg ttgttgatag 1140
aggatcgttt atagaactta ttgattctgg aattgagctt gtgtgatgta ttgtatcatg 1200
tgatcgatag ctgatggatc tatttgagtg aactagcgta cgatcttaag atgagtgtgt 1260
attgtgaact gatgattcga gatcagcaaa acaagatctg atgatatctt cgtcttgtat 1320
gcatcttgaa tttcatgatt ttttattaat tatagctcgc ttagctcaaa ggatagagca 1380
ccacaaaatt ttattgtggt agaaatcggt tcgattccga tagcagctta ctgtgatgaa 1440
tgattttgag atttggtatt tgatatatgt ctactgtgtt gaatgatcgt ttatgcattg 1500
tttaatcgct gcagatttgc attgacaagt agcc 1534
<210> 18
<211> 228
<212> DNA
<213> Arabidopsis chloroplast transit peptide (Arabidopsis thaliana)
<400> 18
atggcgcaag ttagcagaat ctgcaatggt gtgcagaacc catctcttat ctccaatctc 60
tcgaaatcca gtcaacgcaa atctccctta tcggtttctc tgaagacgca gcagcatcca 120
cgagcttatc cgatttcgtc gtcgtgggga ttgaagaaga gtgggatgac gttaattggc 180
tctgagcttc gtcctcttaa ggtcatgtct tctgtttcca cggcgtgc 228
<210> 19
<211> 1368
<212> DNA
<213> 5-enolpyruvylshikimate-3-phosphate synthase gene (Artificial Sequence)
<400> 19
atgcttcacg gtgcaagcag ccgtccagca actgctcgta agtcctctgg tctttctgga 60
accgtccgta ttccaggtga caagtctatc tcccacaggt ccttcatgtt tggaggtctc 120
gctagcggtg aaactcgtat caccggtctt ttggaaggtg aagatgttat caacactggt 180
aaggctatgc aagctatggg tgccagaatc cgtaaggaag gtgatacttg gatcattgat 240
ggtgttggta acggtggact ccttgctcct gaggctcctc tcgatttcgg taacgctgca 300
actggttgcc gtttgactat gggtcttgtt ggtgtttacg atttcgatag cactttcatt 360
ggtgacgctt ctctcactaa gcgtccaatg ggtcgtgtgt tgaacccact tcgcgaaatg 420
ggtgtgcagg tgaagtctga agacggtgat cgtcttccag ttaccttgcg tggaccaaag 480
actccaacgc caatcaccta cagggtacct atggcttccg ctcaagtgaa gtccgctgtt 540
ctgcttgctg gtctcaacac cccaggtatc accactgtta tcgagccaat catgactcgt 600
gaccacactg aaaagatgct tcaaggtttt ggtgctaacc ttaccgttga gactgatgct 660
gacggtgtgc gtaccatccg tcttgaaggt cgtggtaagc tcaccggtca agtgattgat 720
gttccaggtg atccatcctc tactgctttc ccattggttg ctgccttgct tgttccaggt 780
tccgacgtca ccatccttaa cgttttgatg aacccaaccc gtactggtct catcttgact 840
ctgcaggaaa tgggtgccga catcgaagtg atcaacccac gtcttgctgg tggagaagac 900
gtggctgact tgcgtgttcg ttcttctact ttgaagggtg ttactgttcc agaagaccgt 960
gctccttcta tgatcgacga gtatccaatt ctcgctgttg cagctgcatt cgctgaaggt 1020
gctaccgtta tgaacggttt ggaagaactc cgtgttaagg aaagcgaccg tctttctgct 1080
gtcgcaaacg gtctcaagct caacggtgtt gattgcgatg aaggtgagac ttctctcgtc 1140
gtgcgtggtc gtcctgacgg taagggtctc ggtaacgctt ctggagcagc tgtcgctacc 1200
cacctcgatc accgtatcgc tatgagcttc ctcgttatgg gtctcgtttc tgaaaaccct 1260
gttactgttg atgatgctac tatgatcgct actagcttcc cagagttcat ggatttgatg 1320
gctggtcttg gagctaagat cgaactctcc gacactaagg ctgcttga 1368
<210> 20
<211> 643
<212> DNA
<213> terminator of pea RbcS gene (Pisum sativum)
<400> 20
agctttcgtt cgtatcatcg gtttcgacaa cgttcgtcaa gttcaatgca tcagtttcat 60
tgcgcacaca ccagaatcct actgagtttg agtattatgg cattgggaaa actgtttttc 120
ttgtaccatt tgttgtgctt gtaatttact gtgtttttta ttcggttttc gctatcgaac 180
