CN116769739A - G294 mutation-containing plant glutamine synthetase mutant and encoding gene and application thereof - Google Patents
G294 mutation-containing plant glutamine synthetase mutant and encoding gene and application thereof Download PDFInfo
- Publication number
- CN116769739A CN116769739A CN202310883130.3A CN202310883130A CN116769739A CN 116769739 A CN116769739 A CN 116769739A CN 202310883130 A CN202310883130 A CN 202310883130A CN 116769739 A CN116769739 A CN 116769739A
- Authority
- CN
- China
- Prior art keywords
- plant
- glutamine synthetase
- amino acid
- mutant
- pasture
- 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
- 108020002326 glutamine synthetase Proteins 0.000 title claims abstract description 128
- 102000005396 glutamine synthetase Human genes 0.000 title claims abstract description 125
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 64
- 230000035772 mutation Effects 0.000 title abstract description 24
- IAJOBQBIJHVGMQ-UHFFFAOYSA-N 2-amino-4-[hydroxy(methyl)phosphoryl]butanoic acid Chemical compound CP(O)(=O)CCC(N)C(O)=O IAJOBQBIJHVGMQ-UHFFFAOYSA-N 0.000 claims abstract description 86
- 239000005561 Glufosinate Substances 0.000 claims abstract description 86
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 44
- 238000000034 method Methods 0.000 claims abstract description 36
- 241000196324 Embryophyta Species 0.000 claims description 141
- 240000007594 Oryza sativa Species 0.000 claims description 44
- 235000007164 Oryza sativa Nutrition 0.000 claims description 43
- 240000008042 Zea mays Species 0.000 claims description 43
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 42
- 235000009566 rice Nutrition 0.000 claims description 42
- 235000014698 Brassica juncea var multisecta Nutrition 0.000 claims description 40
- 235000006008 Brassica napus var napus Nutrition 0.000 claims description 40
- 240000000385 Brassica napus var. napus Species 0.000 claims description 40
- 235000006618 Brassica rapa subsp oleifera Nutrition 0.000 claims description 40
- 235000004977 Brassica sinapistrum Nutrition 0.000 claims description 40
- 241000209140 Triticum Species 0.000 claims description 40
- 235000021307 Triticum Nutrition 0.000 claims description 40
- 150000001413 amino acids Chemical group 0.000 claims description 36
- 229910052757 nitrogen Inorganic materials 0.000 claims description 24
- 150000007523 nucleic acids Chemical class 0.000 claims description 23
- 241000219198 Brassica Species 0.000 claims description 21
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 21
- 235000016383 Zea mays subsp huehuetenangensis Nutrition 0.000 claims description 21
- 235000005822 corn Nutrition 0.000 claims description 21
- 235000009973 maize Nutrition 0.000 claims description 21
- 229910052700 potassium Inorganic materials 0.000 claims description 21
- 125000000539 amino acid group Chemical group 0.000 claims description 20
- 244000062793 Sorghum vulgare Species 0.000 claims description 19
- 229910052698 phosphorus Inorganic materials 0.000 claims description 18
- 235000013311 vegetables Nutrition 0.000 claims description 18
- 239000013598 vector Substances 0.000 claims description 16
- 229910052739 hydrogen Inorganic materials 0.000 claims description 15
- 108020004707 nucleic acids Proteins 0.000 claims description 15
- 102000039446 nucleic acids Human genes 0.000 claims description 15
- 240000007124 Brassica oleracea Species 0.000 claims description 13
- 235000005254 Allium ampeloprasum Nutrition 0.000 claims description 12
- 240000006108 Allium ampeloprasum Species 0.000 claims description 12
- 235000011331 Brassica Nutrition 0.000 claims description 12
- 241000219112 Cucumis Species 0.000 claims description 12
- 235000015510 Cucumis melo subsp melo Nutrition 0.000 claims description 12
- 241000219104 Cucurbitaceae Species 0.000 claims description 12
- FJJCIZWZNKZHII-UHFFFAOYSA-N [4,6-bis(cyanoamino)-1,3,5-triazin-2-yl]cyanamide Chemical compound N#CNC1=NC(NC#N)=NC(NC#N)=N1 FJJCIZWZNKZHII-UHFFFAOYSA-N 0.000 claims description 12
- 235000019713 millet Nutrition 0.000 claims description 12
- 229910052720 vanadium Inorganic materials 0.000 claims description 11
- 229910052731 fluorine Inorganic materials 0.000 claims description 10
- 235000003351 Brassica cretica Nutrition 0.000 claims description 9
- 235000003343 Brassica rupestris Nutrition 0.000 claims description 9
- QKSKPIVNLNLAAV-UHFFFAOYSA-N bis(2-chloroethyl) sulfide Chemical compound ClCCSCCCl QKSKPIVNLNLAAV-UHFFFAOYSA-N 0.000 claims description 9
- 235000010460 mustard Nutrition 0.000 claims description 9
- 235000010469 Glycine max Nutrition 0.000 claims description 8
- 244000068988 Glycine max Species 0.000 claims description 8
- 235000010582 Pisum sativum Nutrition 0.000 claims description 8
- 240000004713 Pisum sativum Species 0.000 claims description 8
- 241000894006 Bacteria Species 0.000 claims description 7
- 240000002791 Brassica napus Species 0.000 claims description 7
- 235000011293 Brassica napus Nutrition 0.000 claims description 7
- 235000003899 Brassica oleracea var acephala Nutrition 0.000 claims description 7
- 235000011301 Brassica oleracea var capitata Nutrition 0.000 claims description 7
- 235000001169 Brassica oleracea var oleracea Nutrition 0.000 claims description 7
- 108091033409 CRISPR Proteins 0.000 claims description 7
- 229920000742 Cotton Polymers 0.000 claims description 7
- 244000299507 Gossypium hirsutum Species 0.000 claims description 7
- 235000002637 Nicotiana tabacum Nutrition 0.000 claims description 7
- 244000061176 Nicotiana tabacum Species 0.000 claims description 7
- 244000046052 Phaseolus vulgaris Species 0.000 claims description 7
- 235000011684 Sorghum saccharatum Nutrition 0.000 claims description 7
- 238000005516 engineering process Methods 0.000 claims description 7
- 238000010362 genome editing Methods 0.000 claims description 7
- 235000009328 Amaranthus caudatus Nutrition 0.000 claims description 6
- 240000001592 Amaranthus caudatus Species 0.000 claims description 6
- 240000007087 Apium graveolens Species 0.000 claims description 6
- 235000015849 Apium graveolens Dulce Group Nutrition 0.000 claims description 6
- 235000010591 Appio Nutrition 0.000 claims description 6
- 235000017060 Arachis glabrata Nutrition 0.000 claims description 6
- 244000105624 Arachis hypogaea Species 0.000 claims description 6
- 235000010777 Arachis hypogaea Nutrition 0.000 claims description 6
- 235000018262 Arachis monticola Nutrition 0.000 claims description 6
- 244000075850 Avena orientalis Species 0.000 claims description 6
- 235000007319 Avena orientalis Nutrition 0.000 claims description 6
- 235000007558 Avena sp Nutrition 0.000 claims description 6
- 235000000832 Ayote Nutrition 0.000 claims description 6
- 235000011274 Benincasa cerifera Nutrition 0.000 claims description 6
- 244000036905 Benincasa cerifera Species 0.000 claims description 6
- 235000016068 Berberis vulgaris Nutrition 0.000 claims description 6
- 241000335053 Beta vulgaris Species 0.000 claims description 6
- 235000011299 Brassica oleracea var botrytis Nutrition 0.000 claims description 6
- 235000017647 Brassica oleracea var italica Nutrition 0.000 claims description 6
- 240000003259 Brassica oleracea var. botrytis Species 0.000 claims description 6
- 235000000540 Brassica rapa subsp rapa Nutrition 0.000 claims description 6
- 235000002566 Capsicum Nutrition 0.000 claims description 6
- 240000008574 Capsicum frutescens Species 0.000 claims description 6
- 244000067456 Chrysanthemum coronarium Species 0.000 claims description 6
- 241000219109 Citrullus Species 0.000 claims description 6
- 235000012828 Citrullus lanatus var citroides Nutrition 0.000 claims description 6
- 240000008067 Cucumis sativus Species 0.000 claims description 6
- 235000010799 Cucumis sativus var sativus Nutrition 0.000 claims description 6
- 235000009854 Cucurbita moschata Nutrition 0.000 claims description 6
- 240000001980 Cucurbita pepo Species 0.000 claims description 6
- 235000009804 Cucurbita pepo subsp pepo Nutrition 0.000 claims description 6
- 235000002767 Daucus carota Nutrition 0.000 claims description 6
- 244000000626 Daucus carota Species 0.000 claims description 6
- 235000009419 Fagopyrum esculentum Nutrition 0.000 claims description 6
- 240000008620 Fagopyrum esculentum Species 0.000 claims description 6
- 235000016623 Fragaria vesca Nutrition 0.000 claims description 6
- 240000009088 Fragaria x ananassa Species 0.000 claims description 6
- 235000011363 Fragaria x ananassa Nutrition 0.000 claims description 6
- 244000020551 Helianthus annuus Species 0.000 claims description 6
- 235000003222 Helianthus annuus Nutrition 0.000 claims description 6
- 235000007340 Hordeum vulgare Nutrition 0.000 claims description 6
- 240000005979 Hordeum vulgare Species 0.000 claims description 6
- 244000017020 Ipomoea batatas Species 0.000 claims description 6
- 235000002678 Ipomoea batatas Nutrition 0.000 claims description 6
- 235000003228 Lactuca sativa Nutrition 0.000 claims description 6
- 240000008415 Lactuca sativa Species 0.000 claims description 6
- 240000004322 Lens culinaris Species 0.000 claims description 6
- 235000014647 Lens culinaris subsp culinaris Nutrition 0.000 claims description 6
- 235000003956 Luffa Nutrition 0.000 claims description 6
- 244000050983 Luffa operculata Species 0.000 claims description 6
- 235000007688 Lycopersicon esculentum Nutrition 0.000 claims description 6
- 240000003183 Manihot esculenta Species 0.000 claims description 6
- 235000016735 Manihot esculenta subsp esculenta Nutrition 0.000 claims description 6
- 244000088415 Raphanus sativus Species 0.000 claims description 6
- 235000006140 Raphanus sativus var sativus Nutrition 0.000 claims description 6
- 240000000111 Saccharum officinarum Species 0.000 claims description 6
- 235000007201 Saccharum officinarum Nutrition 0.000 claims description 6
- 235000003434 Sesamum indicum Nutrition 0.000 claims description 6
- 244000040738 Sesamum orientale Species 0.000 claims description 6
- 240000003768 Solanum lycopersicum Species 0.000 claims description 6
- 235000002597 Solanum melongena Nutrition 0.000 claims description 6
- 244000061458 Solanum melongena Species 0.000 claims description 6
- 235000009337 Spinacia oleracea Nutrition 0.000 claims description 6
- 244000300264 Spinacia oleracea Species 0.000 claims description 6
- 238000010459 TALEN Methods 0.000 claims description 6
- 244000269722 Thea sinensis Species 0.000 claims description 6
- 235000010749 Vicia faba Nutrition 0.000 claims description 6
- 240000006677 Vicia faba Species 0.000 claims description 6
- 235000002098 Vicia faba var. major Nutrition 0.000 claims description 6
- 241000219977 Vigna Species 0.000 claims description 6
- 240000004922 Vigna radiata Species 0.000 claims description 6
- 235000010721 Vigna radiata var radiata Nutrition 0.000 claims description 6
- 235000011469 Vigna radiata var sublobata Nutrition 0.000 claims description 6
- 235000010726 Vigna sinensis Nutrition 0.000 claims description 6
- 235000009754 Vitis X bourquina Nutrition 0.000 claims description 6
- 235000012333 Vitis X labruscana Nutrition 0.000 claims description 6
- 240000006365 Vitis vinifera Species 0.000 claims description 6
- 235000014787 Vitis vinifera Nutrition 0.000 claims description 6
- 235000012735 amaranth Nutrition 0.000 claims description 6
- 239000004178 amaranth Substances 0.000 claims description 6
- 239000001390 capsicum minimum Substances 0.000 claims description 6
- 244000013123 dwarf bean Species 0.000 claims description 6
- 235000021331 green beans Nutrition 0.000 claims description 6
- 235000021332 kidney beans Nutrition 0.000 claims description 6
- 235000020232 peanut Nutrition 0.000 claims description 6
- 235000015136 pumpkin Nutrition 0.000 claims description 6
- 229910052717 sulfur Inorganic materials 0.000 claims description 6
- 241000234282 Allium Species 0.000 claims description 5
- 235000002732 Allium cepa var. cepa Nutrition 0.000 claims description 5
- 235000007871 Chrysanthemum coronarium Nutrition 0.000 claims description 5
- 235000009811 Momordica charantia Nutrition 0.000 claims description 5
