CN117004583A - 2ODD protein, gene, vector, cell, composition and application thereof - Google Patents
2ODD protein, gene, vector, cell, composition and application thereof Download PDFInfo
- Publication number
- CN117004583A CN117004583A CN202310502130.4A CN202310502130A CN117004583A CN 117004583 A CN117004583 A CN 117004583A CN 202310502130 A CN202310502130 A CN 202310502130A CN 117004583 A CN117004583 A CN 117004583A
- Authority
- CN
- China
- Prior art keywords
- amino acid
- acid sequence
- identity
- seq
- mutated
- 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.)
- Pending
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 166
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 116
- 239000013598 vector Substances 0.000 title claims abstract description 33
- 239000000203 mixture Substances 0.000 title claims abstract description 15
- 239000004009 herbicide Substances 0.000 claims abstract description 52
- 238000000034 method Methods 0.000 claims abstract description 46
- 239000003112 inhibitor Substances 0.000 claims abstract description 43
- 230000002363 herbicidal effect Effects 0.000 claims abstract description 38
- 230000009261 transgenic effect Effects 0.000 claims abstract description 14
- 150000001413 amino acids Chemical group 0.000 claims description 146
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 claims description 139
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 claims description 139
- 235000018102 proteins Nutrition 0.000 claims description 108
- 239000002773 nucleotide Substances 0.000 claims description 69
- 125000003729 nucleotide group Chemical group 0.000 claims description 69
- 230000035772 mutation Effects 0.000 claims description 64
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 claims description 61
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 claims description 55
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 claims description 49
- 101100339555 Zymoseptoria tritici HPPD gene Proteins 0.000 claims description 40
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 claims description 33
- 108010016119 Alpha-Ketoglutarate-Dependent Dioxygenase FTO Proteins 0.000 claims description 32
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 claims description 32
- 102000000383 Alpha-Ketoglutarate-Dependent Dioxygenase FTO Human genes 0.000 claims description 31
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 claims description 30
- 229960000310 isoleucine Drugs 0.000 claims description 30
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 claims description 29
- 229940024606 amino acid Drugs 0.000 claims description 29
- 235000001014 amino acid Nutrition 0.000 claims description 29
- 210000004027 cell Anatomy 0.000 claims description 25
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 24
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 24
- 239000005578 Mesotrione Substances 0.000 claims description 18
- 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 18
- 101001082500 Homo sapiens Histatin-1 Proteins 0.000 claims description 17
- 101001021281 Homo sapiens Protein HEXIM1 Proteins 0.000 claims description 17
- 102100030483 Histatin-1 Human genes 0.000 claims description 16
- 125000000404 glutamine group Chemical group N[C@@H](CCC(N)=O)C(=O)* 0.000 claims description 16
- 125000000741 isoleucyl group Chemical group [H]N([H])C(C(C([H])([H])[H])C([H])([H])C([H])([H])[H])C(=O)O* 0.000 claims description 16
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 claims description 15
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 claims description 14
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 claims description 12
- 239000004473 Threonine Substances 0.000 claims description 12
- 125000000341 threoninyl group Chemical group [H]OC([H])(C([H])([H])[H])C([H])(N([H])[H])C(*)=O 0.000 claims description 9
- 239000004475 Arginine Substances 0.000 claims description 8
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 claims description 8
- 239000004472 Lysine Substances 0.000 claims description 8
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 claims description 8
- -1 trione compound Chemical class 0.000 claims description 8
- 125000001909 leucine group Chemical group [H]N(*)C(C(*)=O)C([H])([H])C(C([H])([H])[H])C([H])([H])[H] 0.000 claims description 6
- 238000010453 CRISPR/Cas method Methods 0.000 claims description 4
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 claims description 4
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 claims description 4
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 claims description 4
- 235000004279 alanine Nutrition 0.000 claims description 4
- 238000010362 genome editing Methods 0.000 claims description 4
- FFSSWMQPCJRCRV-UHFFFAOYSA-N quinclorac Chemical compound ClC1=CN=C2C(C(=O)O)=C(Cl)C=CC2=C1 FFSSWMQPCJRCRV-UHFFFAOYSA-N 0.000 claims description 4
- 108091033409 CRISPR Proteins 0.000 claims description 3
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 claims description 3
- 210000001236 prokaryotic cell Anatomy 0.000 claims description 3
- OFSLKOLYLQSJPB-UHFFFAOYSA-N Cyclosulfamuron Chemical compound COC1=CC(OC)=NC(NC(=O)NS(=O)(=O)NC=2C(=CC=CC=2)C(=O)C2CC2)=N1 OFSLKOLYLQSJPB-UHFFFAOYSA-N 0.000 claims description 2
- 102000016680 Dioxygenases Human genes 0.000 claims description 2
- 108010028143 Dioxygenases Proteins 0.000 claims description 2
- 150000001875 compounds Chemical class 0.000 claims description 2
- IYMLUHWAJFXAQP-UHFFFAOYSA-N topramezone Chemical compound 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 2
- CUJMCPPBTUATEJ-UHFFFAOYSA-N 1-(3-methyl-4-oxido-1-oxoquinoxalin-1-ium-2-yl)ethanone Chemical compound C1=CC=C2[N+](=O)C(C(=O)C)=C(C)N([O-])C2=C1 CUJMCPPBTUATEJ-UHFFFAOYSA-N 0.000 claims 1
- 230000001419 dependent effect Effects 0.000 claims 1
- 238000010353 genetic engineering Methods 0.000 abstract description 2
- 108700026220 vif Genes Proteins 0.000 abstract description 2
- 125000003275 alpha amino acid group Chemical group 0.000 description 208
- 230000003197 catalytic effect Effects 0.000 description 57
- 241000196324 Embryophyta Species 0.000 description 44
- 240000007594 Oryza sativa Species 0.000 description 41
- 235000007164 Oryza sativa Nutrition 0.000 description 40
- 235000009566 rice Nutrition 0.000 description 40
- 239000000243 solution Substances 0.000 description 21
- 240000008042 Zea mays Species 0.000 description 20
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 20
- 125000001493 tyrosinyl group Chemical group [H]OC1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 17
- 101100231902 Arabidopsis thaliana HSL2 gene Proteins 0.000 description 16
- 101100316754 Arabidopsis thaliana VAL3 gene Proteins 0.000 description 16
- 101000871498 Homo sapiens m7GpppX diphosphatase Proteins 0.000 description 15
- 102100033718 m7GpppX diphosphatase Human genes 0.000 description 15
- 108020004705 Codon Proteins 0.000 description 13
- 244000062793 Sorghum vulgare Species 0.000 description 13
- 102000004190 Enzymes Human genes 0.000 description 12
- 108090000790 Enzymes Proteins 0.000 description 12
- 235000011684 Sorghum saccharatum Nutrition 0.000 description 12
- 235000021307 Triticum Nutrition 0.000 description 12
- 241000209140 Triticum Species 0.000 description 12
- 229940088598 enzyme Drugs 0.000 description 12
- 239000000047 product Substances 0.000 description 12
- 239000000758 substrate Substances 0.000 description 12
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 11
- 235000005822 corn Nutrition 0.000 description 11
- 238000003752 polymerase chain reaction Methods 0.000 description 11
- 238000006243 chemical reaction Methods 0.000 description 10
- 235000016383 Zea mays subsp huehuetenangensis Nutrition 0.000 description 9
- 235000009973 maize Nutrition 0.000 description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 9
- 206010064571 Gene mutation Diseases 0.000 description 8
- 230000000694 effects Effects 0.000 description 8
- 239000000499 gel Substances 0.000 description 8
- 238000000746 purification Methods 0.000 description 8
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- 108091028043 Nucleic acid sequence Proteins 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 238000003776 cleavage reaction Methods 0.000 description 5
- 239000008367 deionised water Substances 0.000 description 5
- 229910021641 deionized water Inorganic materials 0.000 description 5
- 238000001962 electrophoresis Methods 0.000 description 5
- 239000012634 fragment Substances 0.000 description 5
- 238000011068 loading method Methods 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- 238000011084 recovery Methods 0.000 description 5
- 230000007017 scission Effects 0.000 description 5
- 238000003786 synthesis reaction Methods 0.000 description 5
- 241000588724 Escherichia coli Species 0.000 description 4
- 230000008901 benefit Effects 0.000 description 4
- 125000000487 histidyl group Chemical group [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C([H])=N1 0.000 description 4
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 4
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 244000068988 Glycine max Species 0.000 description 3
- 235000010469 Glycine max Nutrition 0.000 description 3
- 240000005979 Hordeum vulgare Species 0.000 description 3
- 235000007340 Hordeum vulgare Nutrition 0.000 description 3
- 239000007983 Tris buffer Substances 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- 239000003242 anti bacterial agent Substances 0.000 description 3
- 229940088710 antibiotic agent Drugs 0.000 description 3
- 125000000637 arginyl group Chemical group N[C@@H](CCCNC(N)=N)C(=O)* 0.000 description 3
- 239000007853 buffer solution Substances 0.000 description 3
- 238000006555 catalytic reaction Methods 0.000 description 3
- 244000038559 crop plants Species 0.000 description 3
- 238000007865 diluting Methods 0.000 description 3
- 238000001976 enzyme digestion Methods 0.000 description 3
- 230000013595 glycosylation Effects 0.000 description 3
- 238000006206 glycosylation reaction Methods 0.000 description 3
- 210000004209 hair Anatomy 0.000 description 3
- 230000006698 induction Effects 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 102000039446 nucleic acids Human genes 0.000 description 3
- 108020004707 nucleic acids Proteins 0.000 description 3
- 150000007523 nucleic acids Chemical class 0.000 description 3
- 239000013612 plasmid Substances 0.000 description 3
- 108091008146 restriction endonucleases Proteins 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 3
- 238000001179 sorption measurement Methods 0.000 description 3
- 239000011550 stock solution Substances 0.000 description 3
- 230000009466 transformation Effects 0.000 description 3
- VOYADQIFGGIKAT-UHFFFAOYSA-N 1,3-dibutyl-4-hydroxy-2,6-dioxopyrimidine-5-carboximidamide Chemical compound CCCCn1c(O)c(C(N)=N)c(=O)n(CCCC)c1=O VOYADQIFGGIKAT-UHFFFAOYSA-N 0.000 description 2
- KPGXRSRHYNQIFN-UHFFFAOYSA-N 2-oxoglutaric acid Chemical compound OC(=O)CCC(=O)C(O)=O KPGXRSRHYNQIFN-UHFFFAOYSA-N 0.000 description 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 2
- 229920000936 Agarose Polymers 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 108020004414 DNA Proteins 0.000 description 2
- 108010042407 Endonucleases Proteins 0.000 description 2
- 102000004533 Endonucleases Human genes 0.000 description 2
- YLQBMQCUIZJEEH-UHFFFAOYSA-N Furan Chemical compound C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 2
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 2
- 238000001042 affinity chromatography Methods 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000012131 assay buffer Substances 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 230000006037 cell lysis Effects 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 235000013339 cereals Nutrition 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000005520 cutting process Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000011790 ferrous sulphate Substances 0.000 description 2
- 235000003891 ferrous sulphate Nutrition 0.000 description 2
- 239000004459 forage Substances 0.000 description 2
- 239000003292 glue Substances 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 230000033444 hydroxylation Effects 0.000 description 2
- 238000005805 hydroxylation reaction Methods 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 2
- 229910000359 iron(II) sulfate Inorganic materials 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
- 239000007788 liquid Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 238000010369 molecular cloning Methods 0.000 description 2
- 239000002808 molecular sieve Substances 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 238000001742 protein purification Methods 0.000 description 2
- 230000035939 shock Effects 0.000 description 2
- 238000002741 site-directed mutagenesis Methods 0.000 description 2
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 2
- 235000010378 sodium ascorbate Nutrition 0.000 description 2
- PPASLZSBLFJQEF-RKJRWTFHSA-M sodium ascorbate Substances [Na+].OC[C@@H](O)[C@H]1OC(=O)C(O)=C1[O-] PPASLZSBLFJQEF-RKJRWTFHSA-M 0.000 description 2
- 229960005055 sodium ascorbate Drugs 0.000 description 2
- PPASLZSBLFJQEF-RXSVEWSESA-M sodium-L-ascorbate Chemical compound [Na+].OC[C@H](O)[C@H]1OC(=O)C(O)=C1[O-] PPASLZSBLFJQEF-RXSVEWSESA-M 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 1
- KPGXRSRHYNQIFN-UHFFFAOYSA-L 2-oxoglutarate(2-) Chemical compound [O-]C(=O)CCC(=O)C([O-])=O KPGXRSRHYNQIFN-UHFFFAOYSA-L 0.000 description 1
- 229940122196 4-hydroxyphenylpyruvate dioxygenase inhibitor Drugs 0.000 description 1
- 241000589158 Agrobacterium Species 0.000 description 1
- 241000219195 Arabidopsis thaliana Species 0.000 description 1
- 244000105624 Arachis hypogaea Species 0.000 description 1
- 235000007319 Avena orientalis Nutrition 0.000 description 1
- 244000075850 Avena orientalis Species 0.000 description 1