tgtgaaatgg aaatggatgg agaagagtta atgaatgata tggtcctttt gttcattctc 240
aaattaatat tatttgtttt ttctcttatt tgttgtgtgt tgaatttgaa attataagag 300
atatgcaaac attttgtttt gagtaaaaat gtgtcaaatc gtggcctcta atgaccgaag 360
ttaatatgag gagtaaaaca cttgtagttg taccattatg cttattcact aggcaacaaa 420
tatattttca gacctagaaa agctgcaaat gttactgaat acaagtatgt cctcttgtgt 480
tttagacatt tatgaacttt cctttatgta attttccaga atccttgtca gattctaatc 540
attgctttat aattatagtt atactcatgg atttgtagtt gagtatgaaa atatttttta 600
atgcatttta tgacttgcca attgattgac aacatgcatc aat 643
<210> 21
<211> 1322
<212> DNA
<213> Arabidopsis Ubiquitin (Arabidopsis thaliana promoter of Ubiquitin10 Gene)
<400> 21
gtcgacctgc aggtcaacgg atcaggatat tcttgtttaa gatgttgaac tctatggagg 60
tttgtatgaa ctgatgatct aggaccggat aagttccctt cttcatagcg aacttattca 120
aagaatgttt tgtgtatcat tcttgttaca ttgttattaa tgaaaaaata ttattggtca 180
ttggactgaa cacgagtgtt aaatatggac caggccccaa ataagatcca ttgatatatg 240
aattaaataa caagaataaa tcgagtcacc aaaccacttg ccttttttaa cgagacttgt 300
tcaccaactt gatacaaaag tcattatcct atgcaaatca ataatcatac aaaaatatcc 360
aataacacta aaaaattaaa agaaatggat aatttcacaa tatgttatac gataaagaag 420
ttacttttcc aagaaattca ctgattttat aagcccactt gcattagata aatggcaaaa 480
aaaaacaaaa aggaaaagaa ataaagcacg aagaattcta gaaaatacga aatacgcttc 540
aatgcagtgg gacccacggt tcaattattg ccaattttca gctccaccgt atatttaaaa 600
aataaaacga taatgctaaa aaaatataaa tcgtaacgat cgttaaatct caacggctgg 660
atcttatgac gaccgttaga aattgtggtt gtcgacgagt cagtaataaa cggcgtcaaa 720
gtggttgcag ccggcacaca cgagtcgtgt ttatcaactc aaagcacaaa tacttttcct 780
caacctaaaa ataaggcaat tagccaaaaa caactttgcg tgtaaacaac gctcaataca 840
cgtgtcattt tattattagc tattgcttca ccgccttagc tttctcgtga cctagtcgtc 900
ctcgtctttt cttcttcttc ttctataaaa caatacccaa agcttcttct tcacaattca 960
gatttcaatt tctcaaaatc ttaaaaactt tctctcaatt ctctctaccg tgatcaaggt 1020
aaatttctgt gttccttatt ctctcaaaat cttcgatttt gttttcgttc gatcccaatt 1080
tcgtatatgt tctttggttt agattctgtt aatcttagat cgaagacgat tttctgggtt 1140
tgatcgttag atatcatctt aattctcgat tagggtttca taaatatcat ccgatttgtt 1200
caaataattt gagttttgtc gaataattac tcttcgattt gtgatttcta tctagatctg 1260
gtgttagttt ctagtttgtg cgatcgaatt tgtcgattaa tctgagtttt tctgattaac 1320
ag 1322
<210> 22
<211> 253
<212> DNA
<213> terminator of nopaline synthase gene (Agrobacterium tumefaciens)
<400> 22
gatcgttcaa acatttggca ataaagtttc ttaagattga atcctgttgc cggtcttgcg 60
atgattatca tataatttct gttgaattac gttaagcatg taataattaa catgtaatgc 120
atgacgttat ttatgagatg ggtttttatg attagagtcc cgcaattata catttaatac 180
gcgatagaaa acaaaatata gcgcgcaaac taggataaat tatcgcgcgc ggtgtcatct 240
atgttactag atc 253
<210> 23
<211> 530
<212> DNA
<213> Cauliflower mosaic virus 35S promoter (Cauliflower mosaic virus)
<400> 23
ccatggagtc aaagattcaa atagaggacc taacagaact cgccgtaaag actggcgaac 60