- 108010043645 Transcription Activator-Like Effector Nucleases Proteins 0.000 claims description 5
- 229910052799 carbon Inorganic materials 0.000 claims description 5
- 238000010354 CRISPR gene editing Methods 0.000 claims description 3
- 235000007516 Chrysanthemum Nutrition 0.000 claims description 3
- 244000189548 Chrysanthemum x morifolium Species 0.000 claims description 3
- 241000756137 Hemerocallis Species 0.000 claims description 3
- 238000009395 breeding Methods 0.000 claims description 3
- 230000001488 breeding effect Effects 0.000 claims description 3
- 238000012216 screening Methods 0.000 claims description 3
- 230000002363 herbicidal effect Effects 0.000 claims description 2
- 239000004009 herbicide Substances 0.000 claims description 2
- 230000001131 transforming effect Effects 0.000 claims description 2
- 235000009812 Momordica cochinchinensis Nutrition 0.000 claims 3
- 240000001740 Momordica dioica Species 0.000 claims 3
- 235000018365 Momordica dioica Nutrition 0.000 claims 3
- 235000008553 Allium fistulosum Nutrition 0.000 claims 1
- 244000257727 Allium fistulosum Species 0.000 claims 1
- 235000015134 garland chrysanthemum Nutrition 0.000 claims 1
- 102000004190 Enzymes Human genes 0.000 abstract description 22
- 108090000790 Enzymes Proteins 0.000 abstract description 22
- 230000003197 catalytic effect Effects 0.000 abstract description 6
- 238000010353 genetic engineering Methods 0.000 abstract description 3
- 102100039391 Pseudouridine-5'-phosphatase Human genes 0.000 description 79
- 235000001014 amino acid Nutrition 0.000 description 31
- 229940024606 amino acid Drugs 0.000 description 29
- 125000003275 alpha amino acid group Chemical group 0.000 description 27
- 238000002703 mutagenesis Methods 0.000 description 26
- 231100000350 mutagenesis Toxicity 0.000 description 26
- 235000018102 proteins Nutrition 0.000 description 22
- 210000004027 cell Anatomy 0.000 description 15
- 230000012010 growth Effects 0.000 description 13
- 241000588724 Escherichia coli Species 0.000 description 12
- 239000002773 nucleotide Substances 0.000 description 12
- 125000003729 nucleotide group Chemical group 0.000 description 12
- 229910052760 oxygen Inorganic materials 0.000 description 12
- 108091028043 Nucleic acid sequence Proteins 0.000 description 11
- 239000002609 medium Substances 0.000 description 11
- 108020004705 Codon Proteins 0.000 description 10
- 238000006243 chemical reaction Methods 0.000 description 9
- 230000000694 effects Effects 0.000 description 9
- 239000000243 solution Substances 0.000 description 9
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 8
- 108091026890 Coding region Proteins 0.000 description 8
- 238000012217 deletion Methods 0.000 description 8
- 230000037430 deletion Effects 0.000 description 8
- 239000013604 expression vector Substances 0.000 description 7
- 238000002864 sequence alignment Methods 0.000 description 7
- 230000002950 deficient Effects 0.000 description 6
- 238000003786 synthesis reaction Methods 0.000 description 6
- 230000009466 transformation Effects 0.000 description 6
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 5
- 239000013642 negative control Substances 0.000 description 5
- 108091033319 polynucleotide Proteins 0.000 description 5
- 102000040430 polynucleotide Human genes 0.000 description 5
- 239000002157 polynucleotide Substances 0.000 description 5
- 230000009261 transgenic effect Effects 0.000 description 5
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 4
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 4
- 101150103518 bar gene Proteins 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 230000004060 metabolic process Effects 0.000 description 4
- 101150113864 pat gene Proteins 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 238000006467 substitution reaction Methods 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 108010013043 Acetylesterase Proteins 0.000 description 3
- 108020004414 DNA Proteins 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 238000003776 cleavage reaction Methods 0.000 description 3
- 238000007796 conventional method Methods 0.000 description 3
- 210000003527 eukaryotic cell Anatomy 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 229930195712 glutamate Natural products 0.000 description 3
- 229940049906 glutamate Drugs 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 239000013612 plasmid Substances 0.000 description 3
- 230000007017 scission Effects 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 241000894007 species Species 0.000 description 3
- 238000010998 test method Methods 0.000 description 3
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- 241000589158 Agrobacterium Species 0.000 description 2
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 2
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 2
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 2
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 102000008300 Mutant Proteins Human genes 0.000 description 2
- 108010021466 Mutant Proteins Proteins 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- 235000008322 Trichosanthes cucumerina Nutrition 0.000 description 2
- 244000078912 Trichosanthes cucumerina Species 0.000 description 2
- 244000042324 Trifolium repens Species 0.000 description 2
- 235000013540 Trifolium repens var repens Nutrition 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 239000002168 alkylating agent Substances 0.000 description 2
- 229940100198 alkylating agent Drugs 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 210000003763 chloroplast Anatomy 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 2
- 239000005090 green fluorescent protein Substances 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 238000003780 insertion Methods 0.000 description 2
- 230000037431 insertion Effects 0.000 description 2
- KWGKDLIKAYFUFQ-UHFFFAOYSA-M lithium chloride Chemical compound [Li+].[Cl-] KWGKDLIKAYFUFQ-UHFFFAOYSA-M 0.000 description 2
- 210000004962 mammalian cell Anatomy 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 238000010369 molecular cloning Methods 0.000 description 2
- 231100001160 nonlethal Toxicity 0.000 description 2
- 238000002515 oligonucleotide synthesis Methods 0.000 description 2
- 230000037039 plant physiology Effects 0.000 description 2
- 238000003752 polymerase chain reaction Methods 0.000 description 2
- 238000010561 standard procedure Methods 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 238000011426 transformation method Methods 0.000 description 2
- TXUICONDJPYNPY-UHFFFAOYSA-N (1,10,13-trimethyl-3-oxo-4,5,6,7,8,9,11,12,14,15,16,17-dodecahydrocyclopenta[a]phenanthren-17-yl) heptanoate Chemical compound C1CC2CC(=O)C=C(C)C2(C)C2C1C1CCC(OC(=O)CCCCCC)C1(C)CC2 TXUICONDJPYNPY-UHFFFAOYSA-N 0.000 description 1
- YQNRVGJCPCNMKT-LFVJCYFKSA-N 2-[(e)-[[2-(4-benzylpiperazin-1-ium-1-yl)acetyl]hydrazinylidene]methyl]-6-prop-2-enylphenolate Chemical compound [O-]C1=C(CC=C)C=CC=C1\C=N\NC(=O)C[NH+]1CCN(CC=2C=CC=CC=2)CC1 YQNRVGJCPCNMKT-LFVJCYFKSA-N 0.000 description 1
- ZBMRKNMTMPPMMK-UHFFFAOYSA-N 2-amino-4-[hydroxy(methyl)phosphoryl]butanoic acid;azane Chemical compound [NH4+].CP(O)(=O)CCC(N)C([O-])=O ZBMRKNMTMPPMMK-UHFFFAOYSA-N 0.000 description 1
- -1 294N Inorganic materials 0.000 description 1
- GNKZMNRKLCTJAY-UHFFFAOYSA-N 4'-Methylacetophenone Chemical compound CC(=O)C1=CC=C(C)C=C1 GNKZMNRKLCTJAY-UHFFFAOYSA-N 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 1
- 241001061264 Astragalus Species 0.000 description 1
- 235000010110 Astragalus glycyphyllos Nutrition 0.000 description 1
- NOWKCMXCCJGMRR-UHFFFAOYSA-N Aziridine Chemical compound C1CN1 NOWKCMXCCJGMRR-UHFFFAOYSA-N 0.000 description 1
- 238000010453 CRISPR/Cas method Methods 0.000 description 1
- 101100462537 Caenorhabditis elegans pac-1 gene Proteins 0.000 description 1
- 102100028717 Cytosolic 5'-nucleotidase 3A Human genes 0.000 description 1
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 description 1
- 102100031780 Endonuclease Human genes 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 108010044467 Isoenzymes Proteins 0.000 description 1
- 231100000111 LD50 Toxicity 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 241000219745 Lupinus Species 0.000 description 1
- 240000004658 Medicago sativa Species 0.000 description 1
- 235000017587 Medicago sativa ssp. sativa Nutrition 0.000 description 1
- 241000213996 Melilotus Species 0.000 description 1
- 235000000839 Melilotus officinalis subsp suaveolens Nutrition 0.000 description 1
- 101100117764 Mus musculus Dusp2 gene Proteins 0.000 description 1
- 101710118186 Neomycin resistance protein Proteins 0.000 description 1
- HSNVNALJRSJDHT-UHFFFAOYSA-N P(=O)(=O)[Mo] Chemical compound P(=O)(=O)[Mo] HSNVNALJRSJDHT-UHFFFAOYSA-N 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 240000004371 Panax ginseng Species 0.000 description 1
- 235000005035 Panax pseudoginseng ssp. pseudoginseng Nutrition 0.000 description 1
- 235000003140 Panax quinquefolius Nutrition 0.000 description 1
- 235000010627 Phaseolus vulgaris Nutrition 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 241000255969 Pieris brassicae Species 0.000 description 1
- 241000293869 Salmonella enterica subsp. enterica serovar Typhimurium Species 0.000 description 1
- 241000187747 Streptomyces Species 0.000 description 1
- 241000187391 Streptomyces hygroscopicus Species 0.000 description 1
- 239000004098 Tetracycline Substances 0.000 description 1
- 108010022394 Threonine synthase Proteins 0.000 description 1
- 229910021626 Tin(II) chloride Inorganic materials 0.000 description 1
- 235000015724 Trifolium pratense Nutrition 0.000 description 1
- 244000098338 Triticum aestivum Species 0.000 description 1
- 229930003268 Vitamin C Natural products 0.000 description 1
- 235000007244 Zea mays Nutrition 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 238000007792 addition Methods 0.000 description 1
- 230000005260 alpha ray Effects 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 235000019270 ammonium chloride Nutrition 0.000 description 1
- APUPEJJSWDHEBO-UHFFFAOYSA-P ammonium molybdate Chemical compound [NH4+].[NH4+].[O-][Mo]([O-])(=O)=O APUPEJJSWDHEBO-UHFFFAOYSA-P 0.000 description 1
- 239000011609 ammonium molybdate Substances 0.000 description 1
- 229940010552 ammonium molybdate Drugs 0.000 description 1
- 235000018660 ammonium molybdate Nutrition 0.000 description 1
- 239000012378 ammonium molybdate tetrahydrate Substances 0.000 description 1
- YVBOZGOAVJZITM-UHFFFAOYSA-P ammonium phosphomolybdate Chemical compound [NH4+].[NH4+].[NH4+].[NH4+].[O-]P([O-])=O.[O-][Mo]([O-])(=O)=O YVBOZGOAVJZITM-UHFFFAOYSA-P 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 235000006533 astragalus Nutrition 0.000 description 1
- 229940107666 astragalus root Drugs 0.000 description 1
- FIXLYHHVMHXSCP-UHFFFAOYSA-H azane;dihydroxy(dioxo)molybdenum;trioxomolybdenum;tetrahydrate Chemical compound N.N.N.N.N.N.O.O.O.O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O[Mo](O)(=O)=O.O[Mo](O)(=O)=O.O[Mo](O)(=O)=O FIXLYHHVMHXSCP-UHFFFAOYSA-H 0.000 description 1
- 150000001540 azides Chemical class 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000005250 beta ray Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- JJWKPURADFRFRB-UHFFFAOYSA-N carbonyl sulfide Chemical compound O=C=S JJWKPURADFRFRB-UHFFFAOYSA-N 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 239000012295 chemical reaction liquid Substances 0.000 description 1
- 239000003638 chemical reducing agent Substances 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- DENRZWYUOJLTMF-UHFFFAOYSA-N diethyl sulfate Chemical compound CCOS(=O)(=O)OCC DENRZWYUOJLTMF-UHFFFAOYSA-N 0.000 description 1
- 229940008406 diethyl sulfate Drugs 0.000 description 1
- 102000004419 dihydrofolate reductase Human genes 0.000 description 1
- PXEDJBXQKAGXNJ-QTNFYWBSSA-L disodium L-glutamate Chemical compound [Na+].[Na+].[O-]C(=O)[C@@H](N)CCC([O-])=O PXEDJBXQKAGXNJ-QTNFYWBSSA-L 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 239000004459 forage Substances 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 230000005251 gamma ray Effects 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 230000014509 gene expression Effects 0.000 description 1