- 241000195940 Bryophyta Species 0.000 description 1
- 238000010356 CRISPR-Cas9 genome editing Methods 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 241000195493 Cryptophyta Species 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- 108010053770 Deoxyribonucleases Proteins 0.000 description 1
- 102000016911 Deoxyribonucleases Human genes 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 239000005562 Glyphosate Substances 0.000 description 1
- 244000299507 Gossypium hirsutum Species 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- 244000020551 Helianthus annuus Species 0.000 description 1
- 235000003222 Helianthus annuus Nutrition 0.000 description 1
- 235000007688 Lycopersicon esculentum Nutrition 0.000 description 1
- 240000004658 Medicago sativa Species 0.000 description 1
- 235000017587 Medicago sativa ssp. sativa Nutrition 0.000 description 1
- 102000016943 Muramidase Human genes 0.000 description 1
- 108010014251 Muramidase Proteins 0.000 description 1
- 101710135898 Myc proto-oncogene protein Proteins 0.000 description 1
- 102100038895 Myc proto-oncogene protein Human genes 0.000 description 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
- 244000061176 Nicotiana tabacum Species 0.000 description 1
- 235000002637 Nicotiana tabacum Nutrition 0.000 description 1
- BZQFBWGGLXLEPQ-UHFFFAOYSA-N O-phosphoryl-L-serine Natural products OC(=O)C(N)COP(O)(O)=O BZQFBWGGLXLEPQ-UHFFFAOYSA-N 0.000 description 1
- 108010002747 Pfu DNA polymerase Proteins 0.000 description 1
- 241000209504 Poaceae Species 0.000 description 1
- 241000985694 Polypodiopsida Species 0.000 description 1
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 1
- 235000007238 Secale cereale Nutrition 0.000 description 1
- 244000082988 Secale cereale Species 0.000 description 1
- 229940122055 Serine protease inhibitor Drugs 0.000 description 1
- 101710102218 Serine protease inhibitor Proteins 0.000 description 1
- 240000003768 Solanum lycopersicum Species 0.000 description 1
- 244000061456 Solanum tuberosum Species 0.000 description 1
- 235000002595 Solanum tuberosum Nutrition 0.000 description 1
- 239000005618 Sulcotrione Substances 0.000 description 1
- 238000010459 TALEN Methods 0.000 description 1
- 239000005620 Tembotrione Substances 0.000 description 1
- 108010043645 Transcription Activator-Like Effector Nucleases Proteins 0.000 description 1
- 101710150448 Transcriptional regulator Myc Proteins 0.000 description 1
- 241001464837 Viridiplantae Species 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 229960000583 acetic acid Drugs 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- HWXBTNAVRSUOJR-UHFFFAOYSA-N alpha-hydroxyglutaric acid Natural products OC(=O)C(O)CCC(O)=O HWXBTNAVRSUOJR-UHFFFAOYSA-N 0.000 description 1
- 229940009533 alpha-ketoglutaric acid Drugs 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 238000010009 beating Methods 0.000 description 1
- 238000010170 biological method Methods 0.000 description 1
- UDSAIICHUKSCKT-UHFFFAOYSA-N bromophenol blue Chemical compound C1=C(Br)C(O)=C(Br)C=C1C1(C=2C=C(Br)C(O)=C(Br)C=2)C2=CC=CC=C2S(=O)(=O)O1 UDSAIICHUKSCKT-UHFFFAOYSA-N 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 229950006137 dexfosfoserine Drugs 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 239000012149 elution buffer Substances 0.000 description 1
- 210000002257 embryonic structure Anatomy 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 description 1
- 229960005542 ethidium bromide Drugs 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000012362 glacial acetic acid Substances 0.000 description 1
- XDDAORKBJWWYJS-UHFFFAOYSA-N glyphosate Chemical compound OC(=O)CNCP(O)(O)=O XDDAORKBJWWYJS-UHFFFAOYSA-N 0.000 description 1
- 229940097068 glyphosate Drugs 0.000 description 1
- 235000008216 herbs Nutrition 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 235000021374 legumes Nutrition 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 230000002934 lysing effect Effects 0.000 description 1
- 229960000274 lysozyme Drugs 0.000 description 1
- 235000010335 lysozyme Nutrition 0.000 description 1
- 239000004325 lysozyme Substances 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000000442 meristematic effect Effects 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 235000019713 millet Nutrition 0.000 description 1
- 210000003739 neck Anatomy 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- YFCAPEGQZXEKKG-UHFFFAOYSA-N oxolane-2,3,4-trione Chemical compound O=C1COC(=O)C1=O YFCAPEGQZXEKKG-UHFFFAOYSA-N 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 235000020232 peanut Nutrition 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- BZQFBWGGLXLEPQ-REOHCLBHSA-N phosphoserine Chemical compound OC(=O)[C@@H](N)COP(O)(O)=O BZQFBWGGLXLEPQ-REOHCLBHSA-N 0.000 description 1
- USRGIUJOYOXOQJ-GBXIJSLDSA-N phosphothreonine Chemical compound OP(=O)(O)O[C@H](C)[C@H](N)C(O)=O USRGIUJOYOXOQJ-GBXIJSLDSA-N 0.000 description 1
- DCWXELXMIBXGTH-UHFFFAOYSA-N phosphotyrosine Chemical compound OC(=O)C(N)CC1=CC=C(OP(O)(O)=O)C=C1 DCWXELXMIBXGTH-UHFFFAOYSA-N 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 150000003217 pyrazoles Chemical class 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 238000010188 recombinant method Methods 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000002864 sequence alignment Methods 0.000 description 1
- 239000003001 serine protease inhibitor Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 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 1
- 238000004114 suspension culture Methods 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 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 1
- 238000009210 therapy by ultrasound Methods 0.000 description 1
- 238000011426 transformation method Methods 0.000 description 1
- 241001430294 unidentified retrovirus Species 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
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)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
- C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
-
- 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)
- C12Y113/12—Oxidoreductases acting on single donors with incorporation of molecular oxygen (oxygenases) (1.13) with incorporation of one atom of oxygen (internal monooxygenases or internal mixed function oxidases)(1.13.12)
- C12Y113/12019—2-Oxuglutarate dioxygenase (ethylene-forming) (1.13.12.19)
-
- 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
Abstract
The invention relates to the field of genetic engineering, discloses a 2ODD protein, a gene, a vector, a cell, a composition and application thereof, and a method for improving the herbicide resistance of crops, in particular to a 2ODD protein, a gene for encoding the 2ODD protein, a recombinant vector, a transgenic cell, a composition and application thereof in improving the herbicide resistance of crops, and also discloses a method for improving the herbicide resistance of crops. The 2ODD protein and the coding gene thereof provided by the invention can be used for improving the resistance of crops to HPPD inhibitors.
Description
Technical Field
The invention relates to the field of genetic engineering, in particular to a 2ODD protein, a gene, a vector, a cell, a composition and application thereof.
Background
The gene HIS1 in rice, namely the sensitive gene 1 of the p-hydroxyphenylpyruvate dioxygenase inhibitor (4-hydroxyphenylpyruvate dioxygenase inhibitor sensitive gene No. 1), can code Fe (II)/alpha-ketoglutarate dependent dioxygenase (2 ODD for short), and the enzyme can hydroxylate a trione HPPD inhibitor, so that the rice has resistance to the HPPD inhibitor. In addition, a plurality of HIS1 homologous genes were found in rice plants and designated as HSL (HIS 1-Like). Meanwhile, the presence of the HSL gene is also found in other plants such as barley, wheat, sorghum, maize, arabidopsis thaliana.
At present, research on HIS1 and HSL shows that the protein encoded by the HIS1 and the homologous gene HSL thereof can catalyze trione HPPD inhibitors, and the inhibition activity of catalytic products on HPPD is greatly reduced. Since HPPD is an important herbicide action target, the important function of HIS1 and HSL to detoxify HPPD inhibitors can be used to develop HPPD herbicide resistant crops.
There are 13 major classes of HPPD inhibitor herbicides currently on the market. Among the 20 classes of herbicides worldwide, HPPD inhibitor herbicides have grown faster in recent years and have been attracting attention in the industry, with the low risk of resistance being the leading foundation for this class of products. Based on the better product characteristics of HPPD inhibitor herbicides, the future market still has growing potential.
However, HPPD inhibitor herbicides are mostly used in corn fields and wheat fields, and are not widely applied in commercial crop planting areas such as peanuts, soybeans, rice, sorghum, and the like due to crop safety problems. In addition, there are some highly active HPPD inhibitors in the development stage, which have not been further developed due to the lack of certain crop safety. Whereas herbicide resistant crops have provided tremendous benefits to farmers and the environment over the last 20 years, for example, glyphosate resistant corn and soybean crops.
Therefore, the development of new herbicide-resistant crops, such as HPPD inhibitor-resistant crops, has important theoretical significance and economic value.
Disclosure of Invention
The invention aims to overcome the defect that the inhibiting activity and the crop safety of HPPD inhibitor herbicides in the prior art cannot be considered, and provides Fe (II)/alpha-ketoglutarate dependent dioxygenase protein with resistance to an HPPD inhibitor, and a coding gene and application thereof.
To achieve the above object, a first aspect of the present invention provides an Fe (ii)/α -ketoglutarate-dependent dioxygenase protein having an amino acid sequence selected from at least one of:
(1) A first amino acid sequence derived from at least one of the 141 th site and the 205 th site in the amino acid sequence shown in SEQ ID NO. 1;
(2) The first amino acid sequence is a protein derived from the first amino acid sequence by substituting, deleting or adding one or more amino acids, or a protein shown by an amino acid sequence with a label connected to the amino terminal end and/or the carboxyl terminal end of the first amino acid sequence;
(3) A second amino acid sequence derived from mutation at least 3 positions selected from the group consisting of position 140, position 204, position 298 and position 335 in the amino acid sequence shown in SEQ ID NO. 2;
(4) The second amino acid sequence is a protein derived from the second amino acid sequence by substituting, deleting or adding one or more amino acids, or a protein shown by an amino acid sequence with a label connected to the amino terminal end and/or the carboxyl terminal end of the second amino acid sequence;
(5) A third amino acid sequence derived from mutation at least one site selected from the group consisting of site 206, site 301 and site 339 in the amino acid sequence shown in SEQ ID NO. 3;
(6) The third amino acid sequence is a protein derived from the third amino acid sequence by substituting, deleting or adding one or more amino acids, or a protein shown by an amino acid sequence with a label connected to the amino terminal end and/or the carboxyl terminal end of the third amino acid sequence;
(7) A fourth amino acid sequence derived from mutation at least one site selected from the group consisting of site 142, site 207 and site 301 in the amino acid sequence shown in SEQ ID NO. 4;
(8) The protein derived from the fourth amino acid sequence by substituting, deleting or adding one or more amino acids, or the protein shown by the amino acid sequence with a label connected with the amino terminal and/or the carboxyl terminal of the fourth amino acid sequence;
(9) A fifth amino acid sequence derived from at least one position selected from the 285 position and the 322 position in the amino acid sequence shown in SEQ ID NO. 5;
(10) The protein derived from the fifth amino acid sequence by substituting, deleting or adding one or more amino acids, or the protein shown by the amino acid sequence with a label connected to the amino terminal and/or the carboxyl terminal of the fifth amino acid sequence;
(11) A sixth amino acid sequence derived from mutation at least one site selected from the group consisting of site 142, site 207, site 301 and site 338 in the amino acid sequence shown in SEQ ID NO. 6;
(12) The protein derived from the sixth amino acid sequence by substituting, deleting or adding one or more amino acids, or the protein shown by the amino acid sequence with a label connected to the amino terminal and/or carboxyl terminal of the sixth amino acid sequence;
(13) A seventh amino acid sequence derived from mutation at least 2 positions selected from the group consisting of 140 positions, 205 positions and 336 positions in the amino acid sequence shown in SEQ ID NO. 7;
(14) A protein derived from the seventh amino acid sequence by substituting, deleting or adding one or more amino acids, or a protein shown by an amino acid sequence with a tag connected to the amino terminal and/or carboxyl terminal of the seventh amino acid sequence;
(15) An eighth amino acid sequence derived after mutation at least 2 positions selected from the group consisting of 140 positions, 205 positions and 336 positions in the amino acid sequence shown in SEQ ID NO. 8;
(16) The eighth amino acid sequence is a protein derived from the eighth amino acid sequence by substituting, deleting or adding one or more amino acids, or a protein shown by an amino acid sequence with a tag connected to the amino terminal and/or carboxyl terminal of the eighth amino acid sequence.