agttcataca gagtctctta cgactcaatg acaagaagaa aatcttcgtc aacatggtgg 120
agcacgacac gcttgtctac tccaaaaata tcaaagatac agtctcagaa gaccaaaggg 180
caattgagac ttttcaacaa agggtaatat ccggaaacct cctcggattc cattgcccag 240
ctatctgtca ctttattgtg aagatagtgg aaaaggaagg tggctcctac aaatgccatc 300
attgcgataa aggaaaggcc atcgttgaag atgcctctgc cgacagtggt cccaaagatg 360
gacccccacc cacgaggagc atcgtggaaa aagaagacgt tccaaccacg tcttcaaagc 420
aagtggattg atgtgatatc tccactgacg taagggatga cgcacaatcc cactatcctt 480
cgcaagaccc ttcctctata taaggaagtt catttcattt ggagaggaca 530
<210> 24
<211> 552
<212> DNA
<213> phosphinothricin N-acetyltransferase gene nucleotide sequence (Streptomyces viridochromogenes)
<400> 24
atgtctccgg agaggagacc agttgagatt aggccagcta cagcagctga tatggccgcg 60
gtttgtgata tcgttaacca ttacattgag acgtctacag tgaactttag gacagagcca 120
caaacaccac aagagtggat tgatgatcta gagaggttgc aagatagata cccttggttg 180
gttgctgagg ttgagggtgt tgtggctggt attgcttacg ctgggccctg gaaggctagg 240
aacgcttacg attggacagt tgagagtact gtttacgtgt cacataggca tcaaaggttg 300
ggcctaggat ccacattgta cacacatttg cttaagtcta tggaggcgca aggttttaag 360
tctgtggttg ctgttatagg ccttccaaac gatccatctg ttaggttgca tgaggctttg 420
ggatacacag cccggggtac attgcgcgca gctggataca agcatggtgg atggcatgat 480
gttggttttt ggcaaaggga ttttgagttg ccagctcctc caaggccagt taggccagtt 540
acccagatct ga 552
<210> 25
<211> 195
<212> DNA
<213> Cauliflower mosaic virus 35S terminator (Cauliflower mosaic virus)
<400> 25
ctgaaatcac cagtctctct ctacaaatct atctctctct ataataatgt gtgagtagtt 60
cccagataag ggaattaggg ttcttatagg gtttcgctca tgtgttgagc atataagaaa 120
cccttagtat gtatttgtat ttgtaaaata cttctatcaa taaaatttct aattcctaaa 180
accaaaatcc agtgg 195
Claims (14)
1. A protein, comprising:
(a) has an amino acid sequence shown as SEQ ID NO. 9;
(b) and (b) a protein derived from (a) and having hydroxyphenylpyruvate dioxygenase activity, wherein the amino acid sequence in (a) is substituted and/or deleted and/or one or more amino acids are added.
2. A gene, comprising:
(a) a nucleotide sequence encoding the protein of claim 1; or
(b) A nucleotide sequence which is hybridized with the nucleotide sequence limited by the (a) under strict conditions and encodes protein with hydroxyphenylpyruvate dioxygenase activity; or
(c) Has the nucleotide sequence shown in SEQ ID NO. 10 or SEQ ID NO. 11.
3. An expression cassette comprising the gene of claim 2 under the control of an operably linked regulatory sequence.
4. A recombinant vector comprising the gene of claim 2 or the expression cassette of claim 3.
5. A method of extending the herbicide tolerance of a plant comprising: expressing in a plant the protein of claim 1 or the protein encoded by the expression cassette of claim 3 together with at least one second herbicide tolerance protein different from the protein of claim 1 or the protein encoded by the expression cassette of claim 3.