- 235000008434 ginseng Nutrition 0.000 description 1
- IAJOBQBIJHVGMQ-BYPYZUCNSA-N glufosinate-P Chemical group CP(O)(=O)CC[C@H](N)C(O)=O IAJOBQBIJHVGMQ-BYPYZUCNSA-N 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 238000009830 intercalation Methods 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 230000005486 microgravity Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 1
- 229910000069 nitrogen hydride Inorganic materials 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 125000001477 organic nitrogen group Chemical group 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 230000029553 photosynthesis Effects 0.000 description 1
- 238000010672 photosynthesis Methods 0.000 description 1
- 210000002706 plastid Anatomy 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 210000001236 prokaryotic cell Anatomy 0.000 description 1
- 230000009465 prokaryotic expression Effects 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 239000012460 protein solution Substances 0.000 description 1
- 230000004800 psychological effect Effects 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 238000010188 recombinant method Methods 0.000 description 1
- 235000013526 red clover Nutrition 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000013605 shuttle vector Substances 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 229940073490 sodium glutamate Drugs 0.000 description 1
- 235000011150 stannous chloride Nutrition 0.000 description 1
- 239000001119 stannous chloride Substances 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 239000010902 straw Substances 0.000 description 1
- 230000004960 subcellular localization Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 229960002180 tetracycline Drugs 0.000 description 1
- 229930101283 tetracycline Natural products 0.000 description 1
- 235000019364 tetracycline Nutrition 0.000 description 1
- 150000003522 tetracyclines Chemical class 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 230000014621 translational initiation Effects 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- 241001430294 unidentified retrovirus Species 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 235000019154 vitamin C Nutrition 0.000 description 1
- 239000011718 vitamin C Substances 0.000 description 1
- 210000005253 yeast cell Anatomy 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
Landscapes
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses a plant glutamine synthetase mutant containing G294 mutation, and a coding gene and application thereof, and relates to the technical field of genetic engineering. The method for obtaining the protein with the glufosinate resistance is simple and feasible, has high success rate, and is convenient for people to obtain the protein with the glufosinate resistance quickly and efficiently. The glutamine synthetase mutant provided by the invention is originally derived from plants, has glufosinate resistance after mutation, and has good biological enzyme catalytic activity. Plants transformed with the glutamine synthetase mutant have glufosinate resistance and can grow and develop normally.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a plant glutamine synthetase mutant containing G294 mutation, and a coding gene and application thereof.
Background
Glutamine synthetase (Glutamine synthetase, GS) is a key enzyme in nitrogen metabolism in plants, which catalyzes the condensation of glutamic acid (gin) with NH3 to form glutamine (Glu) in the glutamate synthetase cycle, participating in the metabolism of nitrogen-containing compounds in plants. Depending on distribution and subcellular localization, higher plant GS (class GSII) isoenzymes can be divided into two classes: one which is located in the cytoplasm is called cytoplasmic GS (GS 1), with a molecular weight of 38-40kDa; another type, designated as apoplast GS (GS 2), is located in chloroplasts (or plastids) and has a molecular weight of 44-45kDa.
Glufosinate (glufosinate ammoni μm, trade name Basta) is a glutamine synthetase (GS 1) inhibitor developed by anget corporation (now bayer corporation), and its active ingredient is phosphinothricin (abbreviated PPT), and its chemical name is (RS) -2-amino-4- (hydroxymethylphosphino) ammonium butyrate. The target enzyme for glufosinate is GS, which can normally form lambda-glutamyl phosphate from ATP and glutamate. However, after PPT treatment, PPT is first bound to ATP and phosphorylated PPT occupies 8 reaction centers of GS molecules, so that the spatial configuration of GS is changed and GS activity is inhibited. PPT inhibits all known forms of GS.
As a result of inhibition of GS by glufosinate, it can lead to disturbance of nitrogen metabolism in plants, excessive accumulation of ammonium, disintegration of chloroplasts, inhibition of photosynthesis, and finally death of plants
At present, the main method for cultivating the glufosinate-resistant variety is to introduce the glufosinate-resistant gene from bacteria into crops by using genetic engineering means, so as to cultivate a new transgenic glufosinate-resistant crop variety. The most widely used anti-glufosinate genes in agriculture today are the bar gene from strain Streptomyces hygroscopicus and the pat gene from strain s. The Bar gene and the pat gene have 80% homology, and can code glufosinate acetylase, and the glufosinate acetylase can be inactivated by the enzyme. The glufosinate-resistant variety has great use value, wherein resistant rape, corn and the like are commercially planted in a large area. However, bar and pat genes are derived from microorganisms, not from the crop itself, and are more likely to cause conflicting psychological effects for the consumer.
The glufosinate acetylase enzyme coded by Bar gene and pat gene can acetylate glufosinate to inactivate, but before glufosinate contacts GS, the enzyme can hardly inactivate glufosinate completely, because many GS are distributed on cell membranes, so that when the glufosinate is applied to crops with Bar gene and pat gene, nitrogen metabolism of plants can be interfered to different degrees, and normal growth and development of the plants are affected. Overexpression of wild-type GS in plants can reduce the sensitivity of transgenic plants to glufosinate but is not sufficiently tolerant for commercial use.
In view of this, the present invention has been made.
Disclosure of Invention
The invention aims to provide a plant glutamine synthetase mutant containing G294 mutation, and a coding gene and application thereof, so as to obtain a protein with glufosinate resistance, the coding gene of the protein mutant (namely the plant glutamine synthetase mutant) is integrated into bacteria or cells, and a transformed plant can further select and breed a new plant variety with glufosinate resistance. The obtained plant glutamine synthetase mutant has good biological enzyme catalytic activity.
Term interpretation:
"deletion" is a deletion mutation (del) or "deletion mutation". If the amino acid sequence of the target protein is subjected to deletion mutation corresponding to the 61 st amino acid residue of the reference sequence, the amino acid residue corresponding to the 61 st amino acid residue of the reference sequence is deleted.
The invention is realized in the following way:
in a first aspect, the present invention provides a method for obtaining a protein having glufosinate resistance comprising the steps of:
1) A protein having the reference sequence shown in SEQ ID NO.1 or having an amino acid sequence having at least 85% identity to the reference sequence as a target protein;
2) Comparing the amino acid sequence of the target protein with a reference sequence, and mutating the amino acid sequence of the target protein corresponding to 294 amino acid residues of the reference sequence to A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y or deleting;
3) Proteins with increased glufosinate resistance are selected.
The inventor researches and discovers that the wild type glutamine synthetase of plant origin is compared with a reference sequence, and the amino acid position corresponding to 294 th position of the reference sequence is mutated into A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y or deleted, so that the protein with glufosinate resistance can be obtained by screening. The protein has good biological enzyme catalytic activity.
In an alternative embodiment, when the target protein is derived from rice, the amino acid residue at position 294 of the target protein corresponding to the reference sequence is mutated to C, D, E, F, H, K, N, P, Q, R, S, V, W, Y or deleted.
In an alternative embodiment, when the protein of interest is derived from maize, the amino acid residue of the protein of interest corresponding to amino acid residue 294 of the reference sequence is mutated to C, E, H, I, K, L, M, N, P, R, W, Y or deleted.
In an alternative embodiment, when the protein of interest is derived from wheat, the amino acid residue of the protein of interest corresponding to amino acid residue 294 of the reference sequence is mutated to A, C, D or N.
In an alternative embodiment, when the protein of interest is derived from canola, the amino acid residue of the protein of interest corresponding to amino acid 294 of the reference sequence is mutated to A, C, E, H, K, M, N, Q, R, T or W.
In a second aspect, the present invention provides a plant glutamine synthetase mutant having glufosinate resistance, the plant glutamine synthetase mutant being as follows (1) or (2):
(1): it is obtained by mutating the n-th position of wild glutamine synthetase from plant; the position of the nth bit is determined as follows: the wild type glutamine synthetase is aligned with the reference sequence, the n-th position of the wild type glutamine synthetase corresponds to the 294-th position of the reference sequence, wherein the amino acid sequence of the reference sequence is shown as SEQ ID NO. 1;
The n-th amino acid of the plant glutamine synthetase mutant is A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y or deleted compared with the wild glutamine synthetase;
(2): which has at least 85% identity with the plant glutamine synthetase mutant shown in (1) and is identical to the amino acid at the n-th position of the plant glutamine synthetase mutant shown in (1).
The wild type glutamine synthetase from plant source is compared with reference sequence, the amino acid site corresponding to 294 th site of the reference sequence, i.e. n site, is mutated into A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y or deleted, and the obtained plant glutamine synthetase mutant has glufosinate resistance and maintains its biological enzyme catalytic activity. The recombinant bacteria or plants transformed with the plant glutamine synthetase mutant provided by the invention can normally grow and develop in the presence of glufosinate, and the plant glutamine synthetase mutant not only can be used for cultivating transgenic crops, but also can be used for cultivating glufosinate-resistant non-transgenic plants or transgenic plants such as rice, tobacco, soybean, corn, wheat, rape, cotton, sorghum and the like, and has wide application prospects.
The reference sequence is wild type glutamine synthetase of rice origin.
The sequence alignment method can use Blast website (https:// Blast. Ncbi. Nlm. Nih. Gov/Blast. Cgi) to carry out Protein Blast alignment; the same results can be obtained using other sequence alignment methods or tools well known in the art.
It should be noted that the n-th position of the wild-type glutamine synthetase may be 294 (e.g., corn, soybean, wheat, rape, etc.) on its own sequence, but may not be 294, and the specific position of the n-th position may be determined by alignment of the sequences described above, so long as the position corresponding to the 294-th position of the reference sequence is the n-th position according to the present invention, i.e., the mutation position, after alignment with the reference sequence.
The inventors found that mutation of the wild-type glutamine synthetase of various plants to C or N at position 294 of the corresponding reference sequence can produce a plant glutamine synthetase mutant having glufosinate resistance. Thus, C or N is a common mutant amino acid for a variety of plants, and one skilled in the art would be able to expect that mutation of wild-type glutamine synthetase from a variety of plants would result in a plant glutamine synthetase mutant that is glufosinate resistant. The N-th amino acid of the plant glutamine synthetase mutant is C or N.
All plants have homology to the wild-type glutamine synthetase and essentially identical functions and domains in the plant body. Thus, any plant-derived wild-type glutamine synthetase mutant obtained by the above-described mutation at position 294 has glufosinate resistance. Therefore, the plant glutamine synthetase mutants obtained by mutating wild type glutamine synthetase of any plant origin are all within the scope of the present invention.
In a preferred embodiment of the present invention, the plant is selected from wheat, rice, barley, oat, corn, sorghum, millet, buckwheat, millet, sweet potato, cotton, canola, sesame, peanut, sunflower, radish, carrot, broccoli, tomato, eggplant, capsicum, leek, onion, leek, spinach, celery, amaranth, lettuce, crowndaisy chrysanthemum, grape, strawberry, sugarcane, tobacco, brassica vegetables, cucurbitaceae, leguminous plants, pasture, tea or cassava.
In a preferred embodiment of the invention, the pasture is selected from gramineous pasture or leguminous pasture; leguminous forage includes, but is not limited to: red clover, white clover, alfalfa, arrowhead peas, green peas, lupin yellow, white clover, sweet clover, astragalus root, white milk vetch, white butterfly beans, and the like.
In a preferred embodiment of the invention, the brassica vegetables include, but are not limited to, turnip, cabbage, mustard, cabbage mustard, canola, mustard, cabbage, canola, green vegetables, or beet.
In a preferred embodiment of the present invention, the cucurbitaceae plant includes, but is not limited to, cucumber, pumpkin, wax gourd, luffa, melon, watermelon or melon.
In preferred embodiments of the invention, leguminous plants include, but are not limited to, mung beans, broad beans, peas, lentils, soybeans, kidney beans, cowpeas, or green beans.
Furthermore, it is known and easily achieved by those skilled in the art that a simple amino acid substitution, deletion, or addition is performed in a non-conserved region of the plant glutamine synthetase mutant shown in (1) and the n-th position is maintained as the above-mentioned mutated amino acid, and that the further mutated plant glutamine synthetase mutant has at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% etc.) or more identity with the plant glutamine synthetase mutant shown in (1), and that its functions include the enzyme catalytic activity and glufosinate resistance are equivalent to or slightly decreased or slightly increased or greatly increased as those of the plant glutamine synthetase mutant shown in (1). Therefore, such glutamine synthetases should also fall within the scope of the present invention.
In an alternative embodiment, when the plant is rice, the amino acid at position n of the plant glutamine synthetase mutant is C, D, E, F, H, K, N, P, Q, R, S, V, W, Y or deleted;
in an alternative embodiment, when the plant is maize, the amino acid at position n of the plant glutamine synthetase mutant is C, E, H, I, K, L, M, N, P, R, W, Y or deleted;
in an alternative embodiment, when the plant is wheat, the amino acid at position N of the plant glutamine synthetase mutant is A, C, D or N;
in an alternative embodiment, when the plant is canola, the n-th amino acid of the plant glutamine synthetase mutant is A, C, E, H, K, M, N, Q, R, T or W.
The studies of the present invention have also found that mutation of the n-th position of glutamine synthetase to an amino acid other than C, N for a different plant source also renders the glutamine synthetase glufosinate resistant.
Alternatively, in some embodiments of the invention, when the plant is rice, the rice wild-type glutamine synthetase is SEQ ID No.1:
MASLTDLVNLNLSDTTEKIIAEYIWIGGSGMDLRSKARTLSGPVTDPSKLPKWNYDGSSTGQAPGEDSEVILYPQAIFKDPFRKGNNILVMCDCYTPAGEPIPTNKRHNAAKIFSSPEVASEEPWYGIEQEYTLLQKDINWPLGWPVGGFPGPQGPYYCGIGADKSFGRDIVDSHYKACLYAGINISGINGEVMPGQWEFQVGPSVGISAGDQVWVARYILERITEIAGVVVSFDPKPIPGDWNGAGAHTNYSTKSMRNDGGYEIIKSAIEKLKLRHKEHISAYGEGNERRLTGRHETADINTFSWGVANRGASVRVGRETEQNGKGYFEDRRPASNMDPYIVTSMIAETTIIWKP。
alternatively, in some embodiments of the invention, when the plant is corn, the corn wild-type glutamine synthetase is SEQ ID No.2:
MACLTDLVNLNLSDNTEKIIAEYIWIGGSGMDLRSKARTLSGPVTDPSKLPKWNYDGSSTGQAPGEDSEVILYPQAIFKDPFRRGNNILVMCDCYTPAGEPIPTNKRYNAAKIFSSPEVAAEEPWYGIEQEYTLLQKDTNWPLGWPIGGFPGPQGPYYCGIGAEKSFGRDIVDAHYKACLYAGINISGINGEVMPGQWEFQVGPSVGISSGDQVWVARYILERITEIAGVVVTFDPKPIPGDWNGAGAHTNYSTESMRKEGGYEVIKAAIEKLKLRHREHIAAYGEGNERRLTGRHETADINTFSWGVANRGASVRVGRETEQNGKGYFEDRRPASNMDPYVVTSMIAETTIIWKP。
Alternatively, in some embodiments of the invention, when the plant is wheat, the wheat wild-type glutamine synthetase is SEQ ID No.3:
MALLTDLLNLDLTDSTEKIIAEYIWIGGSGMDLRSKARTLPGPVTDPSKLPKWNYDGSSTGQAPGEDSEVILYPQAIFKDPFRKGNNILVMCDCYTPAGVPIPTNKRYNAAKIFSNPDVAKEEPWYGIEQEYTLLQKDINWPLGWPVGGFPGPQGPYYCSIGADKSFGRDIVDSHYKACLFAGVNISGINGEVMPGQWEFQVGPTVGISAGDQVWVARYLLERITEIAGVVVTFDPKPIPGDWNGAGAHTNYSTESMRKDGGFKVIVDAVEKLKLKHKEHIAAYGEGNERRLTGKHETADINTFSWGVANRGASVRVGRETEQNGKGYFEDRRPASNMDPYVVTSMIAETTILWKP。
alternatively, in some embodiments of the invention, when the plant is canola, the canola wild-type glutamine synthetase is SEQ ID No.4:
MSLLTDLVNLNLSETTDKIIAEYIWVGGSGMDMRSKARTLPGPVSDPSELPKWNYDGSSTGQAPGEDSEVILYPQAIFKDPFRRGNNILVMCDAYTPAGEPIPTNKRHAAAKVFSHPDVVAEVPWYGIEQEYTLLQKDVNWPLGWPIGGFPGPQGPYYCSVGADKSFGRDIVDAHYKACLYAGINISGINGEVMPGQWEFQVGPAVGISAGDEIWVARFILERITEIAGVVVSFDPKPIPGDWNGAGAHCNYSTKSMREDGGYEIIKKAIDKLGLRHKEHIAAYGEGNERRLTGHHETADINTFLWGVANRGASIRVGRDTEKEGKGYFEDRRPASNMDPYIVTSMIAETTILWKP。
the Similarity (Similarity) and Identity (Identity) of the wild-type glutamine synthetases of partial plant origin are shown in the following table, and the partial results of the sequence alignment are shown in FIG. 11, with the arrow indicating amino acid 294.
The above-mentioned Similarity (Similarity) and Identity (Identity) comparison method is as follows: the amino acid sequence of one species is input to the Blast website (https:// Blast. Ncbi. Nlm. Nih. Gov/Blast. Cgi) for Protein Blast alignment, and the Similarity (Similarity) and Identity (Identity) of this species and other species to be aligned are looked up from the alignment.
In a third aspect, the invention also provides an isolated nucleic acid molecule encoding a plant glutamine synthetase mutant as described above.
The term "nucleic acid molecule encoding a mutant of a plant glutamine synthetase as described above" may be a polynucleotide comprising a mutant protein of the present invention, or may be a polynucleotide further comprising additional coding and/or non-coding sequences.
The invention also relates to variants of the above polynucleotides which encode fragments, analogs and derivatives of the polypeptides or muteins having the same amino acid sequence as the invention. Such nucleotide variants include substitution variants, deletion variants and insertion variants. As known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, without substantially altering the function of the mutein encoded thereby.
At present, it is already possible to obtain the DNA sequences encoding the proteins of the invention (or fragments or derivatives thereof) entirely by chemical synthesis. The DNA sequence can then be introduced into a variety of existing DNA molecules (or vectors, for example) and cells known in the art.
Methods of amplifying DNA/RNA using PCR techniques are preferred for obtaining polynucleotides of the invention. In particular, when it is difficult to obtain full-length cDNA from a library, it is preferable to use RACE method (RACE-cDNA end rapid amplification method), and primers for PCR can be appropriately selected according to the sequence information of the present invention disclosed herein and synthesized by a conventional method. The amplified DNA/RNA fragments can be isolated and purified by conventional methods, such as by gel electrophoresis.
In the case where the present invention provides the above amino acid sequence, a nucleic acid sequence encoding the above plant glutamine synthetase mutant is easily obtained by a person skilled in the art based on the degeneracy of codons. For example, a nucleic acid sequence encoding a mutant of a plant glutamine synthetase as described above may be obtained by mutating a corresponding nucleotide in a nucleic acid sequence encoding a wild type glutamine synthetase. This is readily accomplished by one skilled in the art.
For example, the rice wild-type glutamine synthetase has a nucleic acid sequence of SEQ ID NO.5:
ATGGCTTCTCTCACCGATCTCGTCAACCTCAACCTCTCCGACACCACGGAGAAGATCATCGCCGAGTACATATGGATCGGTGGATCTGGCATGGATCTCAGGAGCAAGGCTAGGACTCTCTCCGGCCCTGTGACTGATCCCAGCAAGCTGCCCAAGTGGAACTACGATGGCTCCAGCACCGGCCAGGCCCCCGGCGAGGACAGTGAGGTCATCCTGTACCCACAGGCTATCTTCAAGGACCCATTCAGGAAGGGAAACAACATCCTTGTCATGTGCGATTGCTACACGCCAGCCGGAGAACCGATCCCCACCAACAAGAGGCACAATGCTGCCAAGATCTTCAGCTCCCCTGAGGTTGCTTCTGAGGAGCCCTGGTACGGTATTGAGCAAGAGTACACCCTCCTCCAGAAGGACATCAACTGGCCCCTTGGCTGGCCTGTTGGTGGCTTCCCTGGTCCTCAGGGTCCTTACTACTGTGGTATCGGTGCTGACAAGTCTTTTGGGCGTGATATTGTTGACTCCCACTACAAGGCTTGCCTCTATGCCGGCATCAACATCAGTGGAATCAACGGCGAGGTCATGCCAGGACAGTGGGAGTTCCAAGTTGGCCCGTCTGTCGGCATTTCTGCCGGTGATCAGGTGTGGGTTGCTCGCTACATTCTTGAGAGGATCACCGAGATCGCCGGAGTCGTCGTCTCATTTGACCCCAAGCCCATCCCGGGAGACTGGAACGGTGCTGGTGCTCACACCAACTACAGCACCAAGTCGATGAGGAACGATGGTGGCTACGAGATCATCAAGTCCGCCATTGAGAAGCTCAAGCTCAGGCACAAGGAGCACATCTCCGCCTACGGCGAGGGCAACGAGCGCCGGCTCACCGGCAGGCACGAGACCGCCGACATCAACACCTTCAGCTGGGGAGTTGCCAACCGCGGCGCCTCGGTCCGCGTCGGCCGGGAGACGGAGCAGAACGGCAAGGGCTACTTCGAGGATCGCCGGCCGGCGTCCAACATGGACCCTTACATCGTCACCTCCATGATCGCCGAGACCACCATCATCTGGAAGCCCTGA。
accordingly, on a sequence basis, a mutant encoding rice plant glutamine synthetase as described above can be obtained by performing a corresponding nucleotide mutation at a codon corresponding to 294 of the encoded amino acid sequence.
The coding nucleic acid sequence of the corn wild type glutamine synthetase is SEQ ID NO.6:
ATGGCCTGCCTCACCGACCTCGTCAACCTCAACCTCTCGGACAACACCGAGAAGATCATCGCGGAATACATATGGATCGGTGGATCTGGCATGGATCTCAGGAGCAAAGCAAGGACCCTCTCCGGCCCGGTGACCGATCCCAGCAAGCTGCCCAAGTGGAACTACGACGGCTCCAGCACGGGCCAGGCCCCCGGCGAGGACAGCGAGGTCATCCTGTACCCGCAGGCCATCTTCAAGGACCCATTCAGGAGGGGCAACAACATCCTTGTGATGTGCGATTGCTACACCCCAGCCGGCGAGCCAATCCCCACCAACAAGAGGTACAACGCCGCCAAGATCTTCAGCAGCCCTGAGGTCGCCGCCGAGGAGCCGTGGTATGGTATTGAGCAGGAGTACACCCTCCTCCAGAAGGACACCAACTGGCCCCTTGGGTGGCCCATCGGTGGCTTCCCCGGCCCTCAGGGTCCTTACTACTGTGGAATCGGCGCCGAAAAGTCGTTCGGCCGCGACATCGTGGACGCCCACTACAAGGCCTGCTTGTATGCGGGCATCAACATCAGTGGCATCAACGGGGAGGTGATGCCAGGGCAGTGGGAGTTCCAAGTCGGGCCTTCCGTGGGTATATCTTCAGGCGACCAGGTCTGGGTCGCTCGCTACATTCTTGAGAGGATCACGGAGATCGCCGGTGTGGTGGTGACGTTCGACCCGAAGCCGATCCCGGGCGACTGGAACGGCGCCGGCGCGCACACCAACTACAGCACGGAGTCGATGAGGAAGGAGGGCGGGTACGAGGTGATCAAGGCGGCCATCGAGAAGCTGAAGCTGCGGCACAGGGAGCACATCGCGGCATACGGCGAGGGCAACGAGCGCCGGCTCACCGGCAGGCACGAGACCGCCGACATCAACACGTTCAGCTGGGGCGTGGCCAACCGCGGCGCGTCGGTGCGCGTGGGCCGGGAGACGGAGCAGAACGGCAAGGGCTACTTCGAGGACCGCCGCCCGGCGTCCAACATGGACCCCTACGTGGTCACCTCCATGATCGCCGAGACCACCATCATCTGGAAGCCCTGA。
the coding nucleic acid sequence of the wheat wild type glutamine synthetase is SEQ ID NO.7:
ATGGCGCTCCTCACCGATCTCCTCAACCTCGACCTCACCGACTCCACGGAGAAGATCATCGCCGAGTACATATGGATCGGCGGATCTGGCATGGATCTCAGGAGCAAAGCCAGGACCCTCCCCGGCCCGGTCACCGACCCCAGCAAGCTGCCCAAGTGGAACTACGACGGCTCCAGCACCGGCCAGGCCCCCGGCGAGGACAGCGAGGTCATCCTGTACCCACAGGCCATCTTCAAGGACCCGTTCAGGAAGGGCAACAACATCCTTGTCATGTGCGATTGCTACACCCCAGCTGGAGTGCCAATCCCCACCAACAAGAGATACAACGCTGCCAAGATCTTTAGCAACCCTGATGTTGCCAAGGAGGAGCCATGGTACGGTATCGAGCAGGAGTACACCCTCCTACAGAAGGACATCAACTGGCCTCTCGGCTGGCCTGTTGGTGGATTCCCTGGTCCTCAGGGTCCTTACTACTGTAGTATTGGTGCTGACAAGTCGTTTGGGCGTGACATAGTTGACTCCCACTACAAGGCCTGCCTCTTTGCCGGCGTCAACATCAGTGGCATCAACGGCGAGGTCATGCCCGGACAGTGGGAGTTCCAAGTTGGCCCGACTGTCGGCATCTCTGCTGGTGACCAAGTGTGGGTTGCTCGCTACCTTCTTGAGAGGATCACTGAGATCGCCGGAGTTGTCGTCACATTTGACCCCAAGCCCATCCCAGGCGACTGGAACGGTGCTGGTGCTCACACAAACTACAGTACCGAGTCGATGAGGAAGGACGGCGGGTTCAAGGTCATCGTGGACGCTGTCGAGAAGCTCAAGCTGAAGCACAAGGAGCACATCGCCGCCTACGGCGAGGGCAACGAGCGCCGTCTCACCGGCAAGCACGAAACCGCCGACATCAACACCTTCAGCTGGGGTGTCGCGAACCGTGGCGCGTCGGTGCGCGTGGGACGGGAGACGGAGCAGAACGGCAAGGGCTACTTCGAGGACCGCCGGCCGGCGTCCAACATGGACCCCTACGTGGTCACCTCCATGATCGCCGAGACCACCATCCTGTGGAAGCCCTGA。
the encoding nucleic acid sequence of the wild type rape glutamine synthetase is SEQ ID NO.8:
ATGAGTCTTCTTACAGATCTCGTTAACCTTAACCTCTCAGAGACCACTGACAAAATCATTGCGGAATACATATGGGTTGGAGGTTCAGGAATGGATATGAGAAGCAAAGCCAGGACTCTTCCTGGACCAGTGAGTGACCCTTCGGAGCTACCAAAGTGGAACTATGATGGCTCAAGCACAGGCCAAGCTCCTGGTGAAGACAGTGAAGTCATCTTATACCCTCAAGCCATATTCAAAGATCCTTTCCGTAGAGGCAACAACATTCTTGTCATGTGCGATGCTTACACTCCAGCGGGCGAACCGATCCCAACAAACAAAAGACACGCTGCGGCTAAGGTCTTTAGCCACCCCGATGTTGTAGCTGAAGTGCCATGGTATGGTATTGAGCAAGAGTATACTTTACTTCAGAAAGATGTGAACTGGCCTCTTGGTTGGCCTATTGGCGGCTTCCCCGGTCCTCAGGGACCATACTATTGTAGTGTTGGAGCAGATAAATCTTTTGGTAGAGACATCGTTGATGCTCACTACAAGGCCTGCTTATACGCTGGCATCAATATTAGTGGCATCAACGGAGAAGTCATGCCTGGTCAGTGGGAGTTCCAAGTTGGTCCAGCTGTTGGTATCTCGGCCGGTGATGAAATTTGGGTCGCACGTTTCATTTTGGAGAGGATCACAGAGATTGCTGGTGTGGTGGTATCTTTTGACCCAAAACCGATTCCCGGTGACTGGAATGGTGCTGGTGCTCACTGCAACTATAGTACCAAGTCAATGAGGGAAGATGGTGGTTACGAGATTATTAAGAAGGCAATCGATAAACTGGGACTGAGACACAAAGAACACATTGCAGCTTACGGTGAAGGCAATGAGCGCCGTCTCACGGGTCACCACGAGACTGCTGACATCAACACTTTCCTCTGGGGTGTTGCGAACCGTGGAGCATCAATCCGTGTAGGACGTGACACAGAGAAAGAAGGGAAAGGATACTTTGAGGATAGGAGGCCAGCTTCGAACATGGATCCTTACATTGTGACTTCCATGATTGCAGAGACCACAATCCTCTGGAAACCTTGA。
in a fourth aspect, the invention also provides a vector comprising the nucleic acid molecule described above.
Vectors include, but are not limited to, expression vectors, shuttle vectors, and integration vectors.
In the present invention, the term "expression vector" refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses or other vectors well known in the art. Any plasmid or vector may be used as long as it is replicable and stable in the host. An important feature of expression vectors is that they generally contain an origin of replication, a promoter, a marker gene and translational control elements.
In an alternative embodiment, the expression vector further comprises a ribosome binding site for translation initiation and a transcription terminator.
In addition, the expression vector preferably comprises one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance and Green Fluorescent Protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for E.coli.
Vectors comprising the appropriate DNA sequences as described above, as well as appropriate promoter or control sequences, may be used to transform appropriate host cells to enable expression of the protein.
In a fifth aspect, the invention also provides a recombinant bacterium or recombinant cell comprising the nucleic acid molecule or vector described above.
The recombinant cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: coli, streptomyces; bacterial cells of salmonella typhimurium; fungal cells such as yeast, plant cells (e.g., ginseng cells).
In a preferred embodiment of the invention, the strain is Agrobacterium or E.coli.
In a sixth aspect, the invention also provides the use of a plant glutamine synthetase mutant, a nucleic acid molecule, a vector or a recombinant bacterium or recombinant cell as described above for breeding a plant variety having glufosinate resistance.
In a preferred embodiment of the application of the present invention, the application includes any of the following uses:
(1) Transforming a target plant with a vector containing a coding gene encoding a plant glutamine synthetase mutant;
(2) Modifying the endogenous glutamine synthetase gene of the target plant by a gene editing method to code a plant glutamine synthetase mutant;
(3) Mutagenizing and screening plant cells, tissues, individuals or populations to encode a plant glutamine synthetase mutant;
In an alternative embodiment, the gene editing is selected from CRISPR/Cas9, TALEN technology, or ZFN technology.
In preferred embodiments of the invention for use, the gene editing is selected from CRISPR/Cas9, TALEN technology or ZFN technology.
On the basis of the present invention, it is easy for a person skilled in the art to modify a target plant by a conventional gene editing technique in the art, such as a zinc finger endonuclease (ZFN, zinc-finger nucleic acid) technique, transcription activator-like effector nuclease (TALEN, transcription activator-like effector nucleases) technique or CRISPR/Cas 9), a mutation breeding technique (such as chemical, radiation mutagenesis, etc.), etc., to have a gene encoding the plant glutamine synthetase mutant as described above, thereby obtaining glufosinate resistance and enabling normal growth and development, and to obtain a new variety of plants having glufosinate resistance. Therefore, whatever technology is adopted, the plant glutamine synthetase mutant provided by the invention is utilized to endow the plant with glufosinate resistance, and belongs to the protection scope of the invention.
In an alternative embodiment, the plant is subjected to mutagenesis in a physicochemical mutagenesis mode that is mutagenized to a non-lethal dose to obtain plant material.
The above-mentioned non-lethal dose means that the dose is controlled to be within a range of 20% floating above and below the semi-lethal dose.
Physicochemical mutagenesis modes include combinations of one or more of the following physical mutagenesis and chemical mutagenesis modes: physical mutagenesis includes ultraviolet mutagenesis, X-ray mutagenesis, gamma-ray mutagenesis, beta-ray mutagenesis, alpha-ray mutagenesis, high-energy particle mutagenesis, cosmic ray mutagenesis, microgravity mutagenesis; chemical mutagenesis includes alkylating agent mutagenesis, azide mutagenesis, base analogue mutagenesis, lithium chloride mutagenesis, antibiotic mutagenesis and intercalating dye mutagenesis; alkylating agent mutagenesis includes ethylcyclomate mutagenesis, diethylsulfate mutagenesis, and ethylenimine mutagenesis.