In a second aspect, the present invention provides a gene encoding an Fe (II)/alpha-ketoglutarate-dependent dioxygenase protein, the nucleotide sequence of which is a nucleotide sequence capable of encoding the amino acid sequence of the Fe (II)/alpha-ketoglutarate-dependent dioxygenase protein of the first aspect.
In a third aspect, the present invention provides a recombinant vector comprising the gene according to the second aspect.
In a fourth aspect, the present invention provides a transgenic cell comprising a gene according to the second aspect.
In a fifth aspect the present invention provides a composition comprising a Fe (II)/alpha-ketoglutarate dependent dioxygenase protein as defined in the first aspect.
In a sixth aspect, the present invention provides the use of at least one of the Fe (ii)/α -ketoglutarate-dependent dioxygenase protein according to the first aspect, the gene according to the second aspect, the recombinant vector according to the third aspect, and the transgenic cell according to the fourth aspect for increasing herbicide resistance in crops.
A seventh aspect of the invention provides a method of increasing herbicide resistance in a crop, the method comprising: transferring the recombinant vector of the third aspect into a target plant, so that the target plant expresses the Fe (II)/alpha-ketoglutarate-dependent dioxygenase protein of the first aspect to obtain resistance to herbicide.
In an eighth aspect, the invention provides a method of increasing herbicide resistance in a crop, the method comprising: transferring the recombinant vector in the mutant crop containing the recombinant vector of the third aspect into a target plant by crossing, transferring or backcrossing, so that the target plant expresses the Fe (II)/alpha-ketoglutarate-dependent dioxygenase protein of the first aspect to obtain the resistance to herbicide.
A ninth aspect of the invention provides a method of increasing herbicide resistance in a crop, the method comprising: the Fe (II)/alpha-ketoglutarate dependent dioxygenase gene of the target plant is modified by a CRISPR/Cas gene editing method, so that the target plant expresses the Fe (II)/alpha-ketoglutarate dependent dioxygenase protein of the first aspect so as to obtain the resistance to herbicide.
Compared with the prior art, the Fe (II)/alpha-ketoglutarate dependent dioxygenase protein with resistance to the HPPD inhibitor and the encoding gene thereof have at least the following advantages:
by transferring the gene encoding the present invention into a target plant, the target plant can be made resistant to an HPPD inhibitor (herbicide) without substantially affecting the catalytic activity of the enzyme itself. Therefore, the Fe (II)/alpha-ketoglutarate dependent dioxygenase protein and the encoding gene thereof provided by the invention can be used for cultivating plants with herbicide resistance.
Additional features and advantages of the invention will be set forth in the detailed description which follows.
Detailed Description
The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.
As previously described, a first aspect of the present invention provides a Fe (II)/alpha-ketoglutarate dependent dioxygenase protein having an amino acid sequence selected from at least one of the following:
(1) A first amino acid sequence derived from at least one of the 141 th site and the 205 th site in the amino acid sequence shown in SEQ ID NO. 1;
(2) The first amino acid sequence is a protein derived from the first amino acid sequence by substituting, deleting or adding one or more amino acids, or a protein shown by an amino acid sequence with a label connected to the amino terminal end and/or the carboxyl terminal end of the first amino acid sequence;
(3) A second amino acid sequence derived from mutation at least 3 positions selected from the group consisting of position 140, position 204, position 298 and position 335 in the amino acid sequence shown in SEQ ID NO. 2;
(4) The second amino acid sequence is a protein derived from the second amino acid sequence by substituting, deleting or adding one or more amino acids, or a protein shown by an amino acid sequence with a label connected to the amino terminal end and/or the carboxyl terminal end of the second amino acid sequence;
(5) A third amino acid sequence derived from mutation at least one site selected from the group consisting of site 206, site 301 and site 339 in the amino acid sequence shown in SEQ ID NO. 3;
(6) The third amino acid sequence is a protein derived from the third amino acid sequence by substituting, deleting or adding one or more amino acids, or a protein shown by an amino acid sequence with a label connected to the amino terminal end and/or the carboxyl terminal end of the third amino acid sequence;
(7) A fourth amino acid sequence derived from mutation at least one site selected from the group consisting of site 142, site 207 and site 301 in the amino acid sequence shown in SEQ ID NO. 4;
(8) The protein derived from the fourth amino acid sequence by substituting, deleting or adding one or more amino acids, or the protein shown by the amino acid sequence with a label connected with the amino terminal and/or the carboxyl terminal of the fourth amino acid sequence;
(9) A fifth amino acid sequence derived from at least one position selected from the 285 position and the 322 position in the amino acid sequence shown in SEQ ID NO. 5;
(10) The protein derived from the fifth amino acid sequence by substituting, deleting or adding one or more amino acids, or the protein shown by the amino acid sequence with a label connected to the amino terminal and/or the carboxyl terminal of the fifth amino acid sequence;
(11) A sixth amino acid sequence derived from mutation at least one site selected from the group consisting of site 142, site 207, site 301 and site 338 in the amino acid sequence shown in SEQ ID NO. 6;
(12) The protein derived from the sixth amino acid sequence by substituting, deleting or adding one or more amino acids, or the protein shown by the amino acid sequence with a label connected to the amino terminal and/or carboxyl terminal of the sixth amino acid sequence;
(13) A seventh amino acid sequence derived from mutation at least 2 positions selected from the group consisting of 140 positions, 205 positions and 336 positions in the amino acid sequence shown in SEQ ID NO. 7;
(14) A protein derived from the seventh amino acid sequence by substituting, deleting or adding one or more amino acids, or a protein shown by an amino acid sequence with a tag connected to the amino terminal and/or carboxyl terminal of the seventh amino acid sequence;
(15) An eighth amino acid sequence derived after mutation at least 2 positions selected from the group consisting of 140 positions, 205 positions and 336 positions in the amino acid sequence shown in SEQ ID NO. 8;
(16) The eighth amino acid sequence is a protein derived from the eighth amino acid sequence by substituting, deleting or adding one or more amino acids, or a protein shown by an amino acid sequence with a tag connected to the amino terminal and/or carboxyl terminal of the eighth amino acid sequence.
In the present invention, the constant enzyme activity means that the percentage (relative activity) between the enzyme activity of a protein derived from the first amino acid sequence, the second amino acid sequence, the third amino acid sequence, the fourth amino acid sequence, the fifth amino acid sequence, the sixth amino acid sequence, the seventh amino acid sequence, and the eighth amino acid sequence and the enzyme activity of a wild-type enzyme is not less than 95% (or 96%, or 97%, or 98%, or 99%, or 100%) under the same measurement conditions.
Preferably, the first amino acid sequence is an amino acid sequence selected from at least one of the following:
(1a) An amino acid sequence derived from the histidine at 141 site in the amino acid sequence shown in SEQ ID NO. 1 is mutated into phenylalanine;
(1b) An amino acid sequence which is derived after histidine at 141 site is mutated into phenylalanine and phenylalanine at 205 site is mutated into leucine in the amino acid sequence shown in SEQ ID NO. 1;
preferably, the first amino acid sequence is the amino acid sequence shown in H141F, H F/F205L.
According to a specific embodiment of the present invention, the amino acid sequence derived from the mutation of histidine at position 141 in the amino acid sequence shown in SEQ ID NO. 1 to phenylalanine is designated as H141F.
According to a specific embodiment of the invention, the amino acid sequence shown in SEQ ID NO. 1, in which histidine at position 141 is mutated to phenylalanine and phenylalanine at position 205 is mutated to leucine, is referred to as H141F/F205L.
Preferably, the second amino acid sequence is the amino acid sequence:
(2a) Amino acid sequence derived from the amino acid sequence shown in SEQ ID NO. 2, wherein phenylalanine at 140 site is mutated to histidine, leucine at 204 site is mutated to phenylalanine, phenylalanine at 298 site is mutated to leucine and isoleucine at 335 site is mutated to phenylalanine;
(2b) Amino acid sequence shown in SEQ ID NO. 2, wherein leucine at position 204 is mutated into phenylalanine, phenylalanine at position 298 is mutated into leucine and isoleucine at position 335 is mutated into alanine;
(2c) The amino acid sequence shown in SEQ ID NO. 2 has leucine at position 204 mutated to phenylalanine, phenylalanine at position 298 mutated to leucine and isoleucine at position 335 mutated to tryptophan.
Preferably, the second amino acid sequence is at least one of the amino acid sequences shown in F140H/L204F/F298L/I335F, L F/F298L/I335A, L F/F298L/I335W.
According to a specific embodiment of the invention, the amino acid sequence shown in SEQ ID NO. 2 is derived from the amino acid sequence that phenylalanine at 140 site is mutated to histidine, leucine at 204 site is mutated to phenylalanine, phenylalanine at 298 site is mutated to leucine and isoleucine at 335 site is mutated to phenylalanine, which is called F140H/L204F/F298L/I335F.
According to a specific embodiment of the invention, the amino acid sequence shown in SEQ ID NO. 2, in which the leucine at position 204 is mutated to phenylalanine, the phenylalanine at position 298 is mutated to leucine and the isoleucine at position 335 is mutated to alanine, is referred to as L204F/F298L/I335A.
According to a specific embodiment of the invention, the amino acid sequence shown in SEQ ID NO. 2, in which leucine at position 204 is mutated to phenylalanine, phenylalanine at position 298 is mutated to leucine and isoleucine at position 335 is mutated to tryptophan, is referred to as L204F/F298L/I335W.
Preferably, the third amino acid sequence is the amino acid sequence:
(3a) Amino acid sequence derived from threonine at 206 in the amino acid sequence shown in SEQ ID NO. 3;
(3b) An amino acid sequence derived from the amino acid sequence shown in SEQ ID NO. 3, wherein threonine at position 206 is mutated to phenylalanine and phenylalanine at position 301 is mutated to leucine;
(3c) Amino acid sequence derived from the amino acid sequence shown in SEQ ID NO. 3, wherein threonine at position 206 is mutated to phenylalanine, phenylalanine at position 301 is mutated to leucine, and tyrosine at position 339 is mutated to phenylalanine.
Preferably, the third amino acid sequence is at least one of the amino acid sequences shown as T206F, T F/F301L, T F/F301L/Y339F.
According to a specific embodiment of the present invention, the amino acid sequence derived from the mutation of threonine at position 206 in the amino acid sequence shown in SEQ ID NO. 3 to phenylalanine is referred to as T206F.
According to a specific embodiment of the present invention, the amino acid sequence shown in SEQ ID NO. 3, in which threonine at position 206 is mutated to phenylalanine and phenylalanine at position 301 is mutated to leucine, is referred to as T206F/F301L.
According to a specific embodiment of the invention, the amino acid sequence shown in SEQ ID NO. 3 is derived after mutation of threonine at position 206 into phenylalanine, mutation of phenylalanine at position 301 into leucine and mutation of tyrosine at position 339 into phenylalanine, and is called T206F/F301L/Y339F.
Preferably, the fourth amino acid sequence is the amino acid sequence:
(4a) The tyrosine at 142 site in the amino acid sequence shown in SEQ ID NO. 4 is mutated into amino acid sequence derived after histidine;
(4b) An amino acid sequence derived from the amino acid sequence shown in SEQ ID NO. 4, wherein tyrosine at 142 is mutated to histidine and isoleucine at 207 is mutated to phenylalanine;
(4c) Amino acid sequence shown in SEQ ID NO. 4, wherein tyrosine at 142 is mutated to histidine, isoleucine at 207 is mutated to phenylalanine, and phenylalanine at 301 is mutated to leucine.
Preferably, the fourth amino acid sequence is at least one of the amino acid sequences shown in Y142H, Y H/I207F, Y H/I207F/F301L.
According to a specific embodiment of the present invention, the amino acid sequence shown in SEQ ID NO. 4, wherein the tyrosine at position 142 is mutated to histidine and then derived, is designated Y142H.
According to a specific embodiment of the present invention, the amino acid sequence derived from the amino acid sequence shown in SEQ ID NO. 4, in which tyrosine at position 142 is mutated to histidine and isoleucine at position 207 is mutated to phenylalanine, is designated Y142H/I207F.
According to a specific embodiment of the invention, the amino acid sequence shown in SEQ ID NO. 4, in which the tyrosine at position 142 is mutated to histidine, the isoleucine at position 207 is mutated to phenylalanine and the phenylalanine at position 301 is mutated to leucine, is referred to as Y142H/I207F/F301L.