6. The method of claim 5, wherein said second herbicide tolerance protein is 5-enolpyruvylshikimate-3-phosphate synthase, glyphosate oxidoreductase, glyphosate-N-acetyltransferase, glyphosate decarboxylase, glufosinate acetyltransferase, alpha ketoglutarate-dependent dioxygenase, dicamba monooxygenase, acetolactate synthase, cytochrome proteins and/or protoporphyrinogen oxidase.
7. A method of selecting a transformed plant cell comprising: transforming a plurality of plant cells with the gene of claim 2 or the expression cassette of claim 3 and culturing the cells at a concentration of HPPD inhibitor herbicide that allows growth of transformed cells expressing the gene or the expression cassette, while killing or inhibiting growth of untransformed cells;
preferably, the plant includes monocotyledons and dicotyledons; more preferably, the plant is oat, wheat, barley, millet, corn, sorghum, brachypodium distachyon, rice, tobacco, sunflower, alfalfa, soybean, chickpea, peanut, sugar beet, cucumber, cotton, rape, potato, tomato or arabidopsis;
preferably, the HPPD inhibitor herbicide comprises a pyrazolone HPPD inhibitor herbicide, a triketone HPPD inhibitor herbicide, and/or an isoxazole HPPD inhibitor herbicide; more preferably, the HPPD inhibitor herbicide is topramezone, mesotrione, and/or a diketonitrile.
8. A method of controlling weeds, comprising: applying an effective dose of an HPPD inhibitor herbicide to a field planted with a plant of interest comprising the gene of claim 2 or the expression cassette of claim 3 or the recombinant vector of claim 4;
preferably, the plant of interest includes a monocotyledon and a dicotyledon; more preferably, the plant of interest is oat, wheat, barley, millet, corn, sorghum, brachypodium distachyon, rice, tobacco, sunflower, alfalfa, soybean, chickpea, peanut, sugar beet, cucumber, cotton, rape, potato, tomato or arabidopsis; further preferably, the plant of interest is a glyphosate tolerant plant and the weeds are glyphosate resistant weeds;
preferably, the HPPD inhibitor herbicide comprises a pyrazolone HPPD inhibitor herbicide, a triketone HPPD inhibitor herbicide, and/or an isoxazole HPPD inhibitor herbicide; more preferably, the HPPD inhibitor herbicide is topramezone, mesotrione, and/or a diketonitrile.
9. A method for protecting or conferring tolerance to a HPPD inhibitor herbicide on a plant from damage caused by the HPPD inhibitor herbicide, comprising: introducing the gene of claim 2 or the expression cassette of claim 3 or the recombinant vector of claim 4 into a plant such that the introduced plant produces an amount of an herbicide tolerance protein sufficient to protect it from damage by HPPD inhibitor herbicides;
preferably, the plant includes monocotyledons and dicotyledons; more preferably, the plant is oat, wheat, barley, millet, corn, sorghum, brachypodium distachyon, rice, tobacco, sunflower, alfalfa, soybean, chickpea, peanut, sugar beet, cucumber, cotton, rape, potato, tomato or arabidopsis;
preferably, the HPPD inhibitor herbicide comprises a pyrazolone HPPD inhibitor herbicide, a triketone HPPD inhibitor herbicide, and/or an isoxazole HPPD inhibitor herbicide; more preferably, the HPPD inhibitor herbicide is topramezone, mesotrione, and/or a diketonitrile.
10. A method for producing a plant tolerant to HPPD inhibitor herbicides, comprising introducing into the genome of the plant the gene of claim 2;
preferably, the method of introduction includes a genetic transformation method, a genome editing method, or a gene mutation method;
preferably, the plant includes monocotyledons and dicotyledons; more preferably, the plant is oat, wheat, barley, millet, corn, sorghum, brachypodium distachyon, rice, tobacco, sunflower, alfalfa, soybean, chickpea, peanut, sugar beet, cucumber, cotton, rape, potato, tomato or arabidopsis;
preferably, the HPPD inhibitor herbicide comprises a pyrazolone HPPD inhibitor herbicide, a triketone HPPD inhibitor herbicide, and/or an isoxazole HPPD inhibitor herbicide; more preferably, the HPPD inhibitor herbicide is topramezone, mesotrione, and/or a diketonitrile.