In an alternative embodiment, the plant is selected from wheat, rice, barley, oat, corn, sorghum, millet, buckwheat, millet, sweet potato, cotton, canola, sesame, peanut, sunflower, radish, carrot, broccoli, tomato, eggplant, capsicum, leek, onion, leek, spinach, celery, amaranth, lettuce, crowndaisy, day lily, grape, strawberry, sugarcane, tobacco, brassica vegetables, cucurbitaceae, leguminous plants, pasture, tea, or cassava;
In an alternative embodiment, the pasture is selected from gramineous pasture or leguminous pasture;
in an alternative embodiment, the brassica vegetable is selected from turnip, cabbage, mustard, cabbage mustard, canola, mustard, blue, canola, green vegetables, or beet;
in an alternative embodiment, the cucurbitaceae plant is selected from cucumber, pumpkin, wax gourd, bitter gourd, luffa, melon, watermelon or melon;
in an alternative embodiment, the leguminous plant is selected from mung beans, broad beans, peas, lentils, soybeans, kidney beans, cowpeas or green beans;
in an alternative embodiment, the plant is maize, wheat, rice or canola.
In a seventh aspect, the present invention also provides a method of producing a glufosinate herbicide tolerant plant comprising introducing into the genome of a plant of interest a gene encoding a plant glutamine synthetase mutant as described above.
In an alternative embodiment, the method of introduction is selected from the group consisting of genetic transformation methods, genome editing methods, or genetic mutation methods.
The above genetic transformation methods include, but are not limited to: individuals with glufosinate resistance are produced by selfing or crossing parent plants with genes of glufosinate resistant plant glutamine synthetase mutants with other plant individuals.
In other embodiments, the methods of transformation described above include, but are not limited to, agrobacterium-mediated gene transformation, gene gun transformation, and pollen tube channel.
In a preferred embodiment of the present invention, the plant is selected from wheat, rice, barley, oat, corn, sorghum, millet, buckwheat, millet, sweet potato, cotton, canola, sesame, peanut, sunflower, radish, carrot, broccoli, tomato, eggplant, capsicum, leek, onion, leek, spinach, celery, amaranth, lettuce, crowndaisy chrysanthemum, grape, strawberry, sugarcane, tobacco, brassica vegetables, cucurbitaceae, leguminous plants, pasture, tea or cassava;
in an alternative embodiment, the pasture is selected from gramineous pasture or leguminous pasture;
in an alternative embodiment, the brassica vegetable is selected from turnip, cabbage, mustard, cabbage mustard, canola, mustard, blue, canola, green vegetables, or beet;
in an alternative embodiment, the cucurbitaceae plant is selected from cucumber, pumpkin, wax gourd, bitter gourd, luffa, melon, watermelon or melon;
in an alternative embodiment, the leguminous plant is selected from mung beans, broad beans, peas, lentils, soybeans, kidney beans, cowpeas or green beans;
In an alternative embodiment, the plant is maize, wheat, rice or canola.
The invention has the following beneficial effects:
the method for obtaining the protein with the glufosinate resistance is simple and feasible, has high success rate, and is convenient for people to obtain the protein with the glufosinate resistance quickly and efficiently.
The glutamine synthetase mutant provided by the invention is originally derived from plants, has glufosinate resistance after mutation, and has good biological enzyme catalytic activity. Plants transformed with the glutamine synthetase mutant have glufosinate resistance and can grow and develop normally.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the amino acid sequence part alignment of rice GS1 mutant O294C, O294D, O E, O294F, O H, O294K, O N, O P, O294Q, O294R, O S, O294V, O W, O Y and O294X and wild-type rice GS1 OsGS1_WT provided in example 1;
FIG. 2 shows the results of partial alignment of the amino acid sequences of maize GS1 mutant Z294C, Z E, Z, 294H, Z, I, Z294K, Z, L, Z, M, Z, 294N, Z, P, Z, R, Z, W, Z Y and Z294X provided in example 2 with wild-type maize GS1 ZmGS1_WT;
FIG. 3 is a partial alignment of amino acid sequences of wheat GS1 mutant T294A, T C, T294D, T N and wild-type wheat GS1TaGS1_WT provided in example 2 of the present invention;
FIG. 4 shows the result of the partial alignment of amino acid sequences of the wild type canola GS1 mutant B294A, B294C, B38294H, B294K, B294M, B294N, B Q, B R, B T, B W and wild type canola GS1 BnGS1_WT provided in example 2 of the present invention;
FIG. 5 is a schematic diagram of the structure of pADV7 vector provided in Experimental example 1 of the present invention;
FIG. 6 shows the results of growth of the rice GS1 mutant O294C, O294D, O294E, O294F, O H, O294K, O N, O P, O Q, O294R, O S, O294V, O W, O Y and O294X of the rice GS1 OsGS1_WT of the present invention on a medium containing glufosinate at different concentrations, as provided in Experimental example 1;
FIG. 7 shows the results of E.coli growth on medium containing varying concentrations of glufosinate for corn GS1 mutant Z294C, Z294E, Z294H, Z294I, Z294K, Z L, Z M, Z294N, Z P, Z R, Z294W, Z Y and Z294X and wild-type corn GS1 ZmGS1_WT provided in Experimental example 2;
FIG. 8 shows the results of E.coli growth on medium containing glufosinate at different concentrations of wheat GS1 mutant T294A, T294C, T294D, T N and wild-type wheat GS1 TaGS1_WT provided in Experimental example 3 of the present invention;
FIG. 9 shows the results of E.coli growth on medium containing glufosinate of different concentrations of wild-type canola GS1 mutant B294A, B294C, B294E, B H, B294 6783K, B294M, B N, B294Q, B294R, B T, B W provided by transformation example 4 provided by Experimental example 4;
FIG. 10 shows the enzyme kinetic parameters and glufosinate resistance parameters IC of the rice GS1 mutant O294C, the corn GS1 mutant Z294C, the wheat GS1 mutant T294C, the rape GS1 mutant B294C, the wild-type rice GS1 OsGS1_WT, the wild-type corn GS1 ZmGS1_WT, the wild-type wheat GS1 TaGS1_WT and the wild-type rape GS1 BnGS1_WT provided in Experimental example 5 of the present invention 50 ;
FIG. 11 shows amino acid sequence alignment of wild type glutamine synthetase from different plants; in the figure: osGS1_WT: wild type rice glutamine synthetase; zmgs1_wt: corn wild type glutamine synthetase; taGS1_WT: wheat wild type glutamine synthetase; bnGS1_WT: wild type rape glutamine synthetase.
Detailed Description
Reference now will be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. Indeed, it will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment can be used on another embodiment to yield still a further embodiment.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the assays herein, some methods and materials are now described, unless otherwise indicated, the techniques employed or contemplated herein are standard methods. The materials, methods, and examples are illustrative only and not intended to be limiting.
Unless otherwise indicated, practice of the present invention will employ conventional techniques of plant physiology, plant molecular genetics, cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry and immunology, which are within the ability of one skilled in the art. This technique is well explained in the literature, as is the case for molecular cloning: laboratory Manual (Molecular Cloning: A Laboratory Manual), second edition (Sambrook et al, 1989); oligonucleotide Synthesis (Oligonucleotide Synthesis) (M.J.Gait et al, 1984); plant physiology (pallidum et al, 2017); the methods are described in the following examples (methods of enzymology) (Methods in Enzymology) (Academic Press, inc.), experimental immunology handbook (Handbook of Experimental Immunology) (D.M. Weir and C.C. Blackwell, inc.), contemporary molecular biology methods (Current Protocols in Molecular Biology) (F.M. Ausubel et al, 1987), plant molecular genetics (Monica A. Hughes et al), PCR: polymerase chain reaction (PCR: the Polymerase Chain Reaction) (Mullis et al, 1994), each of which is expressly incorporated herein by reference.
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The features and capabilities of the present invention are described in further detail below in connection with the examples.
Example 1
The rice (Oryza sativa) glutamine synthetase (GS 1) mutant provided in this example is obtained by mutating or deleting the 294-th amino acid residue G of a wild-type rice glutamine synthetase itself (named OsGS 1-WT, the amino acid sequence of which is shown in SEQ ID NO.1, and the encoding nucleotide sequence of which is shown in SEQ ID NO. 5) to C, D, E, F, H, K, N, P, Q, R, S, V, W, Y, and the obtained rice GS1 mutants are named O294C, O294D, O E, O294F, O294H, O K, O294N, O294P, O294Q, O294 97294R, O S, O294V, O W, O Y and O294X, respectively.
The amino acid sequences of the rice GS1 mutant O294C, O, D, O, 294E, O, 294F, O, 294H, O, K, O, 294N, O, P, O, 294Q, O, 294R, O, S, O, V, O, 294W, O Y and O294X are aligned with those of the wild-type rice GS1 as shown in fig. 1, in which: the position indicated by the arrow is the mutation site.
In this example, the coding sequence of each rice GS1 mutant was at the position encoding amino acid 294, the codons for the corresponding amino acids were as shown in the following Table, and the nucleotides at the remaining positions were identical to the corresponding wild-type coding sequence.
Amino acids | C | D | E | F | H |
Codons | TGC | GAT | GAG | TTC | CAC |
Amino acids | K | N | P | Q | R |
Codons | AAG | AAC | CCC | CAG | CGC |
Amino acids | S | V | W | Y | Deletion of |
Codons | TCC | GTC | TGG | TAC | Without any means for |
The rice GS1 mutant O294C, O294D, O294E, O294F, O294H, O294K, O294N, O294P, O294Q, O294S, O V, O294W, O294Y and O294X and the nucleic acid molecules encoding them provided in this example can be obtained by chemical synthesis.
Example 2
The corn (Zea mays) GS1 mutant provided in this example is obtained by mutating or deleting the 294 (corresponding to 294 of the reference sequence (SEQ ID No. 1)) of the wild-type corn GS1 itself (named zmgs1_wt, amino acid sequence shown in SEQ ID No.2, encoding nucleotide sequence of SEQ ID No. 6) from the amino acid residue G to C, E, H, I, K, L, M, N, P, R, W, Y. The resulting maize GS1 mutants were named Z294C, Z294E, Z H, Z I, Z294 38294K, Z L, Z294M, Z294N, Z P, Z294R, Z W, Z Y and Z294X, respectively.
The amino acid sequence alignment of maize GS1 mutant Z294C, Z E, Z H, Z294I, Z294K, Z294L, Z M, Z294N, Z P, Z294R, Z W, Z Y and Z294X with wild-type maize GS1 is shown in figure 2, in which: the position indicated by the arrow is the mutation site.
In this example, the coding sequence of each maize GS1 mutant was at the position encoding amino acid 294, the codons for the corresponding amino acids were as shown in the following Table, and the nucleotides at the remaining positions were identical to the corresponding wild-type coding sequence.
The maize GS1 mutant Z294C, Z, E, Z, H, Z, I, Z, K, Z, L, Z, M, Z, 294N, Z, P, Z, R, Z, 294W, Z Y and Z294X provided in this example, and nucleic acid molecules encoding them, can be obtained by chemical synthesis.
Example 3
The wheat (Triticum aestivum) GS1 mutant provided in this example was obtained by mutating the 294 (corresponding to 294 of the reference sequence (SEQ ID NO. 1)) of the wild-type wheat GS1 itself (designated TaGS 1-WT, the amino acid sequence of which is shown in SEQ ID NO.3, and the coding nucleotide sequence of which is SEQ ID NO. 7) from the amino acid residue G to A, C, D, N. The resulting wheat GS1 mutants were designated T294A, T294C, T294D, T N, respectively.
The amino acid sequence alignment of wheat GS1 mutant T294A, T294C, T D, T294N and wild-type wheat GS1 is shown in fig. 3, where: the position indicated by the arrow is the mutation site.
In this example, the coding sequence of each wheat GS1 mutant was at the position encoding amino acid 294, the codons for the corresponding amino acids were as shown in the following table, and the nucleotides at the remaining positions were identical to the corresponding wild-type coding sequence.