Preferably, the fifth amino acid sequence is the amino acid sequence:
(5a) An amino acid sequence derived from the phenylalanine at 285 site in the amino acid sequence shown in SEQ ID NO. 5 after mutation into leucine;
(5b) The amino acid sequence shown in SEQ ID NO. 5 is derived by mutating 285 phenylalanine to leucine and mutating 322 histidine to phenylalanine.
Preferably, the fifth amino acid sequence is at least one of the amino acid sequences shown in F285L, F L/H322F.
According to a specific embodiment of the present invention, phenylalanine at position 285 of the amino acid sequence shown in SEQ ID NO. 5 is mutated to a leucine-derived amino acid sequence, designated F285L.
According to a specific embodiment of the present invention, the amino acid sequence derived from the amino acid sequence shown in SEQ ID NO. 5, in which phenylalanine at position 285 is mutated to leucine and histidine at position 322 is mutated to phenylalanine, is designated as F285L/H322F.
Preferably, the sixth amino acid sequence is the amino acid sequence:
(6a) The glutamine at 142 site in the amino acid sequence shown in SEQ ID NO. 6 is mutated into histidine to obtain an amino acid sequence;
(6b) An amino acid sequence derived from the amino acid sequence shown in SEQ ID NO. 6, wherein glutamine at position 142 is mutated to histidine and isoleucine at position 207 is mutated to phenylalanine;
(6c) An amino acid sequence shown in SEQ ID NO. 6, wherein the amino acid sequence is derived after glutamine at 142 site is mutated to histidine, isoleucine at 207 site is mutated to phenylalanine and phenylalanine at 301 site is mutated to leucine;
(6d) Amino acid sequence derived from the amino acid sequence shown in SEQ ID NO. 6, wherein glutamine at 142 site is mutated to histidine, isoleucine at 207 site is mutated to phenylalanine, phenylalanine at 301 site is mutated to leucine and arginine at 338 site is mutated to phenylalanine;
(6e) The amino acid sequence shown in SEQ ID NO. 6 is derived by mutating glutamine at 142 site into histidine and mutating arginine at 338 site into phenylalanine.
Preferably, the sixth amino acid sequence is at least one of the amino acid sequences shown in Q142H, Q H/I207F, Q H/I207F/F301L, Q H/I207F/F301L/R338F, Q H/R338F.
According to a specific embodiment of the present invention, the amino acid sequence derived from the mutation of glutamine at position 142 in the amino acid sequence shown in SEQ ID NO. 6 to histidine is referred to as Q142H.
According to a specific embodiment of the present invention, the amino acid sequence derived from the amino acid sequence shown in SEQ ID NO. 6, in which glutamine at position 142 is mutated to histidine and isoleucine at position 207 is mutated to phenylalanine, is referred to as Q142H/I207F.
According to a specific embodiment of the invention, the amino acid sequence shown in SEQ ID NO. 6, in which glutamine at position 142 is mutated to histidine, isoleucine at position 207 is mutated to phenylalanine and phenylalanine at position 301 is mutated to leucine, is referred to as Q142H/I207F/F301L.
According to a specific embodiment of the invention, the amino acid sequence shown in SEQ ID NO. 6 is derived after mutation of glutamine at position 142 into histidine, mutation of isoleucine at position 207 into phenylalanine, mutation of phenylalanine at position 301 into leucine and mutation of arginine at position 338 into phenylalanine, and is called Q142H/I207F/F301L/R338F.
According to a specific embodiment of the invention, the amino acid sequence shown in SEQ ID NO. 6, wherein the amino acid sequence is derived from the fact that the glutamine at the 142 site is mutated to histidine and the arginine at the 338 site is mutated to phenylalanine, is called Q142H/R338F.
Preferably, the seventh amino acid sequence is the amino acid sequence:
(7a) An amino acid sequence derived from the amino acid sequence shown in SEQ ID NO. 7, wherein the glutamine at the 140 site is mutated to histidine and the tyrosine at the 205 site is mutated to phenylalanine;
(7b) The amino acid sequence shown in SEQ ID NO. 7 is derived after the mutation of glutamine at 140 site to histidine, the mutation of tyrosine at 205 site to phenylalanine and the mutation of lysine at 336 site to phenylalanine.
Preferably, the seventh amino acid sequence is at least one of the amino acid sequences shown in Q140H/Y205F, Q H/Y205F/K336F.
According to a specific embodiment of the invention, the amino acid sequence derived from the amino acid sequence shown in SEQ ID NO. 7, in which glutamine at position 140 is mutated to histidine and tyrosine at position 205 is mutated to phenylalanine, is referred to as the Q140H/Y205F sequence.
According to a specific embodiment of the invention, the amino acid sequence shown in SEQ ID NO. 7 is derived after mutation of glutamine at position 140 to histidine, mutation of tyrosine at position 205 to phenylalanine and mutation of lysine at position 336 to phenylalanine, and is called a Q140H/Y205F/K336F sequence.
Preferably, the eighth amino acid sequence is the amino acid sequence:
(8a) An amino acid sequence derived from the amino acid sequence shown in SEQ ID NO. 8, wherein the glutamine at the 140 site is mutated to histidine and the tyrosine at the 205 site is mutated to phenylalanine;
(8b) The amino acid sequence shown in SEQ ID NO. 8 is derived after the mutation of glutamine at 140 site into histidine, the mutation of tyrosine at 205 site into phenylalanine and the mutation of lysine at 336 site into phenylalanine.
Preferably, the eighth amino acid sequence is at least one of the amino acid sequences shown in Q140H/Y205F, Q H/Y205F/K336F.
According to a specific embodiment of the present invention, the amino acid sequence derived from the amino acid sequence shown in SEQ ID NO. 8, in which glutamine at position 140 is mutated to histidine and tyrosine at position 205 is mutated to phenylalanine, is referred to as Q140H/Y205F.
According to a specific embodiment of the invention, the amino acid sequence shown in SEQ ID NO. 8 is derived after mutation of glutamine at position 140 to histidine, mutation of tyrosine at position 205 to phenylalanine and mutation of lysine at position 336 to phenylalanine, and is called Q140H/Y205F/K336F.
In the present invention, the method for obtaining the above-mentioned protein is well known to those skilled in the art, and for example, the protein may be obtained directly by a chemical synthesis method, or may be obtained by obtaining a gene encoding the protein and then obtaining the protein by a biological expression method, with knowledge of the amino acid sequence of the protein.
The protein provided by the invention can also be modified. The modified (typically without altering the primary structure, i.e., without altering the amino acid sequence) forms include: chemically derivatized forms of proteins such as acetylated or hydroxylated in vivo or in vitro. Modified forms also include glycosylation such as those produced by glycosylation modification during synthesis and processing of the protein or during further processing steps, such modification may be accomplished by exposing the protein to an enzyme that performs glycosylation (e.g., mammalian glycosylase or deglycosylase). Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine).
In the present invention, the protein may be additionally modified with a tag common in the art for convenience of purification, and may be obtained by ligating a common purification tag shown in Table 1 (e.g., at least one of GST, poly-His, FLAG, his-SUMO and c-myc) at the amino-terminal and/or carboxyl-terminal of the protein, for example. The label does not influence the activity of the protein provided by the invention, and whether the label is added or not can be selected according to the requirement in the actual application process.
TABLE 1
As described above, the second aspect of the present invention provides a gene encoding an Fe (II)/alpha-ketoglutarate-dependent dioxygenase protein, the nucleotide sequence of which is a nucleotide sequence capable of encoding the amino acid sequence of the Fe (II)/alpha-ketoglutarate-dependent dioxygenase protein of the first aspect.
Preferably, the nucleotide sequence of the gene is a nucleotide sequence selected from at least one of the following:
(1) A nucleotide sequence as shown in SEQ ID NO. 11;
(2) A nucleotide sequence having at least 90% identity, preferably at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to SEQ ID No. 11;
(3) A nucleotide sequence as shown in SEQ ID NO. 21;
(4) A nucleotide sequence having at least 90% identity, preferably at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to SEQ ID No. 21;
(5) A nucleotide sequence as shown in SEQ ID NO. 31;
(6) A nucleotide sequence having at least 90% identity, preferably at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to SEQ ID No. 31;
(7) A nucleotide sequence as shown in SEQ ID NO. 41;
(8) A nucleotide sequence having at least 90% identity, preferably at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to SEQ ID No. 41;
(9) A nucleotide sequence shown as SEQ ID NO. 51;
(10) A nucleotide sequence having at least 90% identity, preferably at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to SEQ ID No. 51;
(11) A nucleotide sequence as shown in SEQ ID NO. 61;
(12) A nucleotide sequence having at least 90% identity, preferably at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to SEQ ID No. 61;
(13) A nucleotide sequence shown as SEQ ID NO. 71;
(14) A nucleotide sequence having at least 90% identity, preferably at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to SEQ ID No. 71;
(15) A nucleotide sequence as shown in SEQ ID NO. 81;
(16) A nucleotide sequence having at least 90% identity, preferably at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to SEQ ID No. 81.
In the present invention, it is well known in the art that of the 20 different amino acids constituting a protein, other than Met (ATG) or Trp (TGG) are each encoded by a single codon, the 18 other amino acids are each encoded by 2-6 codons (Sambrook et al, molecular cloning, cold spring harbor laboratory Press, new York, U.S., second edition, 1989, see appendix D of page 950). That is, due to the degeneracy of the genetic code, the nucleotide sequence of the gene encoding the same protein may differ, since the substitution of the third nucleotide in the triplet codon, which determines most of the codons of one amino acid, does not change the composition of the amino acid.
According to the known codon table, the amino acid sequence SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 7. SEQ ID NO:8 and specific mutation patterns of proteins, it is entirely possible to deduce nucleotide sequences of genes encoding them and obtain the nucleotide sequences by biological methods (e.g., PCR methods, mutation methods) or chemical synthesis methods, and thus, the partial nucleotide sequences should be included in the scope of the present invention.
Likewise, the nucleotide sequence SEQ ID NO provided by the present invention: 11. SEQ ID NO: 21. SEQ ID NO: 31. SEQ ID NO: 41. SEQ ID NO: 51. SEQ ID NO: 61. SEQ ID NO: 71. SEQ ID NO:81 and protein can also be carried out by methods known in the art, for example by the method of Sambrook et al (molecular cloning, cold spring harbor laboratory Press, new York, U.S. second edition, 1989), by modifying the sequence of SEQ ID NO: 11. SEQ ID NO: 21. SEQ ID NO: 31. SEQ ID NO: 41. SEQ ID NO: 51. SEQ ID NO: 61. SEQ ID NO: 71. SEQ ID NO:81, the amino acid sequence provided by the invention is obtained.
In the present invention, the above protein and nucleotide sequence SEQ ID NO: 11. SEQ ID NO: 21. SEQ ID NO: 31. SEQ ID NO: 41. SEQ ID NO: 51. SEQ ID NO: 61. SEQ ID NO: 71. SEQ ID NO:81, one skilled in the art can readily obtain a gene encoding a protein provided by the present invention. Illustratively, one can set forth in SEQ ID NO:11, including, but not limited to ZFN site-directed mutagenesis methods, TALEN site-directed mutagenesis methods, and/or CRISPR-Cas9, etc.
In the present invention, as described in the first aspect, correspondingly, the 5 'end and/or the 3' end of the nucleotide sequence provided by the present invention may be further linked with a coding sequence of a common purification tag shown in table 1.
The nucleotide sequence provided by the invention can be obtained by a Polymerase Chain Reaction (PCR) amplification method, a recombination method or an artificial synthesis method. Illustratively, templates and primers can be readily obtained by one skilled in the art from the nucleotide sequences provided herein, which are obtained by amplification using PCR. After obtaining the nucleotide sequence, the amino acid sequence can be obtained in large quantities by recombinant methods. The nucleotide sequence obtained is usually cloned into a vector, transferred into genetically engineered bacteria, and then isolated from the proliferated host cells by a conventional method to obtain the nucleotide sequence. Furthermore, the nucleotide sequence may be synthesized by methods of artificial chemical synthesis known in the art.
As described above, the third aspect of the present invention provides a recombinant vector comprising the gene according to the second aspect.
In the present invention, the "vector" used in the recombinant vector may be any of various vectors known in the art, such as various plasmids, cosmids, phages, retroviruses, etc. commercially available, and may be selected according to the specific circumstances, and may be pGWC, pB2GW7.0, pET-28a, etc., for example. The recombinant vector construction may be carried out by performing cleavage with various endonucleases capable of having cleavage sites at the vector multiple cloning sites to obtain a linear plasmid, and ligating the gene fragments cleaved with the same endonucleases to obtain a recombinant plasmid.
As described above, the fourth aspect of the present invention provides a transgenic cell comprising the gene of the second aspect.
Preferably, the transgenic cell is a prokaryotic cell.
In the present invention, the recombinant vector may be transformed, transduced or transfected into host cells by methods known in the art, such as calcium chloride chemical transformation, high voltage shock transformation, preferably shock transformation. The host cell can be a prokaryotic cell or a eukaryotic cell, and can be selected according to practical conditions. The cells may be DH 5. Alpha. Strain, agrobacterium strain GV3101, etc.
As previously mentioned, the fifth aspect of the present invention provides a composition comprising the Fe (II)/alpha-ketoglutarate dependent dioxygenase protein of the first aspect.
The composition provided by the invention contains the Fe (II)/alpha-ketoglutarate-dependent dioxygenase protein as an active ingredient, wherein the content of the protein is 50-90 wt% based on the total weight of the composition. The composition may further contain a solvent (e.g., a protein protectant such as glycerol, saccharide, and protease inhibitor) known to those skilled in the art, and the like.
As described above, the sixth aspect of the present invention provides the use of at least one of the Fe (II)/alpha-ketoglutarate dependent dioxygenase protein of the first aspect, the gene of the second aspect, the recombinant vector of the third aspect, and the transgenic cell of the fourth aspect for increasing herbicide resistance in crops.
Preferably, the herbicide is an HPPD inhibitor.
Preferably, the herbicide is at least one of a trione compound, a pyrazole compound, an isoxazole compound, a diketopolynitrile compound and a heterocyclic amide compound.
More preferably, the herbicide is at least one of mesotrione, cyclosulfamuron, furansulcotrione, bicyclosulcotrione, quinclorac (Y13161), methylquinclorac (Y13287), Y18024, Y16550 and topramezone, and the molecular structural formula is as follows:
as previously mentioned, a seventh aspect of the present invention provides a method of increasing herbicide resistance in a crop, the method comprising: transferring the recombinant vector of the third aspect into a target plant, so that the target plant expresses the Fe (II)/alpha-ketoglutarate-dependent dioxygenase protein of the first aspect to obtain resistance to herbicide.
As previously mentioned, the eighth aspect of the present invention provides a method of increasing herbicide resistance in a crop, the method comprising: transferring the recombinant vector in the mutant crop containing the recombinant vector of the third aspect into a target plant by crossing, transferring or backcrossing, so that the target plant expresses the Fe (II)/alpha-ketoglutarate-dependent dioxygenase protein of the first aspect to obtain the resistance to herbicide.
As previously mentioned, a ninth aspect of the present invention provides a method of increasing herbicide resistance in a crop, the method comprising: the HIS1/HSL gene of the target plant was engineered by CRISPR/Cas gene editing method such that the target plant expressed the Fe (ii)/α -ketoglutarate dependent dioxygenase protein of the aforementioned first aspect to obtain resistance to herbicides.
In the invention, the transfer of the recombinant vector into a target plant specifically means: the nucleotide sequence of the protein with herbicide resistance provided by the invention is transferred into target plants by a transgenic method, so that the target plants obtain the resistance to HPPD inhibitor herbicides; the nucleotide sequence of the protein provided by the invention can be transferred into target plants by methods of hybridization, transfer, backcross and the like, so that the target plants can obtain the resistance to HPPD inhibitor herbicides; in addition, the HIS1/HSL genes of the target plants can be directly modified by CRISPR/Cas and other gene editing technologies, so that the target plants can obtain the resistance to HPPD inhibitor herbicides. More specifically, the protein can be used as a parent material, crossed with other elite plant varieties and further backcrossed to further transform herbicide resistant traits into other target plant varieties.
The transgenic methods employed in the present invention are well known to those skilled in the art, and include direct or indirect transformation methods, including chemical induction, liposome, gene gun, electroporation, microinjection, and the like.
In the present invention, the term "plant" is used in its broadest sense and examples of plants include, but are not limited to, vascular bundle plants, vegetables, foodstuffs, flowers, trees, herbs, shrubs, grasses, vines, ferns, mosses, fungi, algae, etc., as well as clones and plant parts for asexual propagation (e.g., cuttings, beads, shoots, rhizomes, subterranean stems, cluster stems, root necks, bulbs, tubers, rhizomes, plants/tissues produced in tissue culture, etc.). The term "plant" further encompasses whole plants, plant parents and progeny, and plant parts, including seeds, shoots, stems, leaves, roots (including tubers), flowers, florets, fruits, stems, inflorescences, stamens, anthers, stigmas, columns, ovaries, petals, sepals, carpels, root tips, root crowns, root hairs, leaf hairs, seed hairs, pollen grains, microspores, cotyledons, hypocotyls, epicotyls, xylem, phloem, parenchyma, endosperm, companion cells, guard cells, and any other known organ, tissue and cell, and tissue and organ of a plant. The term "plant" also encompasses plant cells, suspension cultures, calli, embryos, meristematic regions, gametophytes, sporophytes, pollen and microspores. Among these, all mentioned above include the genes/nucleic acids of interest provided by the present invention.
Plants which are particularly suitable for use in the method of the invention include all plants belonging to the superfamily kingdom (Viridiplantae), in particular monocotyledonous and dicotyledonous plants, including food crops, forage or forage legumes, ornamental plants, trees or shrubs.
According to a preferred embodiment of the invention, the plant is a crop plant. Examples of crop plants include, in particular, rice, wheat, maize, sorghum, soybean, sunflower, rape, alfalfa, cotton, tomato, potato or tobacco. More preferably, the crop plant is a cereal, such as rice, wheat, maize, sorghum, barley, millet, rye or oats.
The invention has the advantages that the protein, the gene, the recombinant vector, the transgenic cells, the composition and the plants which can replace the prior art for enabling crops to have herbicide resistance are obtained, the application and the method for obtaining the crops with the herbicide resistance are realized, the crop varieties with the herbicide resistance can be obtained through the transgenic or non-transgenic method, and the weed control in paddy fields and dry fields can be cultivated more efficiently, so that the yield and the yield stability of the crops can be improved.
In the examples below, room temperature is 25.+ -. 2 ℃ unless otherwise indicated.
In the following examples, unless otherwise specified, the experimental methods used were all conventional.
In the examples below, materials, reagents and the like referred to are commercially available unless otherwise specified.
Agarose gel recovery kit was purchased from TIANGEN company;
restriction enzymes and matched solutions were purchased from Takara corporation;
the ligation kit was purchased from New England BioLabs.
Example 1
Synthesis of coding Gene and nucleotide sequence
The following coding genes and nucleotide sequences were synthesized by the nucleotide sequences provided by the present invention of the wuhan Jin Kairui bioengineering limited company.
(1)SEQ ID NO：11
According to SEQ ID NO:11, and synthesizing a coding gene of the protein shown in the table 2 and a nucleotide sequence shown in SEQ ID NO:11, and a nucleotide sequence shown in seq id no.
TABLE 2
| Proteins | Coding gene | Mutation site codon |
| SEQ ID NO：1 | WT (wild type) | / |
| H141F | H141F | TTT |
| H141F/F205L | H141F/F205L | TTT/CTG |
(2)SEQ ID NO：21
According to SEQ ID NO:21 to synthesize the coding gene of the protein shown in table 3 and the nucleotide sequence shown in SEQ ID NO:21, and a nucleotide sequence shown in seq id no.
TABLE 3 Table 3
| Proteins | Coding gene | Mutation site codon |
| SEQ ID NO：2 | WT (wild type) | / |
| F140H/L204F/F298L/I335F | F140H/L204F/F298L/I335F | CAT/TTT/CTG/TTT |
| L204F/F298L/I335A | L204F/F298L/I335A | TTT/CTG/GCG |
| L204F/F298L/I335W | L204F/F298L/I335W | TTT/CTG/TGG |
(3)SEQ ID NO：31
According to SEQ ID NO:31 to synthesize the coding gene of the protein shown in table 4 and the nucleotide sequence shown in SEQ ID NO: 31.
TABLE 4 Table 4
| Proteins | Coding gene | Mutation site codon |
| SEQ ID NO：3 | WT (wild type) | / |
| T206F | T206F | TTT |
| T206F/F301L | T206F/F301L | TTT/CTG |
| T206F/F301L/Y339F | T206F/F301L/Y339F | TTT/CTG/TTT |
(4)SEQ ID NO：41
According to SEQ ID NO:41 into the protein shown in table 5, and the nucleotide sequence shown in SEQ ID NO:41, and a nucleotide sequence shown in seq id no.
TABLE 5
| Proteins | Coding gene | Mutation site codon |
| SEQ ID NO：4 | WT (wild type) | / |
| Y142H | Y142H | CAT |
| Y142H/I207F | Y142H/I207F | CAT/TTT |
| Y142H/I207F/F301L | Y142H/I207F/F301L | CAT/TTT/CTG |
(5)SEQ ID NO：51
According to SEQ ID NO:51 to synthesize the coding gene of the protein shown in table 6 and the nucleotide sequence shown in SEQ ID NO:51, and a nucleotide sequence shown in seq id no.
TABLE 6
| Proteins | Coding gene | Mutation site codon |
| SEQ ID NO：5 | WT (wild type) | / |
| F285L | F285L | CTG |
| F285L/H322F | F285L/H322F | CTG/TTT |
(6)SEQ ID NO：61
According to SEQ ID NO:61 to synthesize the coding gene of the protein shown in table 7 and the nucleotide sequence shown in SEQ ID NO:61, and a nucleotide sequence shown in seq id no.
TABLE 7
| Proteins | Coding gene | Mutation site codon |
| SEQ ID NO：6 | WT (wild type) | / |
| Q142H | Q142H | CAT |
| Q142H/I207F | Q142H/I207F | CAT/TTT |
| Q142H/I207F/F301L | Q142H/I207F/F301L | CAT/TTT/CTG |
| Q142H/I207F/F301L/R338F | Q142H/I207F/F301L/R338F | CAT/TTT/CTG/TTT |
| Q142H/R338F | Q142H/R338F | CAT/TTT |
(7)SEQ ID NO：71
According to SEQ ID NO:71 to synthesize the coding gene of the protein shown in table 8 and the nucleotide sequence shown in SEQ ID NO: 71.
TABLE 8
| Proteins | Coding gene | Mutation site codon |
| SEQ ID NO：7 | WT (wild type) | / |
| Q140H/Y205F | Q140H/Y205F | CAT/TTT |
| Q140H/Y205F/K336F | Q140H/Y205F/K336F | CAT/TTT/TTT |
(8)SEQ ID NO：81
According to SEQ ID NO:81 to synthesize the coding gene of the protein shown in table 9 and the nucleotide sequence shown in SEQ ID NO:81, and a nucleotide sequence shown in seq id no.
TABLE 9
| Proteins | Coding gene | Mutation site codon |
| SEQ ID NO：8 | WT (wild type) | / |
| Q140H/Y205F | Q140H/Y205F | CAT/TTT |
| Q140H/Y205F/K336F | Q140H/Y205F/K336F | CAT/TTT/TTT |
Example 2
Construction of expression vectors
(1) Amplification of target Gene
Respectively with SEQ ID NO: 11. SEQ ID NO: 21. SEQ ID NO: 31. SEQ ID NO: 41. SEQ ID NO: 51. SEQ ID NO: 61. SEQ ID NO: 71. SEQ ID NO:81 as templates, wild-type and mutant gene sequences of HIS1/HSL as shown in tables 2-9 were obtained by PCR reaction and ligated into the corresponding vectors.
The PCR reaction system and PCR program settings are shown in tables 10 and 11, respectively, below:
table 10
| Composition of PCR reaction system | Addition (volume) |
| 10 XPCR buffer | 5μL |
| 5×dNTP(10mM) | 1μL |
| Stencil plate | 1μL |
| Forward primer | 1.0μL |
| Directional primers | 1.0μL |
| PfuDNA polymerase | 0.5μL |
| dd H 2 O | To a total volume of 50. Mu.L |
TABLE 11
| Step (a) | Temperature (. Degree. C.) | Time(s) |
| 1 | 95 | 240 |
| 2 | 95 | 45 |
| 3 | 55 | 45 |
| 4 | 72 | 400 |
| 5 | 72 | 600 |
| 6 | 4 | Preservation of |
The extension time at 72℃depends, among other things, on the specific fragment length and the efficiency of the enzyme used. Steps 2-4 of the above procedure were repeated for 25 cycles.
(2) Nucleic acid agarose gel electrophoresis and gel recovery PCR product
a. Weighing 1g of agarose, adding 100mL of 1 xTAE solution (prepared into 50 times stock solution, the preparation method comprises the steps of weighing 242g of Tris and 18.6g of EDTA in a 1L beaker, adding about 800mL of deionized water, stirring, adding 57.1mL of glacial acetic acid, fully dissolving, adjusting pH to 8.3 by NaOH, adding deionized water to a volume of 1L, preserving at room temperature, the same), heating in a microwave oven, cooling until agarose particles are completely dissolved, adding 5 mu L of ethidium bromide solution after not scalding hands, mixing uniformly, pouring into a gel making groove with a comb, and cooling and solidifying;
b. Pulling off the comb before use, placing the gel into an electrophoresis tank, and adding a proper amount of 1 xTAE solution; adding the sample into the hole, covering the hole with a cover to start electrophoresis, and stopping electrophoresis when bromophenol blue moves to 1/3 distance from the bottom;
c. cutting off the target strip, putting the target strip into a clean 1.5mLEP tube and recycling the glue; the kit used for the recovery of the glue was purchased from TIANGEN company (cat No. DP 209); the gel is operated according to the amount of 100mg of gel corresponding to 300 mu L of sol solution PN (provided by a kit), and incubated in a water bath at 50 ℃, and the gel is evenly mixed upside down continuously during the incubation, so as to help the dissolution of the gel;
d. adding the solution in the step c into a pretreated adsorption column (provided by a kit), standing at room temperature for 5min to fully combine the solution, and centrifuging at 12000rpm for 1min;
e. pouring out the liquid in the collecting pipe, repeating the step d once to increase the recovery rate of the product;
f. 500. Mu.L of rinse solution PW (provided by the kit) was added to the purification column and centrifuged at 12000rpm for 1min; repeating this operation step once;
g. centrifuging the adsorption column at 13000rpm for 2min, and removing PW solution as clean as possible;
h. placing the purification column into a new EP tube, heating at 65deg.C for 10min, volatilizing PW solution;
i. dripping 50 μl of the eluting solution (provided by kit) into the middle of the adsorption column, standing at 65deg.C for 2min, centrifuging at 13000rpm for 2min, and eluting DNA; repeating this operation step once;
j. Finally, the recovered PCR product is preserved at-20 ℃.
(3) Enzyme digestion reaction
After the DNA with correct size fragments is obtained through electrophoresis verification, double enzyme digestion is carried out on the vector and the PCR product (enzyme digestion solution is purchased together with restriction enzyme). The cleavage system is shown in Table 12:
table 12
| Component (A) | Volume (mu L) |
| PCR products/vectors | 43 |
| 10 XCutsmart solution | 5 |
| BamHI | 1 |
| XhoI | 1 |
After incubation for 3h at 37 ℃, electrophoresis is carried out and recovered by adopting a gel recovery kit; to increase the efficiency of vector cleavage, 1.5. Mu.L of restriction enzyme was added and the 5-hour cleavage time was prolonged.
(4) Ligation reaction
The gene fragments recovered were ligated according to the following scheme, and the ligation reagent Solution1 (containing T4DNA ligase and its associated buffer) was purchased from NewEngland BioLabs (cat#M0202S), and incubated at a constant temperature of 16℃for 5 hours or more. The connection system is shown in Table 13:
TABLE 13
| Composition of the components | Volume (mu L) |
| Solution 1 | 8 |
| Target fragment | 7 |
| Carrier body | 1 |
(5) Transformation
After the ligation was completed, the ligation product was transformed into E.coli JM109 competent cells (purchased from Promega Co., product No. ST 1105) and cultured as follows:
a. taking out a tube of competent cells, and placing the tube of competent cells on ice to melt for 10min;
b. adding the connection product into competent cells, mixing, and standing on ice for 30min;
c. Heat-beating at 42 deg.C for 90s, and placing on ice for 2min;
d. 200. Mu.L of Luria-Bertani (LB) medium (without antibiotics) was added to the tube and incubated at 37℃for 30min at 220 rpm; taking a solid LB plate with corresponding resistance to room temperature for preheating;
e. the bacterial liquid was taken out, spread on a plate and cultured in an incubator at 37℃for 16 hours.
(6) Sequencing
Selecting some of the monoclonal on the 16h plate, transferring to a clean plate, culturing at 37 ℃ for 5h, and then sequencing by the Wuhan Jin Kairui bioengineering Co., ltd; after the sequencing result is fed back, sequence alignment is performed to confirm correct cloning.
Example 3
Expression and purification of target proteins
(1) Expression of
Firstly, small extraction is carried out on the protein, the optimal expression conditions, the purification strategy and the like are searched, and then a large amount of extraction is carried out. Protein expression was performed in E.coli BL21 (DE 3) cells (available from Kangbody Life technologies Co., ltd., product number KTSM 109) as follows:
a. the monoclonal is selected on a plate cultivated for 16 hours and added into 100mL LB culture medium containing the required resistance antibiotics, and cultivated for 5 hours at 37 ℃ and 220rpm, namely, obvious turbidity is observed;
b. expanding the small bottle strains in the step a into a large bottle LB culture medium (containing antibiotics) according to the volume ratio of 1:100, and culturing for 3 hours at 37 ℃ and 220 rpm; after the OD600 value reaches 0.7h, the temperature is reduced to 20 ℃, 0.2mM isopropyl-beta-D-thiogalactoside (IPTG) is added for induction, and the induction time is 14h;
c. The bacteria were collected for protein purification by centrifugation at 4000rpm at 4℃for 10 min.
(2) Purification
a. The collected escherichia coli is resuspended by using a cell lysis solution, and the volume ratio of the cell lysis solution to LB culture solution for collecting the escherichia coli is 30mL: 1L; stirring on a magnetic stirrer for 20min to homogenize the cells, adding serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF) 1mM, lysozyme for lysing bacterial cell walls 40g/mL, deoxyribonuclease I1 μg/mL for decomposing nucleic acid and cofactor MgCl 10mM 2 The method comprises the steps of carrying out a first treatment on the surface of the Then carrying out ultrasonic crushing;
b. centrifuging at 13000rpm and 4deg.C for 1 hr after ultrasonic treatment, and collecting supernatant for affinity chromatography; protein purification was performed in a constant temperature chamber at 4 ℃;
c. pouring the supernatant after centrifugation into a treated Ni column for full combination; washing with solutions containing imidazole at different concentrations (10 mM, 30mM, 50 mM) to remove the hybrid protein; finally eluting the target protein by using imidazole solution with high concentration (250 mM); then, performing result analysis by SDS-PAGE;
d. diluting the protein after affinity chromatography, loading the diluted protein into an ion exchange column, and performing linear gradient (NaCl from 0 to 500 mM) elution by using a buffer solution containing NaCl (the buffer solution is Tris and the pH value is 7.0) after loading, wherein the step is completed by means of a protein purifier; performing result analysis by SDS-PAGE, combining and concentrating proteins with better properties and purity, and using the proteins for the next molecular sieve chromatography;
e. Centrifuging at 13000rpm for 5min to remove part of precipitated proteins and impurities before loading molecular sieve chromatography, and delivering the sample into a loading ring by using a syringe; then eluting the protein with elution buffer (containing 100mM NaCl, tris, pH 7.0) and collecting 0.5mL per tube; detecting and analyzing the protein by SDS-PAGE, combining the proteins to be stored, and then sub-packaging and freezing; the protein for activity measurement was stored with 30% by volume glycerol.
Test example 1
Catalytic Activity Studies of HPPD inhibitors
And on the premise of ensuring that the concentration of each protein and the concentration of the HPPD inhibitor are the same, testing the catalysis rate and the catalysis degree of each protein on the HPPD inhibitor by adopting a High Performance Liquid Chromatography (HPLC).
The specific testing method comprises the following steps:
a. buffer solution, substrate and cofactor preparation:
live HEPES buffer: preparing a stock solution with the concentration of 1M, adjusting the pH value to 7.0 by using sodium hydroxide, diluting to 20mM before use, and filtering by using a filter membrane with the thickness of 0.22 mu M;
preparation of the substrate (i.e., HPPD inhibitor): preparing a stock solution with the concentration of 10mM by using DMSO, and diluting by using a live assay buffer solution before use;
preparation of sodium ascorbate: deionized water was used to prepare a concentration of 20 mM. Preserving at-80 deg.C;
Preparing ferrous sulfate: deionized water was used to prepare a concentration of 1 mM. Preserving at-80 deg.C;
preparation of alpha-ketoglutaric acid: the concentration was made up to 50mM with deionized water. Preserving at-80 deg.C;
b. in the test, a live assay buffer (final concentration: 20 mM), a substrate (final concentration: 50. Mu.M), sodium ascorbate (final concentration: 2 mM), ferrous sulfate (final concentration: 100. Mu.M) and alpha-ketoglutarate (final concentration: 1 mM) were mixed, and finally an enzyme (final concentration: 0.5 mg/mL) was added thereto in a total volume of 200. Mu.L and reacted at 30℃for 10 hours;
c. adding 20 mu L of 10% trifluoroacetic acid aqueous solution to stop the reaction, centrifuging 12000g for 10min, and adding 180 mu L into a sample bottle to be detected;
d. when the sample was measured by HPLC, the loading was 60. Mu.L, the area of the absorption peak of the remaining substrate at 286nm was measured, and the amount of substrate consumed in the different reactions was calculated from the standard curve of the substrate (i.e., control), thereby evaluating the catalytic activity of each protein. Each sample was repeated three times.
e. Determination of a standard curve for substrate (HPPD inhibitor): using the same reaction system as described above (but without the addition of enzyme), different substrate concentrations, e.g., 0.1. Mu.M, 0.5. Mu.M, 1. Mu.M, 10. Mu.M, 50. Mu.M, were set simultaneously, thereby obtaining the relationship between the substrate concentration and the peak area.
Control: the term "inhibitor" refers to the amount of substrate remaining in the reaction system when the inhibitor is not catalyzed, but the enzyme is not added.
Sample: in the enzyme reaction system, inhibitors of the same volume and the same concentration (50. Mu.M) were added, and the catalytic efficiency of the enzyme on the inhibitors was observed.
Catalytic efficiency= (1- (peak area of substrate in sample)/(peak area of substrate in control)) ×100%, the larger the number, the better the catalytic effect.
Class 2 representative HPPD inhibitor herbicides were tested in this test example:
triones: mesotrione (Mesotrione), cyclotrione (Tembotrione), bicyclosultone stock (BBC-OH), fursultone (terfuryltrione), quinclorac (Y13287), Y18024,
pyrazoles: the results of catalysis of rice HIS1 protein and rice, wheat, barley, sorghum and corn HSL proteins on the HPPD inhibitor are tested respectively by using quinclorac original drug (Y16550) and bract (Topramzone); the specific results are shown in tables 14 to 21.
Table 14: catalytic results of rice HIS1 mutants on different HPPD inhibitors
In Table 14, H141F refers to a mutant in which the 141 th site of rice HIS1 is mutated from histidine to phenylalanine; H141F/F205L refers to a mutant of rice HIS1, wherein the 141 th site is mutated from histidine to phenylalanine and the 205 th site is mutated from phenylalanine to leucine.
Table 15: catalytic results of rice HSL1 mutants on different HPPD inhibitors
In Table 15, L204F/F298L/I335A refers to a mutant in which the 204 th site of rice HSL1 was mutated from leucine to phenylalanine, the 298 th site was mutated from phenylalanine to leucine and the 335 th site was mutated from isoleucine to alanine; L204F/F298L/I335W refers to a mutant of rice HSL1, wherein the 204 th site is mutated from leucine to phenylalanine, the 298 th site is mutated from phenylalanine to leucine and the 335 th site is mutated from isoleucine to tryptophan; F140H/L204F/F298L/I335F refers to a mutant in which phenylalanine at position 140 of rice HSL1 is mutated to histidine, leucine at position 204 is mutated to phenylalanine, phenylalanine at position 298 is mutated to leucine and isoleucine at position 335 is mutated to phenylalanine.
Table 16: catalytic results of rice HSL2 mutants on different HPPD inhibitors
In Table 16, T206F refers to a mutant in which threonine at position 206 of rice HSL2 was mutated to phenylalanine; T206F/F301L refers to a mutant of rice HSL2 in which threonine at position 206 is mutated to phenylalanine and phenylalanine at position 301 is mutated to leucine; T206F/F301L/Y339F refers to a mutant in which threonine at position 206 of rice HSL2 is mutated to phenylalanine, phenylalanine at position 301 is mutated to leucine and tyrosine at position 339 is mutated to phenylalanine.
Table 17: catalytic results of rice HSL4 mutants on different HPPD inhibitors
In Table 17, Y142H refers to a mutant in which tyrosine at position 142 of rice HSL4 was mutated to histidine; Y142H/I207F refers to a mutant of rice HSL4, in which tyrosine at position 142 is mutated to histidine and isoleucine at position 207 is mutated to phenylalanine; Y142H/I207F/F301L refers to a mutant in which tyrosine at position 142 of rice HSL4 is mutated to histidine, isoleucine at position 207 is mutated to phenylalanine and phenylalanine at position 301 is mutated to leucine.
Table 18: catalytic results of wheat HSL2 mutants on different HPPD inhibitors
In table 18, F285L refers to a mutant in which phenylalanine at position 285 of wheat HSL2 is mutated to leucine; F285L/H322F refers to a mutant of wheat HSL2 in which phenylalanine at position 285 is mutated to leucine and histidine at position 322 is mutated to phenylalanine.
Table 19: catalytic results of maize HSL1A mutants on different HPPD inhibitors
In Table 19, Q142H/R338F refers to a mutant in which glutamine at position 142 of maize HSL1A was mutated to histidine and arginine at position 338 was mutated to phenylalanine; Q142H/I207F/F301L/R338F refers to a mutant in which glutamine at position 142 of maize HSL1A is mutated to histidine, isoleucine at position 207 is mutated to phenylalanine, phenylalanine at position 301 is mutated to leucine and arginine at position 338 is mutated to phenylalanine.
Table 20: catalytic results of maize HSL1B mutants on different HPPD inhibitors
| Mesotrione | BBC-OH | Y16550 | |
| Wild type | 8 | 95 | 6 |
| Q140H/Y205F | 18 | 100 | 9 |
| Q140H/Y205F/K336F | 19 | 100 | 2 |
In Table 20, Q140H/Y205F refers to a mutant in which glutamine at position 140 of maize HSL1B was mutated to histidine and tyrosine at position 205 was mutated to phenylalanine; Q140H/Y205F/K336F refers to a mutant in which glutamine at position 140 of maize HSL1B is mutated to histidine, tyrosine at position 205 is mutated to phenylalanine and lysine at position 336 is mutated to phenylalanine.
Table 21: catalytic results of sorghum HSL1 mutants on different HPPD inhibitors
| Mesotrione | BBC-OH | Furan sulcotrione | Y16550 | |
| Wild type | 20 | 21 | 0 | 0 |
| Q140H/Y205F | 15 | 98 | 7 | 7 |
| Q140H/Y205F/K336F | 26 | 100 | 15 | 10 |
In Table 21, Q140H/Y205F refers to a mutant in which glutamine at position 140 of sorghum HSL1 was mutated to histidine and tyrosine at position 205 was mutated to phenylalanine; Q140H/Y205F/K336F refers to the amino acid sequence of sorghum HSL1 with the mutation of glutamine at position 140 to histidine, tyrosine at position 205 to phenylalanine and lysine at position 336 to phenylalanine.
From the results of the above test, the hydroxylation reactivity of the mutant forms of rice HIS1, rice HSL2, rice HSL4, wheat HSL2, corn HSL1A, corn HSL1B and sorghum HSL1 with the HPPD inhibitor was significantly improved relative to the Wild Type (WT).
Specifically, the single-point mutation H141F of the rice HIS1 has the catalytic efficiency of 31% to the bract and 37% to the Y16550, and the catalytic efficiency is respectively improved by 9% and 26% compared with the wild type; the double point mutation H141F/F205L of rice HIS1 has a catalytic efficiency of 27% for bract and 26% for Y16550, which are improved by 5% and 15% respectively compared with the wild type.
Specifically, the three-point mutation L204F/F298L/I335A of rice HSL1 has a catalytic efficiency of 87% for BBC-OH, 72% for Y13287, 21% for Y18024, 69% for Y16550, and an improvement of 38%, 72%, 21% and 52% respectively compared with the wild type;
the three-point mutation L204F/F298L/I335W of the rice HSL1 has the catalytic efficiency of 76% for mesotrione, 100% for BBC-OH, 64% for Y13287, 48% for Y18024 and 42% for Y16550, and the catalytic efficiency is respectively improved by 36%, 51%, 64%, 48% and 35% compared with the wild type;
the four-point mutation F140H/L204F/F298L/I335F of rice HSL1 has a catalytic efficiency of 85% for mesotrione, 100% for BBC-OH, 92% for Y13287 and 96% for Y18024, which are respectively 45%, 51%, 92% and 96% higher than the wild type.
Specifically, the single-point mutation T206F of rice HSL2 has the catalytic efficiency of 20% for mesotrione, 26% for BBC-OH, 8% for furansultone, 9% for Y13287, 16% for Y18024, 11% for Y16550, and 11%, 26%, 8%, 9%, 16% and 6% improvement compared with the wild type;
the double point mutation T206F/F301L of rice HSL2 has the catalytic efficiency of 47 percent for mesotrione, 100 percent for BBC-OH, 25 percent for furansultone, 83 percent for Y13287, 30 percent for Y18024, 17 percent for Y16550, which are respectively improved by 38 percent, 100 percent, 25 percent, 83 percent, 30 percent and 12 percent compared with the wild type;
the three-point mutation T206F/F301L/Y339F of rice HSL2 has the catalytic efficiency of 44% for mesotrione, 100% for BBC-OH, 21% for furantrione, 86% for Y13287, 27% for Y18024, 12% for bract, 16% for Y16550, and the catalytic efficiency is respectively improved by 35%, 100%, 21%, 86%, 27%, 12% and 11% compared with the wild type.
Specifically, the single-point mutation Y142H of the rice HSL4 has the catalytic efficiency of 30% on fursulcotrione, 75% on Y13287 and 8% on bract, and compared with the wild type, the catalytic efficiency of the rice HSL4 is respectively improved by 13%, 2% and 6%;
the catalytic efficiency of the double point mutation Y142H/I207F of the rice HSL4 on fursultone is 27%, the catalytic efficiency on Y13287 is 76%, the catalytic efficiency on bract is 5%, and compared with a wild type, the catalytic efficiency is respectively improved by 10%, 3% and 3%;
the three-point mutation Y142H/I207F/F301L of rice HSL4 has the catalytic efficiency of 22% for mesotrione and 4% for bract, and compared with the wild type, the catalytic efficiency is respectively improved by 1% and 2%.
Specifically, the single-point mutation F285L of wheat HSL2 has a catalytic efficiency of 10% on BBC-OH, a catalytic efficiency of 32% on Y13287 and a catalytic efficiency of 34% on Y18024, and the catalytic efficiency is respectively improved by 4%, 32% and 34% compared with the wild type;
the catalytic efficiency of the double point mutation 285L/H322F of wheat HSL2 on BBC-OH is 17%, the catalytic efficiency on Y13287 is 12%, the catalytic efficiency on Y18024 is 9%, and compared with the wild type, the catalytic efficiency is respectively improved by 11%, 12% and 9%.
Specifically, the catalytic efficiency of the double point mutation Q142H/R338F of the corn HSL1A on mesotrione is 21%, the catalytic efficiency on the furansultone is 77%, and compared with a wild type, the catalytic efficiency of the furansultone is respectively improved by 8% and 28%;
the four-point mutation Q142H/I207F/F301L/R338F of the corn HSL1A has the catalytic efficiency of 15 percent of mesotrione, and compared with a wild type, the catalytic efficiency of the mesotrione is improved by 2 percent.
Specifically, the double point mutation Q140H/Y205F of the corn HSL1B has the catalytic efficiency of 18% for mesotrione, 100% for BBC-OH and 9% for Y16550, and the catalytic efficiency is respectively improved by 10%, 5% and 3% compared with the wild type;
the catalytic efficiency of the corn HSL1B three-point mutation Q140H/Y205F/K336F on mesotrione is 19%, the catalytic efficiency on BBC-OH is 100%, and compared with the wild type, the catalytic efficiency is respectively improved by 11% and 5%.
Specifically, the catalytic efficiency of the double point mutation Q140H/Y205F of the sorghum HSL1 on BBC-OH is 98%, the catalytic efficiency on fursultone is 7%, and the catalytic efficiency on Y16550 is 7%, compared with the wild type, the catalytic efficiency is respectively improved by 77%, 7% and 7%;
the three-point mutation Q140H/Y205F/K336F of sorghum HSL1 has the catalytic efficiency of 26% for mesotrione, 100% for BBC-OH, 15% for furansultone and 10% for Y16550, which are respectively improved by 6%, 79%, 15% and 10% compared with the wild type.
The hydroxylation activity of the rice HIS1, the rice HSL2, the rice HSL4, the wheat HSL2, the sorghum HSL1, the corn HSL1A and the corn HSL1 mutant on the HPPD inhibitor is enhanced, namely, the Fe (II)/alpha-ketoglutarate dependent dioxygenase protein provided by the invention can detoxify the HPPD inhibitor. Therefore, the Fe (II)/alpha-ketoglutarate dependent dioxygenase provided by the invention can be used for improving herbicide resistance of crops.
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, a number of simple variants of the technical solution of the invention are possible, including combinations of the individual technical features in any other suitable way, which simple variants and combinations should likewise be regarded as being disclosed by the invention, all falling within the scope of protection of the invention.
Claims (10)
1. An Fe (ii)/α -ketoglutarate-dependent dioxygenase protein characterized by having an amino acid sequence selected from at least one of the following:
(1) A first amino acid sequence derived from at least one of the 141 th site and the 205 th site in the amino acid sequence shown in SEQ ID NO. 1;
(2) The first amino acid sequence is a protein derived from the first amino acid sequence by substituting, deleting or adding one or more amino acids, or a protein shown by an amino acid sequence with a label connected to the amino terminal end and/or the carboxyl terminal end of the first amino acid sequence;
(3) A second amino acid sequence derived from mutation at least 3 positions selected from the group consisting of position 140, position 204, position 298 and position 335 in the amino acid sequence shown in SEQ ID NO. 2;
(4) The second amino acid sequence is a protein derived from the second amino acid sequence by substituting, deleting or adding one or more amino acids, or a protein shown by an amino acid sequence with a label connected to the amino terminal end and/or the carboxyl terminal end of the second amino acid sequence;
(5) A third amino acid sequence derived from mutation at least one site selected from the group consisting of site 206, site 301 and site 339 in the amino acid sequence shown in SEQ ID NO. 3;
(6) The third amino acid sequence is a protein derived from the third amino acid sequence by substituting, deleting or adding one or more amino acids, or a protein shown by an amino acid sequence with a label connected to the amino terminal end and/or the carboxyl terminal end of the third amino acid sequence;
(7) A fourth amino acid sequence derived from mutation at least one site selected from the group consisting of site 142, site 207 and site 301 in the amino acid sequence shown in SEQ ID NO. 4;
(8) The protein derived from the fourth amino acid sequence by substituting, deleting or adding one or more amino acids, or the protein shown by the amino acid sequence with a label connected with the amino terminal and/or the carboxyl terminal of the fourth amino acid sequence;
(9) A fifth amino acid sequence derived from at least one position selected from the 285 position and the 322 position in the amino acid sequence shown in SEQ ID NO. 5;
(10) The protein derived from the fifth amino acid sequence by substituting, deleting or adding one or more amino acids, or the protein shown by the amino acid sequence with a label connected to the amino terminal and/or the carboxyl terminal of the fifth amino acid sequence;
(11) A sixth amino acid sequence derived from mutation at least one site selected from the group consisting of site 142, site 207, site 301 and site 338 in the amino acid sequence shown in SEQ ID NO. 6;
(12) The protein derived from the sixth amino acid sequence by substituting, deleting or adding one or more amino acids, or the protein shown by the amino acid sequence with a label connected to the amino terminal and/or carboxyl terminal of the sixth amino acid sequence;
(13) A seventh amino acid sequence derived from mutation at least 2 positions selected from the group consisting of 140 positions, 205 positions and 336 positions in the amino acid sequence shown in SEQ ID NO. 7;
(14) A protein derived from the seventh amino acid sequence by substituting, deleting or adding one or more amino acids, or a protein shown by an amino acid sequence with a tag connected to the amino terminal and/or carboxyl terminal of the seventh amino acid sequence;
(15) An eighth amino acid sequence derived after mutation at least 2 positions selected from the group consisting of 140 positions, 205 positions and 336 positions in the amino acid sequence shown in SEQ ID NO. 8;
(16) The eighth amino acid sequence is a protein derived from the eighth amino acid sequence by substituting, deleting or adding one or more amino acids, or a protein shown by an amino acid sequence with a tag connected to the amino terminal and/or carboxyl terminal of the eighth amino acid sequence.