11. A method of growing a plant tolerant to a HPPD inhibitor herbicide, comprising:
growing at least one plant propagule comprising in its genome the gene of claim 2 or the expression cassette of claim 3;
growing the plant propagule into a plant;
applying an effective dose of an HPPD inhibitor herbicide to a plant growing environment comprising at least said plant, harvesting plants having reduced plant damage and/or increased plant yield as compared to other plants not having the gene of claim 2 or the expression cassette of claim 3;
preferably, the plant includes monocotyledons and dicotyledons; more preferably, the plant is oat, wheat, barley, millet, corn, sorghum, brachypodium distachyon, rice, tobacco, sunflower, alfalfa, soybean, chickpea, peanut, sugar beet, cucumber, cotton, rape, potato, tomato or arabidopsis;
preferably, the HPPD inhibitor herbicide comprises a pyrazolone HPPD inhibitor herbicide, a triketone HPPD inhibitor herbicide, and/or an isoxazole HPPD inhibitor herbicide; more preferably, the HPPD inhibitor herbicide is topramezone, mesotrione, and/or a diketonitrile.
12. A method for obtaining a processed agricultural product, comprising treating a harvest of HPPD inhibitor herbicide tolerant plants obtained by the method of claim 11 to obtain a processed agricultural product.
13. A planting system for controlling weed growth comprising combining a HPPD inhibitor herbicide and a plant growing environment in which at least one plant of interest is present, said plant of interest comprising a gene of claim 2 or an expression cassette of claim 3;
preferably, the plant of interest includes a monocotyledon and a dicotyledon; more preferably, the plant of interest is oat, wheat, barley, millet, corn, sorghum, brachypodium distachyon, rice, tobacco, sunflower, alfalfa, soybean, chickpea, peanut, sugar beet, cucumber, cotton, rape, potato, tomato or arabidopsis; further preferably, the plant of interest is a glyphosate tolerant plant and the weeds are glyphosate resistant weeds;
preferably, the HPPD inhibitor herbicide comprises a pyrazolone HPPD inhibitor herbicide, a triketone HPPD inhibitor herbicide, and/or an isoxazole HPPD inhibitor herbicide; more preferably, the HPPD inhibitor herbicide is topramezone, mesotrione, and/or a diketonitrile.
14. Use of a protein according to claim 1 for conferring herbicide tolerance to a plant HPPD inhibitor;
preferably, the plant includes monocotyledons and dicotyledons; more preferably, the plant is oat, wheat, barley, millet, corn, sorghum, brachypodium distachyon, rice, tobacco, sunflower, alfalfa, soybean, chickpea, peanut, sugar beet, cucumber, cotton, rape, potato, tomato or arabidopsis;
preferably, the HPPD inhibitor herbicide comprises a pyrazolone HPPD inhibitor herbicide, a triketone HPPD inhibitor herbicide, and/or an isoxazole HPPD inhibitor herbicide; more preferably, the HPPD inhibitor herbicide is topramezone, mesotrione, and/or a diketonitrile.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210330228.1A CN114854702B (en) | 2020-03-19 | 2020-03-19 | Herbicide tolerance protein, coding gene and application thereof |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210330228.1A CN114854702B (en) | 2020-03-19 | 2020-03-19 | Herbicide tolerance protein, coding gene and application thereof |
CN202010194585.0A CN111304178B (en) | 2020-03-19 | 2020-03-19 | Herbicide tolerance protein, coding gene and application thereof |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202010194585.0A Division CN111304178B (en) | 2020-03-19 | 2020-03-19 | Herbicide tolerance protein, coding gene and application thereof |
Publications (2)
Publication Number | Publication Date |
---|---|
CN114854702A true CN114854702A (en) | 2022-08-05 |
CN114854702B CN114854702B (en) | 2024-04-19 |
Family
ID=71157254