Amino acids | A | C | D | N |
Codons | GCT | TGC | GAT | AAC |
The wheat GS1 mutant T294A, T294C, T294D, T N and the nucleic acid molecules encoding them provided in this example can be obtained by chemical synthesis.
Example 4
The mutant of canola (Brassica napus) GS1 provided in this example is obtained by mutating the 294 (corresponding to 294 of the reference sequence (SEQ ID No. 1)) of the wild-type canola GS1 itself (named bngs1_wt, having the amino acid sequence shown in SEQ ID No.5, and having the encoding nucleotide sequence of SEQ ID No. 8) from the amino acid residue G to A, C, E, H, K, M, N, Q, R, T, W. The resulting canola GS1 mutants were designated B294A, B294C, B294E, B H, B294K, B294M, B294N, B294Q, B294R, B294T, B W, respectively.
The amino acid sequence alignment of canola GS1 mutant B294A, B C, B294E, B H, B294K, B294M, B N, B294Q, B294R, B294T, B W and wild-type canola GS1 is shown in fig. 4, in which: the position indicated by the arrow is the mutation site.
In this example, the coding sequence of each canola GS1 mutant is at the position encoding amino acid 294, the codons for the corresponding amino acids are shown in the following table, and the nucleotides at the remaining positions are identical to the corresponding wild-type coding sequence.
The rape GS1 mutant B294A, B294C, B E, B294H, B294K, B294M, B294N, B294Q, B294R, B294T, B W and the nucleic acid molecules encoding them provided in this example can be obtained by chemical synthesis.
Experimental example 1
This experimental example separately detects glufosinate resistance of rice GS1 mutants O294C, O294D, O E, O294F, O H, O294K, O N, O P, O294Q, O294R, O S, O294V, O W, O Y and O294X provided in example 1 as follows:
according to the sequence of the nucleic acid molecule provided in example 1, coding genes encoding rice GS1 mutant O294C, O294D, O294E, O294F, O H, O294K, O294N, O294P, O294Q, O294R, O294S, O823 294W, O Y and O294X were synthesized by chemical synthesis, enzyme cleavage sites (Pac 1 and Sbf 1) were introduced into both ends, and after enzyme cleavage, the resulting mixture was ligated to an expression vector (e.g., pADV7 vector, the structure of which was shown in FIG. 5) subjected to the same enzyme cleavage treatment, and then glutamine synthetase-deficient E.coli, which was obtained by knocking out glutamine synthetase based on E.coli DH 5. Alpha. By Sichuan Yuxing straw biotechnology Co. After verification, positive clones are selected, inoculated to M9 culture medium containing glufosinate with different concentrations for growth, and defective E.coli growth is observed. The wild-type rice GS1 mutant was used as a negative control, and glufosinate resistance was examined with the GS1 mutant O294C (G294C, amino acid G mutation at position 294 of rice GS1 to C), O294D (G294D), O294E (G294E), O294F (G294F), O294H (G294H), O294K (O294K), O294N (G294N), O294P (G294P), O294Q (G294Q), O294R (G294R), O294S (G294S), O294V (G294V), O294W (G294W), O294Y (G294Y), and O294X (G294X). The results are shown in FIG. 6.
On a medium containing 0mM glufosinate (KP 0), the defective strains encoding the wild-type rice GS1 (OsGs1_WT) and the rice GS1 mutant O294C, O D, O294E, O294F, O H, O294K, O294N, O P, O294Q, O294R, O294S, O294V, O294W, O294Y and O294X were transformed to grow normally, indicating that GS1 encoded by O294C, O294D, O294E, O F, O294K, O294N, O294P, O294Q, O294Q, O Y and O294X both have normal GS1 enzyme activity;
coli transformed with wild-type rice GS1 could not grow on a medium containing 10mM glufosinate (KP 10), but E.coli transformed with rice mutant O294C, O D, O294E, O294F, O H, O294K, O294N, O P, O294Q, O294R, O294S, O294V, O294W, O Y and O294X showed significantly better growth than the negative control, it was demonstrated that the single mutant containing O294C, O294D, O294E, O F, O294K, O294N, O294P, O294Q, O294Q, O294Q, O Y and O294X was significantly better against glufosinate than the wild type.
These results demonstrate that the single mutants of O294C, O294D, O E, O294F, O H, O294K, O N, O P, O294Q, O294R, O S, O294V, O294W, O Y and O294X both have good resistance to glufosinate.
Experimental example 2
Referring to the test method of experimental example 1, the corn GS1 mutant provided in example 2 was tested for glufosinate tolerance.
Specifically, glufosinate resistance was examined for Z294C (G294C, mutation of amino acid G at position 294 of maize GS1 to C), Z294E (G294E), Z294H (G294H), Z294I (G294I), Z294K (G294K), Z294L (G294L), Z294M (G294M), Z294N (G294N), Z294P (G294P), Z294R (G294R), Z294W (G294W), Z294Y (G294Y), and Z294X (G294X), respectively. The results are shown in FIG. 7.
As can be seen from the results of fig. 7:
transformation of a defective strain encoding the wild-type maize GS1 (zmgs1_wt) and maize GS1 mutant Z294C, Z E, Z294H, Z294I, Z294L, Z M, Z K, Z294L, Z M, Z N, Z294P, Z294R, Z294W, Z Y and Z294X both grown normally on medium containing 0mM glufosinate (KP 0), indicating that the GS1 encoded by Z294C, Z294E, Z H, Z294I, Z294K, Z294L, Z M, Z294N, Z294P, Z R, Z294W, Z Y and Z294X both had normal GS1 enzyme activity;
coli transformed with wild-type maize GS1 was essentially incapable of growth on 2mM glufosinate (KP 2), but E.coli transformed with maize mutant Z294C, Z294E, Z294H, Z294K, Z294L, Z294M, Z294N, Z294P, Z294R, Z W, Z Y and Z294X grew significantly better than the negative control, it was demonstrated that the single mutant containing Z294C, Z294E, Z H, Z294I, Z294K, Z294L, Z294M, Z294N, Z P, Z294R, Z294W, Z Y and Z294X was significantly better against glufosinate than the wild type. Coli transformed with maize GS1 mutant Z294C, Z294E, Z294H, Z294I, Z294K, Z294L, Z M, Z294N, Z294P, Z294R, Z W, Z Y and Z294X all grew significantly on higher glufosinate concentrations (10 mm, kp10) medium.
These results demonstrate that the single mutants of Z294C, Z294E, Z294H, Z I, Z294K, Z L, Z294M, Z294N, Z P, Z294R, Z294W, Z Y and Z294X provided in example 2 have resistance to glufosinate.
Experimental example 3
Referring to the test method of experimental example 1, glufosinate resistance of wheat GS1 mutants T294A (G294A, amino acid G at position 294 of wheat GS1 was mutated to a), T294C (G294C), T294D (G294D), and T294N (G294N) provided in example 3 was tested. The results are shown in FIG. 8.
As can be seen from the results of fig. 8:
transformation of defective strains encoding the wild type wheat GS1 (tags1_wt) and the wheat GS1 mutant T294A, T294C, T D, T294N, both grown normally, on a medium containing 0mM glufosinate (KP 0), indicating that the GS1 encoded by T294A, T294C, T294D, T N has normal GS1 enzyme activity;
coli transformed with wild-type wheat GS1 was essentially incapable of growth on medium containing 10mM glufosinate (KP 10), but the growth of the e.coli transformed with wheat mutant T294A, T294C, T D, T N was significantly better than the negative control, indicating that the single mutant containing T294A, T294C, T294D, T N was significantly better than the wild-type.
These results demonstrate that the single mutants of T294A, T294C, T294D, T N all have resistance to glufosinate.
Experimental example 4
Referring to the test method of experimental example 1, glufosinate resistance of canola GS1 mutant B294A (G294A, amino acid G at position 294 of canola GS1 was mutated to a), B294C (G294C), B294E (G294E), B294H (G294H), B294K (G294K), B294M (G294M), B294N (G294N), B294Q (G294Q), B294R (G294R), B294T (G294T), B294W (G294W) provided in example 4 was tested. The results are shown in FIG. 9.
As can be seen from the results of fig. 9:
on a medium containing 0mM glufosinate (KP 0), defective strains encoding wild type canola GS1 (BnGS 1-WT) and canola GS1 mutant B294A, B294C, B294E, B294H, B K, B294M, B294 4639 294R, B294T, B W were transformed to grow normally, shows that the GS1 encoded by B294A, B C, B294 38394H, B294K, B294M, B Q, B294R, B294T, B W has normal GS1 enzyme activity;
coli transformed with wild-type canola GS1 was substantially incapable of growth on 2mM glufosinate (KP 2), but E.coli transformed with canola mutant B294A, B294C, B294E, B294H, B K, B294M, B294N, B294Q, B294R, B294T, B W grew significantly better than the negative control, it was demonstrated that the single mutant containing B294A, B C, B294 38394H, B294K, B294M, B Q, B294R, B294T, B W was significantly better in its ability to resist glufosinate than the wild type.
These results demonstrate that the single mutants of B294A, B294C, B E, B294H, B294K, B294M, B294N, B294Q, B294R, B294T, B W all have resistance to glufosinate.
Experimental example 5
The enzyme kinetic parameters of the O294C provided in example 1, the Z294C provided in example 2, the T294C provided in example 3, the B294C mutant provided in example 4 and the enzyme kinetic parameters in the presence of glufosinate were tested against wild-type rice gs1 osgs1_wt, wild-type maize gs1zmgs1_wt, wild-type wheat gs1 tags1_wt and wild-type canola gs1 bngs1_wt as follows:
1. and (3) constructing a carrier:
the nucleic acid sequence encoding the mutant is cloned into a prokaryotic expression vector pET32a, and the cloning is verified by sequencing.
2.6His protein purification:
the mutant enzyme protein was purified by 6His and the concentration was determined using the Bradford protein concentration determination kit using standard methods and the protein was stored in a protein stock solution.
3. Enzyme activity determination:
(1) Instrument and reagents: enzyme-labeled instrument (De-Fe: HBS-1096A), glufosinate, substrate L-sodium glutamate (CAS: 6106-04-3).
(2) Principle of measurement
Glutamine Synthetase (GS) is the first enzyme to convert inorganic nitrogen to organic nitrogen, catalyzes the binding of ammonia from different sources to glutamate to form glutamine, and liberates Pi, as shown in the following specific reaction scheme:
The invention adopts a phosphomolybdic blue method to measure GS enzyme activity, and the principle of the method is as follows: under suitable acidic conditions, phosphoric acid (Pi) reacts with ammonium molybdate to form ammonium phosphomolybdate, which in turn is reduced by reducing agents (e.g., vitamin C, stannous chloride, etc.) to blue phosphomolybdenum blue, the blue shade of which is proportional to the phosphorus content.
(3) The operation steps are as follows:
the reaction was carried out in a Tris-HCl buffer system at pH7.5 in a total reaction volume of 30uL. The glutamine synthetase enzyme activity determination reaction liquid comprises the following components: 200mM Tris-HCl (pH 7.5), 1.67mM ATP,30mM ammonium chloride, 20mM MgCl 2 10mM sodium L-glutamate. After 30ul of reaction solution is uniformly mixed, preheating is carried out for 5min at 35 ℃, mutant protein solution is added for reaction, after reaction is carried out for 30min at 35 ℃, 100 μl of color reaction D solution [ D=2A+B ] is added; and (3) solution A: 12% (W/V) ascorbic acid in 1mol/L hydrochloric acid solution, solution B: 2% (W/V) aqueous solution of ammonium molybdate tetrahydrate]Color was developed, left to stand for 5 minutes, and 100. Mu.l of a reaction termination F solution (2% sodium citrate, 2% acetic acid aqueous solution) was added thereto, and left to stand for 15 minutes, 200. Mu.l of the light absorption value was measured at 660 nm.
The results are shown in fig. 10, and can be seen from the results of fig. 10:
the Km values of the GS1 mutants were slightly higher than those of the wild-type controls osgs1_wt, zmgs1_wt, tags1_wt and bngs1_wt, indicating that the GS mutants reduced sensitivity to glufosinate inhibitors while slightly reduced sensitivity to normal substrates. V of GS1 mutant max All were higher than the wild type control, indicating that these mutants had improved enzymatic ability.
Wild type controls were sensitive to glufosinate, IC 50 IC of mutants at 0.006mM, 0.002mM, 0.007mM, respectively 50 The IC's of O294C, Z C, T C and B294C are all significantly higher than the wild-type control 50 Far higher than the wild-type control, indicating that the mutant is less sensitive to glufosinate. From mutant IC 50 And wild type IC 50 It can also be seen in the multiple relationship of O294C, Z, 294C, T C and B294C 50 Corresponding to wild type GS1 IC 50 These values also indicate that the enzyme activity of the mutants is much higher than that of the wild-type control, 94.667, 2941.667, 169, 8.143 times. These data illustrate the mechanism of mutant resistance to glufosinate by enzyme kinetics.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A method for obtaining a protein having glufosinate resistance comprising the steps of:
1) A protein having the reference sequence shown in SEQ ID NO.1 or having an amino acid sequence having at least 85% identity to the reference sequence as a target protein;
2) Comparing the amino acid sequence of the target protein with the reference sequence, and mutating or deleting the amino acid residue of the target protein corresponding to 294 th amino acid residue of the reference sequence;
3) Selecting a protein with enhanced glufosinate resistance;
preferably, when the target protein is derived from rice, the target protein is mutated to C, D, E, F, H, K, N, P, Q, R, S, V, W, Y or deleted corresponding to the 294 amino acid residue of the reference sequence;
preferably, when the target protein is derived from corn, the target protein is mutated to C, E, H, I, K, L, M, N, P, R, W, Y or deleted corresponding to amino acid residue 294 of the reference sequence;
preferably, when the target protein is derived from wheat, the target protein is mutated to A, C, D or N corresponding to amino acid residue 294 of the reference sequence;
preferably, when the target protein is derived from rape, the amino acid residue at position 294 of the target protein corresponding to the reference sequence is mutated to A, C, E, H, K, M, N, Q, R, T or W.
2. A plant glutamine synthetase mutant having glufosinate resistance, wherein said plant glutamine synthetase mutant is represented by (1) or (2):
(1): it is obtained by mutating the n-th position of wild glutamine synthetase from plant; the position of the nth bit is determined as follows: the wild type glutamine synthetase is aligned with a reference sequence, and the nth position of the wild type glutamine synthetase corresponds to the 294 th position of the reference sequence, wherein the amino acid sequence of the reference sequence is shown as SEQ ID NO. 1;
the n-th amino acid of the plant glutamine synthetase mutant is A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y or deleted compared with the wild type glutamine synthetase;
(2): which has at least 85% identity with the plant glutamine synthetase mutant shown in (1) and is identical to the amino acid at the n-th position of the plant glutamine synthetase mutant shown in (1).
3. The plant glutamine synthetase mutant according to claim 2, wherein the amino acid at the N-th position of said plant glutamine synthetase mutant is C or N.
4. The plant glutamine synthetase mutant of claim 2, wherein said plant is selected from the group consisting of wheat, rice, barley, oat, corn, sorghum, millet, buckwheat, millet, sweet potato, cotton, canola, sesame, peanut, sunflower, radish, carrot, broccoli, tomato, eggplant, capsicum, leek, welsh onion, leek, spinach, celery, amaranth, lettuce, crowndaisy, day lily, grape, strawberry, sugarcane, tobacco, brassica vegetable, cucurbitaceae, leguminous plant, pasture, tea, or cassava;
preferably, the pasture is selected from gramineous pasture or leguminous pasture;
preferably, the brassica vegetable is selected from turnip, cabbage, mustard, cabbage mustard, blue, canola, green vegetables or beet;
preferably, the cucurbitaceae plant is selected from cucumber, pumpkin, white gourd, balsam pear, luffa, melon, watermelon or melon;
preferably, the leguminous plant is selected from mung beans, broad beans, peas, lentils, soybeans, kidney beans, cowpeas or green beans;
preferably, when the plant is rice, the n-th amino acid of the plant glutamine synthetase mutant is C, D, E, F, H, K, N, P, Q, R, S, V, W, Y or deleted;
Preferably, when the plant is maize, the amino acid at the n-th position of the plant glutamine synthetase mutant is C, E, H, I, K, L, M, N, P, R, W, Y or deleted;
preferably, when the plant is wheat, the N-th amino acid of the plant glutamine synthetase mutant is A, C, D or N;
preferably, when the plant is canola, the n-th amino acid of the plant glutamine synthetase mutant is A, C, E, H, K, M, N, Q, R, T or W.
5. An isolated nucleic acid molecule encoding a plant glutamine synthetase mutant according to any one of claims 2 to 4.
6. A vector comprising the nucleic acid molecule of claim 5.
7. A recombinant bacterium or recombinant cell comprising the nucleic acid molecule of claim 5 or the vector of claim 6.
8. Use of a plant glutamine synthetase mutant of any one of claims 2-4, a nucleic acid molecule of claim 5, a vector of claim 6 or a recombinant bacterium or recombinant cell of claim 7 for breeding a plant variety having glufosinate resistance;
preferably, the application comprises any one of the following uses:
(1) Transforming a plant of interest with a vector comprising a gene encoding a mutant of said plant glutamine synthetase;
(2) Modifying an endogenous glutamine synthetase gene of a target plant by a gene editing method to code the plant glutamine synthetase mutant;
(3) Mutagenizing and screening plant cells, tissues, individuals or populations to encode said plant glutamine synthetase mutants;
preferably, the gene editing is selected from CRISPR/Cas9, TALEN technology or ZFN technology;
preferably, the plant is selected from wheat, rice, barley, oat, corn, sorghum, millet, buckwheat, millet, sweet potato, cotton, canola, sesame, peanut, sunflower, radish, carrot, broccoli, tomato, eggplant, capsicum, leek, onion, leek, spinach, celery, amaranth, lettuce, crowndaisy chrysanthemum, grape, strawberry, sugarcane, tobacco, brassica vegetable, cucurbitaceae, leguminous plant, pasture, tea or cassava;
preferably, the pasture is selected from gramineous pasture or leguminous pasture;
preferably, the brassica vegetable is selected from turnip, cabbage, mustard, cabbage mustard, blue, canola, green vegetables or beet;
Preferably, the cucurbitaceae plant is selected from cucumber, pumpkin, white gourd, balsam pear, luffa, melon, watermelon or melon;
preferably, the leguminous plant is selected from mung beans, broad beans, peas, lentils, soybeans, kidney beans, cowpeas or green beans;
preferably, the plant is maize, wheat, rice or canola.
9. A method of producing a glufosinate herbicide tolerant plant comprising introducing into the genome of a plant of interest a gene encoding a plant glutamine synthetase mutant of any one of claims 2-4.
10. The method of producing a glufosinate herbicide-tolerant plant of claim 9, wherein the plant is selected from the group consisting of wheat, rice, barley, oat, corn, sorghum, millet, buckwheat, millet, sweet potato, cotton, canola, sesame, peanut, sunflower, radish, carrot, broccoli, tomato, eggplant, capsicum, leek, onion, leek, spinach, celery, amaranth, lettuce, garland chrysanthemum, daylily, grape, strawberry, sugarcane, tobacco, brassica vegetables, cucurbitaceae, leguminous, pasture, tea, or cassava;
Preferably, the pasture is selected from gramineous pasture or leguminous pasture;
preferably, the brassica vegetable is selected from turnip, cabbage, mustard, cabbage mustard, blue, canola, green vegetables or beet;
preferably, the cucurbitaceae plant is selected from cucumber, pumpkin, white gourd, balsam pear, luffa, melon, watermelon or melon;
preferably, the leguminous plant is selected from mung beans, broad beans, peas, lentils, soybeans, kidney beans, cowpeas or green beans;
preferably, the plant is maize, wheat, rice or canola.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310883130.3A CN116769739A (en) | 2023-07-17 | 2023-07-17 | G294 mutation-containing plant glutamine synthetase mutant and encoding gene and application thereof |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310883130.3A CN116769739A (en) | 2023-07-17 | 2023-07-17 | G294 mutation-containing plant glutamine synthetase mutant and encoding gene and application thereof |
Publications (1)
Publication Number | Publication Date |
---|---|
CN116769739A true CN116769739A (en) | 2023-09-19 |
Family
ID=88013375
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202310883130.3A Pending CN116769739A (en) | 2023-07-17 | 2023-07-17 | G294 mutation-containing plant glutamine synthetase mutant and encoding gene and application thereof |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN116769739A (en) |
-
2023
- 2023-07-17 CN CN202310883130.3A patent/CN116769739A/en active Pending
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN112574967B (en) | Glutamine synthetase mutant with glufosinate resistance of plant origin, nucleic acid molecule and application | |
CN113604443B (en) | Glutamine synthetase mutant and application thereof in cultivation of glufosinate-resistant plant variety | |
CN107828794A (en) | A kind of method for creating of Rice Salt gene OsRR22 mutant, its amino acid sequence encoded, plant and the mutant | |
CN102282262B (en) | Auxotrophic agrobacterium for plant transformation and methods thereof | |
CN107964543A (en) | Rice herbicide resistance ALS muteins, nucleic acid and its application | |
Melino et al. | RNA catabolites contribute to the nitrogen pool and support growth recovery of wheat | |
WO2023040565A1 (en) | Glutamine synthetase mutant having glufosinate-ammonium resistance, nucleic acid molecule and use | |
Valenzuela et al. | Streptomycin resistance in Clavibacter michiganensis subsp. michiganensis strains from Chile is related to an rpsL gene mutation | |
CN109825638A (en) | A kind of Rice Salt gene OsRR22 guidance primer, application and target spot carrier and target spot support preparation method | |
CN109912702A (en) | Application of the protein OsARE1 in regulation plant low nitrogen resisting | |
WO2023207669A1 (en) | Method for acquiring protein with glufosinate resistance and glutamine synthetase mutant | |
WO2023071438A1 (en) | Glutamine synthetase mutant and application | |
WO2023087812A1 (en) | Glutamine synthetase mutant having glufosinate-ammonium resistance and application thereof | |
Csonka et al. | Biosynthesis of proline | |
CN116769739A (en) | G294 mutation-containing plant glutamine synthetase mutant and encoding gene and application thereof | |
CN112437811B (en) | Recombinant NAD synthetase, gene and application thereof | |
CN117363633A (en) | Nucleic acid molecule, vector, recombinant bacterium, GS mutant and application thereof | |
CN116875568A (en) | G61 mutation-containing plant glutamine synthetase mutant and encoding gene and application thereof | |
Bobo et al. | Identification and characterization of a soybean protein with adenylyl cyclase activity. | |
CN114807064B (en) | Method for obtaining protein with glufosinate resistance and mutant thereof | |
Onishchuk et al. | Identification of new genes of nodule bacteria Sinorhizobium meliloti involved in the control of efficiency of symbiosis with alfalfa Medicago sativa | |
Yesland et al. | Anticodon bases C34 and C35 are major, positive, identity elements in Saccharomyces cerevisiae tRNA Trp | |
Chompoo et al. | RT-PCR based detection of resistance conferred by an insensitive GS in glufosinate-resistant maize cell lines | |
Xie et al. | No Significant Impact of Transgenic Cry1Ab/1Ac Cotton on Rhizosphere‐Soil Enzyme Activities and Bacterial Communities | |
Gao et al. | Characterization of novel nitrate reductase-deficient mutants for transgenic Dunaliella salina systems |
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 |