2. The Fe (ii)/a-ketoglutarate dependent dioxygenase protein according to claim 1, wherein the first amino acid sequence is an amino acid sequence selected from at least one of the following:
(1a) An amino acid sequence derived from the histidine at 141 site in the amino acid sequence shown in SEQ ID NO. 1 is mutated into phenylalanine;
(1b) An amino acid sequence which is derived after histidine at 141 site is mutated into phenylalanine and phenylalanine at 205 site is mutated into leucine in the amino acid sequence shown in SEQ ID NO. 1;
preferably, the first amino acid sequence is an amino acid sequence shown as H141F, H F/F205L;
preferably, the second amino acid sequence is the amino acid sequence:
(2a) Amino acid sequence derived from the amino acid sequence shown in SEQ ID NO. 2, wherein phenylalanine at 140 site is mutated to histidine, leucine at 204 site is mutated to phenylalanine, phenylalanine at 298 site is mutated to leucine and isoleucine at 335 site is mutated to phenylalanine;
(2b) Amino acid sequence shown in SEQ ID NO. 2, wherein leucine at position 204 is mutated into phenylalanine, phenylalanine at position 298 is mutated into leucine and isoleucine at position 335 is mutated into alanine;
(2c) Amino acid sequence shown in SEQ ID NO. 2, wherein leucine at 204 site is mutated to phenylalanine, phenylalanine at 298 site is mutated to leucine, and isoleucine at 335 site is mutated to tryptophan;
preferably, the second amino acid sequence is at least one of the amino acid sequences shown in F140H/L204F/F298L/I335F, L F/F298L/I335A, L F/F298L/I335W; preferably, the third amino acid sequence is the amino acid sequence:
(3a) Amino acid sequence derived from threonine at 206 in the amino acid sequence shown in SEQ ID NO. 3;
(3b) An amino acid sequence derived from the amino acid sequence shown in SEQ ID NO. 3, wherein threonine at position 206 is mutated to phenylalanine and phenylalanine at position 301 is mutated to leucine;
(3c) Amino acid sequence shown in SEQ ID NO. 3, wherein threonine at 206 is mutated to phenylalanine, phenylalanine at 301 is mutated to leucine, and tyrosine at 339 is mutated to phenylalanine;
preferably, the third amino acid sequence is at least one of the amino acid sequences shown as T206F, T F/F301L, T F/F301L/Y339F;
preferably, the fourth amino acid sequence is the amino acid sequence:
(4a) The tyrosine at 142 site in the amino acid sequence shown in SEQ ID NO. 4 is mutated into amino acid sequence derived after histidine;
(4b) An amino acid sequence derived from the amino acid sequence shown in SEQ ID NO. 4, wherein tyrosine at 142 is mutated to histidine and isoleucine at 207 is mutated to phenylalanine;
(4c) Amino acid sequence shown in SEQ ID NO. 4, wherein tyrosine at 142 site is mutated to histidine, isoleucine at 207 site is mutated to phenylalanine, and phenylalanine at 301 site is mutated to leucine;
Preferably, the fourth amino acid sequence is at least one of the amino acid sequences shown in Y142H, Y H/I207F, Y H/I207F/F301L;
preferably, the fifth amino acid sequence is the amino acid sequence:
(5a) An amino acid sequence derived from the phenylalanine at 285 site in the amino acid sequence shown in SEQ ID NO. 5 after mutation into leucine;
(5b) An amino acid sequence derived from the amino acid sequence shown in SEQ ID NO. 5, wherein phenylalanine at 285 site is mutated to leucine and histidine at 322 site is mutated to phenylalanine;
preferably, the fifth amino acid sequence is at least one of the amino acid sequences shown in F285L, F L/H322F;
preferably, the sixth amino acid sequence is the amino acid sequence:
(6a) The glutamine at 142 site in the amino acid sequence shown in SEQ ID NO. 6 is mutated into histidine to obtain an amino acid sequence;
(6b) An amino acid sequence derived from the amino acid sequence shown in SEQ ID NO. 6, wherein glutamine at position 142 is mutated to histidine and isoleucine at position 207 is mutated to phenylalanine;
(6c) An amino acid sequence shown in SEQ ID NO. 6, wherein the amino acid sequence is derived after glutamine at 142 site is mutated to histidine, isoleucine at 207 site is mutated to phenylalanine and phenylalanine at 301 site is mutated to leucine;
(6d) Amino acid sequence derived from the amino acid sequence shown in SEQ ID NO. 6, wherein glutamine at 142 site is mutated to histidine, isoleucine at 207 site is mutated to phenylalanine, phenylalanine at 301 site is mutated to leucine and arginine at 338 site is mutated to phenylalanine;
(6e) An amino acid sequence derived from the amino acid sequence shown in SEQ ID NO. 6, wherein glutamine at 142 site is mutated to histidine and arginine at 338 site is mutated to phenylalanine;
preferably, the sixth amino acid sequence is at least one of the amino acid sequences shown in Q142H, Q H/I207F, Q H/I207F/F301L, Q H/I207F/F301L/R338F, Q H/R338F;
preferably, the seventh amino acid sequence is the amino acid sequence:
(7a) An amino acid sequence derived from the amino acid sequence shown in SEQ ID NO. 7, wherein the glutamine at the 140 site is mutated to histidine and the tyrosine at the 205 site is mutated to phenylalanine;
(7b) An amino acid sequence which is derived from the amino acid sequence shown in SEQ ID NO. 7, wherein the glutamine at the 140 site is mutated to histidine, the tyrosine at the 205 site is mutated to phenylalanine and the lysine at the 336 site is mutated to phenylalanine;
Preferably, the seventh amino acid sequence is at least one of the amino acid sequences shown in Q140H/Y205F, Q H/Y205F/K336F;
preferably, the eighth amino acid sequence is the amino acid sequence:
(8a) An amino acid sequence derived from the amino acid sequence shown in SEQ ID NO. 8, wherein the glutamine at the 140 site is mutated to histidine and the tyrosine at the 205 site is mutated to phenylalanine;
(8b) An amino acid sequence which is derived from the amino acid sequence shown in SEQ ID NO. 8, wherein the glutamine at the 140 site is mutated to histidine, the tyrosine at the 205 site is mutated to phenylalanine and the lysine at the 336 site is mutated to phenylalanine;
preferably, the eighth amino acid sequence is at least one of the amino acid sequences shown in Q140H/Y205F, Q H/Y205F/K336F.
3. A gene encoding a Fe (ii)/α -ketoglutarate-dependent dioxygenase protein, characterized in that the nucleotide sequence of the gene is a nucleotide sequence capable of encoding the amino acid sequence of the Fe (ii)/α -ketoglutarate-dependent dioxygenase protein of claim 1 or 2;
preferably, the nucleotide sequence of the gene is a nucleotide sequence selected from at least one of the following:
(1) A nucleotide sequence as shown in SEQ ID NO. 11;
(2) A nucleotide sequence having at least 90% identity, preferably at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to SEQ ID No. 11;
(3) A nucleotide sequence as shown in SEQ ID NO. 21;
(4) A nucleotide sequence having at least 90% identity, preferably at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to SEQ ID No. 21;
(5) A nucleotide sequence as shown in SEQ ID NO. 31;
(6) A nucleotide sequence having at least 90% identity, preferably at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to SEQ ID No. 31;
(7) A nucleotide sequence as shown in SEQ ID NO. 41;
(8) A nucleotide sequence having at least 90% identity, preferably at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to SEQ ID No. 41;
(9) A nucleotide sequence shown as SEQ ID NO. 51;
(10) A nucleotide sequence having at least 90% identity, preferably at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to SEQ ID No. 51;
(11) A nucleotide sequence as shown in SEQ ID NO. 61;
(12) A nucleotide sequence having at least 90% identity, preferably at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to SEQ ID No. 61;
(13) A nucleotide sequence shown as SEQ ID NO. 71;
(14) A nucleotide sequence having at least 90% identity, preferably at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to SEQ ID No. 71;
(15) A nucleotide sequence as shown in SEQ ID NO. 81;
(16) A nucleotide sequence having at least 90% identity, preferably at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to SEQ ID No. 81.
4. A recombinant vector comprising the gene according to claim 3.
5. A transgenic cell comprising the gene of claim 3;
preferably, the transgenic cell is a prokaryotic cell.
6. A composition comprising the Fe (ii)/α -ketoglutarate-dependent dioxygenase protein according to claim 1 or 2.
7. Use of at least one of the Fe (ii)/α -ketoglutarate-dependent dioxygenase protein of claim 1 or 2, the gene of claim 3, the recombinant vector of claim 4, the transgenic cell of claim 5 for increasing herbicide resistance in crops;
preferably, the herbicide is an HPPD inhibitor;
preferably, the herbicide is at least one of a trione compound, a pyrazole compound, an isoxazole compound, a diketopolynitrile compound and a heterocyclic amide compound;
More preferably, the herbicide is at least one of mesotrione, cyclosulfamuron, fursultone, bicyclosultone, quinclorac, mequindox, Y18024, Y16550 and topramezone.
8. A method of increasing herbicide resistance in a crop, the method comprising: transferring the recombinant vector of claim 4 into a target plant, so that the target plant expresses the Fe (ii)/α -ketoglutarate-dependent dioxygenase protein of claim 1 or 2 to obtain resistance to herbicides.
9. A method of increasing herbicide resistance in a crop, the method comprising: transferring the recombinant vector in the mutant crop containing the recombinant vector of claim 4 into a target plant by crossing, transferring or backcrossing, so that the target plant expresses the Fe (ii)/α -ketoglutarate-dependent dioxygenase protein of claim 1 or 2 to obtain resistance to herbicides.
10. A method of increasing herbicide resistance in a crop, the method comprising: the HIS1/HSL gene of the target plant is engineered by CRISPR/Cas gene editing method such that the target plant expresses the Fe (ii)/α -ketoglutarate dependent dioxygenase protein of claim 1 or 2 to obtain resistance to herbicides.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210488261 | 2022-05-06 | ||
| CN2022104882617 | 2022-05-06 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117004583A true CN117004583A (en) | 2023-11-07 |
Family
ID=88575138
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310502130.4A Pending CN117004583A (en) | 2022-05-06 | 2023-05-06 | 2ODD protein, gene, vector, cell, composition and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117004583A (en) |
-
2023
- 2023-05-06 CN CN202310502130.4A patent/CN117004583A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP3523657B2 (en) | Imidazolinone resistant AHAS mutant | |
| US8049063B2 (en) | Rice bentazon and sulfonylurea herbicide resistant gene Cyp81a6 | |
| CA2492711A1 (en) | Herbicide-resistant plants, and polynucleotides and methods for providing same | |
| JP2011101653A (en) | Method and composition for expression of transgene in plant | |
| CN111574605B (en) | Application of rice gene OsLAT5 in regulation of absorption and accumulation of diquat | |
| EP0478502A2 (en) | Histidinol dehydrogenase protein, DNA and muteins and transgenic plants thereof | |
| US6639130B2 (en) | Plant sterol reductases and uses thereof | |
| CN110093325B (en) | Tobacco mitochondria protoporphyrinogen oxidase mutant and coding gene and application thereof | |
| WO1997048793A9 (en) | Plant sterol reductases and uses thereof | |
| JP3283850B2 (en) | Flower regulation gene and flower regulation method | |
| CN111560055B (en) | Application of rice gene OsLAT3 in regulation of absorption and accumulation of diquat | |
| US7241878B1 (en) | Modified cellulose synthase gene from Arabidopsis thaliana confers herbicide resistance to plants | |
| WO2021047377A1 (en) | Application of tpst gene in regulation of traits of plant | |
| CN117004583A (en) | 2ODD protein, gene, vector, cell, composition and application thereof | |
| CN114774377B (en) | HPPD proteins, genes, vectors, cells, compositions, uses thereof and methods for increasing herbicide resistance in crops | |
| CN117586973A (en) | HSL protein, gene, vector, cell, composition, application and method for improving herbicide resistance of crops | |
| CN114616333A (en) | Abiotic stress tolerant plants and methods | |
| NL2030468B1 (en) | Use of soybean protein kinase gene gmstk_irak | |
| CN113122566B (en) | Role of HAK gene in regulating and controlling plant traits | |
| WO2012028673A1 (en) | Spontaneous organogenesis in plants | |
| CN111373035B (en) | Rape triazole-resistant pyrimidine sulfonamide herbicide gene and application thereof | |
| WO2023115030A2 (en) | Lodging resistance in eragrostis tef | |
| CN114174518A (en) | Abiotic stress tolerant plants and methods | |
| JPWO2005026345A1 (en) | Plant dwarfing gene | |
| CN115160425A (en) | Rice mutant protein with high dinitroaniline herbicide resistance and corresponding gene |
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 |