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202010194585.0A Active CN111304178B (en) | 2020-03-19 | 2020-03-19 | Herbicide tolerance protein, coding gene and application thereof |
CN202210330228.1A Active CN114854702B (en) | 2020-03-19 | 2020-03-19 | Herbicide tolerance protein, coding gene and application thereof |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202010194585.0A Active CN111304178B (en) | 2020-03-19 | 2020-03-19 | Herbicide tolerance protein, coding gene and application thereof |
Country Status (1)
Country | Link |
---|---|
CN (2) | CN111304178B (en) |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114774377B (en) * | 2021-03-24 | 2024-01-12 | 华中师范大学 | HPPD proteins, genes, vectors, cells, compositions, uses thereof and methods for increasing herbicide resistance in crops |
CN113980990B (en) * | 2021-11-26 | 2022-07-26 | 中国农业科学院生物技术研究所 | Transgenic insect-resistant herbicide-resistant corn and cultivation method thereof |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110628733A (en) * | 2019-09-17 | 2019-12-31 | 北京大北农生物技术有限公司 | Herbicide tolerance protein, coding gene and application thereof |
CN110656094A (en) * | 2019-09-17 | 2020-01-07 | 北京大北农生物技术有限公司 | Herbicide tolerance protein, coding gene and application thereof |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110628732B (en) * | 2019-09-17 | 2022-04-15 | 北京大北农生物技术有限公司 | Herbicide tolerance protein, coding gene and application thereof |
-
2020
- 2020-03-19 CN CN202010194585.0A patent/CN111304178B/en active Active
- 2020-03-19 CN CN202210330228.1A patent/CN114854702B/en active Active
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110628733A (en) * | 2019-09-17 | 2019-12-31 | 北京大北农生物技术有限公司 | Herbicide tolerance protein, coding gene and application thereof |
CN110656094A (en) * | 2019-09-17 | 2020-01-07 | 北京大北农生物技术有限公司 | Herbicide tolerance protein, coding gene and application thereof |
Non-Patent Citations (1)
Title |
---|
SRIDEVI NAKKA等: ""Physiological and Molecular Characterization of Hydroxyphenylpyruvate Dioxygenase (HPPD)-inhibitor Resistance in Palmer Amaranth (Amaranthus palmeri S.Wats.)"", 《FRONTIERS IN PLANT SCIENCE》, vol. 8, pages 7 - 8 * |
Also Published As
Publication number | Publication date |
---|---|
CN111304178A (en) | 2020-06-19 |
CN114854702B (en) | 2024-04-19 |
CN111304178B (en) | 2022-06-03 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
AU2016399292B2 (en) | Herbicide tolerant protein, encoding gene and use thereof | |
CA2838523C (en) | Herbicide-resistant protein, encoding gene and use thereof | |
CA2975762C (en) | Herbicide-resistant protein, encoding gene and use thereof | |
CA2838517C (en) | Herbicide-resistant protein, encoding gene and use thereof | |
WO2021051265A1 (en) | Mutant hydroxyphenylpyruvate dioxygenase polypeptide, encoding gene thereof and use thereof | |
CN110628733B (en) | Herbicide tolerance protein, coding gene and application thereof | |
CA2975773C (en) | Herbicide-resistant protein, encoding gene and use thereof | |
CN111304178B (en) | Herbicide tolerance protein, coding gene and application thereof | |
CA2976060C (en) | Herbicide-resistant protein, encoding gene and use thereof | |
CN111334484B (en) | Herbicide tolerance protein, coding gene and application thereof | |
CN110656094B (en) | Herbicide tolerance protein, coding gene and application thereof | |
CN110628732B (en) | Herbicide tolerance protein, coding gene and application thereof | |
CN115340987B (en) | Herbicide tolerance protein, coding gene and application thereof | |
WO2023108495A1 (en) | Mutated hydroxyphenylpyruvate dioxygenase polypeptide, and coding gene and use thereof | |
WO2022237541A1 (en) | Use of protoporphyrinogen oxidase | |
CA3236161A1 (en) | Mutated hydroxyphenylpyruvate dioxygenase polypeptide, and coding gene and use thereof |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant |