WO2017177765A1 - Applications de gène de type mutant d'als dans la résistance aux herbicides - Google Patents

Applications de gène de type mutant d'als dans la résistance aux herbicides Download PDF

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WO2017177765A1
WO2017177765A1 PCT/CN2017/074079 CN2017074079W WO2017177765A1 WO 2017177765 A1 WO2017177765 A1 WO 2017177765A1 CN 2017074079 W CN2017074079 W CN 2017074079W WO 2017177765 A1 WO2017177765 A1 WO 2017177765A1
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als
mutant
rice
nucleotide
gene
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张保龙
陈天子
凌溪铁
王金彦
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江苏省农业科学院
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8274—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for herbicide resistance
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    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A01N47/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid
    • A01N47/40—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having a double or triple bond to nitrogen, e.g. cyanates, cyanamides
    • A01N47/42—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having a double or triple bond to nitrogen, e.g. cyanates, cyanamides containing —N=CX2 groups, e.g. isothiourea
    • A01N47/44—Guanidine; Derivatives thereof
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae

Definitions

  • the invention belongs to the field of vegetable proteins and plant herbicides.
  • the present invention relates to a rice acetolactate synthase (ALS) mutein which confers properties to plants, particularly rice anti-acetolactate synthase inhibitor herbicides.
  • ALS rice acetolactate synthase
  • the present invention discloses the sequences of the proteins and their use in the field of plant herbicide resistance.
  • ALS Inflammatory lactic acid synthase
  • AHAS acetohydroxyacid synthase
  • AHAS acetohydroxyacid synthase
  • EC 4.1.3.18 Inhibitor herbicides use ALS as a target to cause weed death, mainly including Sulfonylureas (SU), Imidazolinones (IMI), Triazolopyrimidines (TP), Pyrimidinylthio(or oxy)-benzoates, PTB; pyrimidinyl-carboxyherbicides; PCs and sulfonamide carbonyls 13 compounds such as Sulfonylamino-carbonyltriazolinones (SCT).
  • Sulfonylureas SU
  • IMI Imidazolinones
  • TP Triazolopyrimidines
  • TP Pyrimidinylthio(or oxy)-benzoates
  • PTB Pyrimidinylthio(or oxy
  • ALS inhibitor herbicides have the characteristics of strong selectivity, wide herbicidal spectrum, low toxicity and high efficiency, and have been widely used in large areas. However, these herbicides also cause phytotoxicity to crops which generally do not have the resistance to herbicides, which greatly limits their use time and space for use. For example, it is necessary to use herbicides for a period of time before crops are planted to avoid crops suffering from medicines. harm. Breeding resistant (resistance) herbicide crop varieties can reduce crop phytotoxicity and broaden the use of herbicides.
  • the mature ALS protein consists of approximately 670 amino acids and its sequence is highly conserved across species. ALS protein in Gly 95, Ala 96, Ala 122, Pro 171, Pro 196, Pro 197, Ala 205, Asp 376, Trp 537, Trp 548, Trp 552, Trp 557, Trp 563, Trp 574, Ser 621, Ser 627 Mutations in amino acid positions such as Ser 638, Ser 653, Gly 654, and Val 669 (calculated as the ALS amino acid position of the model plant Arabidopsis thaliana) can produce ALS inhibitor herbicide resistance, which is found in a variety of crops (including corn). , wheat, rice, rape, sunflower, etc.), model plants Arabidopsis and hundreds of weeds have been reported.
  • known anti-ALS inhibitor mutation sites in rice include Gly 95, Ala 96, Ala 122, Trp 548, Ser 627, Ser 653 and Gly 654.
  • the level of ALS mutant herbicide resistance is related to the location of the ALS amino acid mutation and also to the number of amino acids after mutation and the number of mutant amino acids.
  • the first technical problem to be solved by the present invention is to provide an ALS mutant gene.
  • the technical problem to be solved by the present invention is to provide the ALS protein encoded by the above ALS mutant gene.
  • the technical problem to be solved by the present invention is to provide an expression cassette, a recombinant vector or a cell containing the above ALS mutant gene.
  • the technical problem to be solved by the present invention is to provide the use of the above ALS mutant gene, protein, expression cassette, recombinant vector or cell for preparing a green plant herbicide.
  • a technical problem to be solved by the present invention is to provide a method of obtaining a green plant having herbicide resistance.
  • the final technical problem to be solved by the present invention is to provide a method for identifying green plants having herbicide resistance.
  • the present inventors have long-term and continuous screening of EMS mutant plants of the elite restorer line 9311, and found a series of ALS mutant proteins, including some known ALS mutant proteins and new ALS as described above. Mutant proteins that are insensitive to ALS inhibitor herbicides, thereby rendering plants resistant to ALS inhibitor herbicides.
  • the use of the present invention in plant breeding can be used to grow plants having herbicide resistance, especially crops, and the development of these proteins and their coding genes in transgenic or non-transgenic plants such as rice.
  • an ALS mutant gene which changes from G to nucleotide C and nucleotide 339 at nucleotide 75 of the rice ALS gene sequence. From A to nucleotide C and nucleotide 710 from C to nucleotide T.
  • the nucleotide sequence of the above ALS mutant gene is shown in SEQ ID No: 3.
  • the ALS protein encoded by the above ALS mutant gene is derived from rice, and the amino acid is mutated at Gln25, Gln113, Ala237 (mutant plant 2) compared to wild type rice ALS sensitive to ALS inhibitor herbicides (eg, Genbank accession number BAB20812.1). Or its nucleotide at position 75 of the ALS gene sequence in rice changes from G to nucleotide C, nucleotide 339 changes from A to nucleotide C, and the nucleotide sequence of the ALS mutant gene As shown in SEQ ID No: 1, the amino acid was mutated at the Gln25, Gln113 (mutant plant 1) site (the amino acid sequence of which is shown in SEQ ID NO. 2).
  • the above ALS protein has an amino acid sequence as shown in SEQ ID NO.
  • the present invention also encompasses an expression cassette, a recombinant vector or a cell comprising the above-described ALS mutant gene.
  • the present invention also encompasses the use of the above-described ALS mutant gene, protein, expression cassette, recombinant vector or cell in green plant herbicide resistance.
  • the above green plants are rice, tobacco, Arabidopsis, cotton, and the like.
  • the present invention also includes a method of obtaining a green plant having herbicide resistance, comprising the steps of:
  • the green plant is allowed to express the ALS protein.
  • the above methods include transgenic, hybrid, backcross or asexual propagation steps.
  • a method of identifying a green plant as claimed in the above method comprising the steps of:
  • Figure 1 shows a resistant rice mutant obtained by screening a herbicide
  • Figure 2 PCR amplification of the 5' end of the ALS gene; the first lane is Marker; Marker molecular weight from top to bottom is 5kb, 3kb, 2kb, 1kb, 750bp, 500bp, 250bp, 100bp, and the second lane is 9311 wild type rice.
  • DNA, lanes 3 to 13 are DNA of herbicide resistant mutant 1 and the other lanes are DNA of herbicide resistant mutant 2; the target fragment is 1162 bp in length;
  • Figure 3 PCR amplification of the 3' end of the ALS gene; the first lane is Marker; Marker molecular weight from top to bottom is 5kb, 3kb, 2kb, 1kb, 750bp, 500bp, 250bp, 100bp, and the second lane is 9311 wild type rice.
  • DNA, lanes 3 to 13 are DNA of herbicide resistant mutant 1 and the other lanes are DNA of herbicide resistant mutant 2; the target fragment is 1207 bp in length;
  • 4A and 4B are the inhibition of ALS enzyme activity of wild type and herbicide resistant mutant plant 1 and mutant plant 2, respectively;
  • Figure 5 is a graph showing the determination of the absorbance of the ALS enzyme activity of wild type and herbicide resistant rice mutant mutant 1 and mutant 2;
  • 6A and 6B are the inhibition of ALS activity of the sulfasulfonate herbicide on wild type and herbicide resistant rice mutant mutant plant 1 and mutant plant 2, respectively;
  • Fig. 7 Determination of the absorbance of the inhibition of ALS enzyme activity of wild type and herbicide resistant rice mutant mutant plant 1 and mutant plant 2 by the sulfasulfuron herbicide.
  • Figure 8A Mutant mutated plant 1 ALS gene overexpression vector BamHI / SacI double enzyme digestion verification map; the first lane is Marker; Marker molecular weight from top to bottom are 19329, 7743, 6223, 4254, 3472, 2690, 1882 1489, 925 bp, the second lane is the undigested recombinant vector, and the third lane is the gene fragment and the plasmid fragment DNA produced by double digestion of the recombinant vector by BamHI/SacI; the excess of the ALS gene of the mutant mutant plant 2 of Fig.
  • the expression vector BamHI/SacI double restriction enzyme verification map the first lane is the untransformed recombinant vector, the second lane is Marker, and the molecular weight is 5 kb, 3 kb, 2 kb, 1 kb, 750 bp, 500 bp, 250 bp from top to bottom.
  • 100 bp, lanes 3 and 4 are the gene fragments and plasmid fragment DNA produced by double digestion of the recombinant vector by BamHI/SacI, and the size of the gene fragment is in accordance with expectations, which proves that the vector is successfully constructed;
  • Fig. 9 PCR detection map of transgenic ALS gene rice; there are 21 lanes in total, lanes 1st, 9th lane, and 19th lane are Marker; Marker molecular weight is 5kb, 3kb, 2kb, 1kb, 750bp from top to bottom, 500 bp, 250 bp, 100 bp, lanes 8 and 18 are wild-type DNA, as a negative control, lanes 20 and 21 are plasmid DNA, and as a positive control, lanes 3 to 7 are transgenic DNA of mutant plant 1, other The lane is the transgenic DNA of mutant plant 2.
  • Indica type conventional rice 9311 seed (purchased from Jiangsuzhou agricultural germplasm resources protection and utilization platform) (this is M0, soaked in water for 2 hours) 150kg divided 6 times with 0.5-1.0% (w / w) methanesulfonic acid B
  • the ester (EMS) was immersed for 6-9 hours at room temperature, and the seeds were shaken every 1 hour; the EMS solution was discarded, the seeds were immersed in tap water for 5 times for 5 minutes, and then the seeds were washed with tap water overnight, and the next day, the field was sown.
  • carry out conventional fertilizer management (this is M1). After the plants are mature, the seeds are mixed, dried, and preserved in winter. Sowing the fields the following year.
  • the spray is 3 mL of ridges/L water ("Hundred Ridge" is produced by BASF of Germany.
  • the water-based imidazolinone herbicide is recommended to have a minimum concentration of 1 mL of ridges/1.5 to 3 L of water. After 30 days, it is also a normal green plant that is an imidazolinone herbicide-resistant rice mutant (Fig. 1). .
  • a total of 370 M2 plants with herbicide resistance were obtained. These resistant plants were managed by conventional fertilizer and water. 320 M2 plants were able to be normal. After the seeds were mature, the plants were harvested, dried, and preserved in winter.
  • mutant plant 1 and mutant plant 2 were selected from the herbicide-resistant rice mutant plants obtained in the above Example 1, and the genomic DNA was extracted and sent to Shanghai Hanyu Biotechnology Co., Ltd. for genome sequencing.
  • the sequencing results were compared with the 9311 reference genomic sequence (http://rise2.genomics.org.cn/page/rice/download.jsp), and it was found that the above herbicide-resistant rice mutants had multiple sites on the ALS gene. Mutation, in which the herbicide-resistant mutant 1 is mutated at the 75th and 339th bases of the ALS gene, and changes from G to C, and A to C, respectively, resulting in the 25th and 113th positions of the corresponding encoded amino acid sequence.
  • the nucleotide sequence of the ALS gene which changes from glutamine to histidine and glutamine to histidine, that is, the herbicide-tolerant mutant plant is shown in SEQ ID NO. 1, and encodes the amino acid sequence of the ALS protein.
  • SEQ ID NO. 2 the herbicide-resistant mutant plant 2 mutated at the 75th, 339th, and 710th bases of the ALS gene, and changed from G to C, A to C, and C to T, resulting in The 25th, 113th, and 237th positions of the corresponding encoded amino acid sequence are changed from glutamine to histidine, glutamine to histidine, alanine to proline, that is, herbicide resistant mutant plants.
  • the nucleotide sequence of the ALS gene is shown in SEQ ID NO. 3, and the amino acid sequence of the encoded ALS protein is set forth in SEQ ID NO. .
  • the mutant plant 1 (9311M1) of the present invention is classified as a herbicide-resistant mutant of the genus conventional rice 9311 (Oryza sativa indica Group cultivar 9311), which has been deposited with the Chinese microbial strain on March 30, 2016. Management Committee General Microbiology Center (CGMCC), Address: No. 3, Beichen West Road, Chaoyang District, Beijing, China Institute of Microbiology, Chinese Academy of Sciences, Zip Code: 100101, with the accession number CGMCC No.12265.
  • CGMCC Management Committee General Microbiology Center
  • the mutant plant 2 (9311M2) of the present invention is classified as a herbicide-resistant mutant of the genus conventional rice 9311 (Oryza sativa indica Group cultivar 9311), which has been deposited with the Chinese microbial strain preservation on March 30, 2016.
  • Management Committee General Microbiology Center (CGMCC) Address: No. 3, Beichen West Road, Chaoyang District, Beijing, China Institute of Microbiology, Chinese Academy of Sciences, Zip Code: 100101, with the accession number CGMCC No.12266.
  • the preserved rice seeds are in the same condition as the common rice seeds. They are suitable for high temperature, high humidity and short sunshine, and the soil requirements are not strict. They can be used in 20-32 ° C, moist soil (pH 5.5-7.5) and normal sunshine conditions.
  • the leaves of the herbicide-resistant rice mutants 9311M1 and 9311M2 were taken and genomic DNA was extracted.
  • the specific primers for amplifying the 3' end of the ALS gene according to the chromosomal sequence of the 9311 rice wild type ALS gene were: forward primer 3F5'-GGTCTTGCGTCTGGTTGGCGAGT-3', reverse primer 3R 5'-CTCTTTATGGGTCATTCAGGTCAA-3'
  • the specific primer designed to amplify the 5' end of the ALS gene is: forward primer 5F 5'-ATCCGAGCCACACATCGCCTCAC-3', reverse primer 5R5'-AGCAACAGGTCAGCCTTATCCAC-3'.
  • the sequences amplified by the two pairs of specific primers have a 230 bp overlap and can be spliced into a complete ALS gene sequence.
  • the 5' end sequence and the 3' end sequence of the ALS gene were amplified using Takara PrimerSTAR Max DNA Polymerase polymerase (purchased from Takara), and the reaction system was as follows:
  • the PCR amplification reaction procedure uses a two-step process, annealing and extension together, using 68 degrees.
  • PCR product 2 ⁇ l was detected by 1% agarose gel electrophoresis and found to have the expected size of the fragment (Fig. 2, Fig. 3).
  • the remaining PCR product was cleaned and recovered by PCR cleaning kit (purchased from Axygen) and cloned to The pMD19-T vector (purchased from Takara) was then transformed into E. coli. Each transformation randomly picked 12 E. coli monoclonals for PCR detection, and took 6 monoclonal clones positive for PCR, and sent them to Kingsray Biotechnology Co., Ltd. for sequencing to obtain the mutant ALS gene sequence.
  • the seeds harvested from 9311M1 and 9311M2 were sown out of the seedlings (this is M3).
  • M3 rice seedlings grow to the 3-4 leaf stage, spray 4 mL of ridges/L water (the recommended minimum concentration is 1 mL of ridges/3 L of water). , equivalent to 12 times the concentration).
  • the resistant seedling M3 was normal green and could continue to grow to 20-30 cm.
  • the M3 resistant strains were normal green plants, and the wild type rice sprayed with the same concentration of herbicides had all died, indicating that the mutant rice was resistant to at least 12-fold concentration of Baitailong herbicide.
  • the seeds harvested from 9311M1 and 9311M2 were sown and emerged (this is M3).
  • M3 rice seedlings grow to the 3-4 leaf stage, 4.5 g/L of sulfimsulfuron (water dispersible granules produced by Sailang Biotechnology Co., Ltd.) was sprayed.
  • the active ingredient is 75%; the recommended minimum concentration is 0.5625g/L, which is equivalent to 8 times the recommended concentration).
  • the resistant seedling M3 was normal green and could continue to grow to 20-30 cm.
  • the M3 resistant strain was a normal green plant, and the wild type rice sprayed with the same concentration of herbicide had all died, indicating that the mutant rice was at least 8-fold resistant to the sulfasulfuron herbicide.
  • the inventors performed the ALS enzyme activity assay.
  • the measurement method refer to the method of Singh et al. (Singh B. K., Stidham M. A., Shaner D. L. Assay of acetohydroxyacid synthase. Analytical Biochemistry, 1988, 171: 173-179.). Specifically, 0.2 g of wild type, 9311M1 and 9311M2 M3 plants were taken, ground in a mortar with liquid nitrogen, and 2 mL of extract (100 mM K2HPO4, pH 7.5, 10 mM sodium pyruvate, 5 mM EDTA, 1 mM ammonia) was added.
  • the mixture was incubated at 37 ° C for 30 minutes for color development (ALS catalyzed the formation of acetolactate by two pyruvic acids, decarboxylation of acetolactate to form 3-hydroxybutanone, and then formed a pink complex with creatine and 1-naphthol, the complex
  • ALS catalyzed the formation of acetolactate by two pyruvic acids, decarboxylation of acetolactate to form 3-hydroxybutanone, and then formed a pink complex with creatine and 1-naphthol, the complex
  • the maximum absorption value at 530 nm followed by measuring the absorbance at 530 nm, ALS activity is expressed by the absorbance of A530, and the level of absorbance of A530 reflects the level of ALS activity.
  • the experiment used water as the control, and the wild type, the 9311M1 strain and the 9311M2 strain each tested 5 individual plants.
  • A530 absorbance measurement results showed that when the ALS extract of wild type, 9311M1 and 9311M2 had no ALS inhibitor, their A530 absorbance values were between 1.2 and 1.4, indicating the ALS activity of wild type and mutant. There was no significant difference (Fig. 4, Fig. 5).
  • the wild type A530 had an absorbance of only 0.3, and the 9310M1 and 9311M2 A530 had an absorbance of about 1.1, which is the wild type ALS enzyme activity. It is only about 25% of the control, while the ALS activity of 9311M1 and 9311M2 is still about 80% (Fig. 4, Fig. 5).
  • the ALS activity of the mutant is more than three times that of the wild type, indicating the mutant ALS of 9311M1 and 9311M2. It is not sensitive to ridges and gives resistance.
  • the A530 absorbance measurement showed that when the ALS extract of wild type, 9311M1 and 9311M2 did not contain the ALS inhibitor, sulfasulfuron, their A530 absorbance values were between 1.3 and 1.5, indicating wild type and mutant. There was no significant difference in ALS enzyme activity (Fig. 6, Fig. 7). After adding the ALS inhibitor, sulfasulfuron, the wild type A530 had an absorbance of only 0.2, and the 9310M1 and 9311M2 A530 had an absorbance of 0.7-0.8. That is, the wild-type ALS enzyme activity is only about 16% of the control, while the ALS enzyme activity of the 9311M1 and 9311M2 is still about 54% (Fig. 6, Fig. 7), and the ALS activity of the mutant is more than three times that of the wild type. Mutant ALS indicating that 9311M1 and 9311M2 were insensitive to mesulfuron-methyl, conferring resistance.
  • the mutant ALS gene was amplified from the genomic DNA of the above rice mutants 9311M1 and 9311M2 by PCR, and after sequencing, the double enzyme digestion was performed with BamHI and SacI, respectively.
  • the ALS gene fragment and the plant expression vector pCAMBIA1301 plasmid (purchased from pcambia) were transformed, and the digested product was ligated with T4-DNase (purchased from TaKaRa), and the ligated product was transformed into Escherichia coli.
  • DNA was extracted from the recombinant plasmid and verified by double digestion with BamHI and SacI to generate a large plasmid fragment and a small gene fragment (Fig. 8), demonstrating that the nucleotide sequence is the ALS gene shown in SEQ ID NO. 1 or 3.
  • the plasmid was cloned into the plant expression vector pCAMBIA1301 (purchased from pcambia).
  • the constructed plasmid vector was transformed into Agrobacterium EHA105, and the cells were cultured.
  • the conventional Agrobacterium-mediated transformation of indica rice Nipponbare purchased from the Jiangsuzhou agricultural germplasm resources protection and utilization platform
  • the progeny plants were grown to the 3-4 leaf stage, and the transgenic plants were detected by PCR.
  • the PCR detection primers were forward primer 35SF 5'-ATGGTTAGAGAGGCTTACGC-3', reverse primer 5R5'-AGCAACAGGTCAGCCTTATCCAC-3', and the amplified fragment encompassed the CaMV35S promoter and the 5' end sequence of the ALS gene, and the size was about 2 kb.
  • the PCR amplification reaction system was referred to Example 3.
  • the PCR amplification reaction procedure was carried out in a two-step process, annealing and extension together, using 68 degrees.
  • the amplification procedure was as follows: pre-denaturation: 98 ° C for 3 min; 30 cycles: denaturation 98 ° C for 10 sec; extension 68 ° C for 2 min; incubation: 72 ° C for 10 min.
  • the ALS enzyme activity of the transgenic rice was found to be significantly higher than that of the non-transgenic rice. After 30 days, the transgenic rice was found to be in good growth condition, while the non-transgenic Nipponbare rice was all dead.
  • the mutant ALS gene was amplified from the genomic DNA of the above rice mutants 9311M1 and 9311M2 by PCR, and after sequencing, the ALS gene having the nucleotide sequence shown in SEQ ID NO. 1 or 3 was cloned according to the method of Example 7.
  • the plant expression vector pCAMBIA2301 plasmid (purchased from pcambia) was used.
  • the positive clones were selected to transform Agrobacterium tumefaciens EHA105, and the Agrobacterium tumefaciens-mediated transformation method was used to transform the tobacco leaves. After the transgenic plants were harvested, the progeny plants grew to 3-4 leaf stage, and after PCR positive, spray 4mL. Bailutong/L water (12 times recommended concentration) or sprayed 4.5g/L of sulfometuron (8 times recommended concentration), after 7 days, the ALS activity was determined by the method described in Example 6, and the transgenic tobacco was found. The ALS enzyme activity was significantly higher than that of non-transgenic tobacco; after 30 days, the transgenic tobacco was found to be in good growth condition, while the non-transgenic tobacco was all dead.
  • the mutant ALS gene was amplified from the genomic DNA of the above rice mutants 9311M1 and 9311M2 by PCR, and after sequencing, the ALS gene having the nucleotide sequence as shown in SEQ ID NO. 1 or 3 was cloned by the method of Example 7.
  • Example 10 obtained by genetically modified cotton
  • the mutant ALS gene was amplified from the genomic DNA of the above rice mutants 9311M1 and 9311M2 by PCR, and after sequencing, the ALS gene having the nucleotide sequence as shown in SEQ ID NO. 1 or 3 was cloned by the method of Example 7.
  • pCAMBIA2301 plasmid purchased from pcambia company
  • select positive clones to transform Agrobacterium LAB4404 culture the cells, transform cotton hypocotyls and cotyledons by Agrobacterium-mediated method, and obtain the length of the progeny plants after the transgenic plants are harvested.
  • 3 mL of ridges/L water (9 times recommended concentration) was sprayed on the seedlings. After 30 days, non-GM cottons were found to die, while the transgenic cottons grew well.

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Abstract

L'invention concerne un gène de type mutant d'acétolactate synthase (ALS). Le 75ème nucléotide du gène de type mutant d'ALS dans une séquence de gène d'ALS du riz mute du nucléotide G vers le nucléotide C, le 339ème nucléotide du gène de type mutant d'ALS dans la séquence du gène d'ALS du riz mute du nucléotide A au nucléotide C et le 710ème nucléotide du gène de type mutant d'ALS dans la séquence du gène d'ALS mute du nucléotide C au nucléotide T. L'invention concerne également des applications d'une protéine mutante d'ALS codée par le gène de type mutant d'ALS dans la résistance aux herbicides. La protéine provient d'une plante mutante du riz résistant aux herbicides inhibiteurs de l'ALS. Par comparaison avec la séquence d'ALS de type sauvage du riz, la séquence de la protéine du gène de type mutant d'ALS mute au niveau des sites Gln25, Gln113 et Ala237. La plante verte exprimant la séquence de protéine mutante d'ALS peut résister aux herbicides inhibiteurs de l'acétolactate synthétase, en particulier aux herbicides imidazolones et sulfonylurées.
PCT/CN2017/074079 2016-04-12 2017-02-20 Applications de gène de type mutant d'als dans la résistance aux herbicides WO2017177765A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113717957A (zh) * 2021-11-04 2021-11-30 科稷达隆(北京)生物技术有限公司 一种als突变蛋白及其应用

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105695493A (zh) * 2016-04-12 2016-06-22 江苏省农业科学院 一种als突变型基因在抗除草剂方面的应用
CN106119370B (zh) * 2016-07-04 2019-10-22 山东省农业科学院植物保护研究所 抗als抑制剂类除草剂马唐的pcr检测方法及试剂盒
CN106591334B (zh) * 2016-12-19 2019-10-22 江苏省农业科学院 一种小麦als突变型基因及其在抗除草剂方面的应用
CN107217044A (zh) * 2017-08-03 2017-09-29 江苏省农业科学院 使植物具有除草剂抗性的水稻als突变型蛋白及其应用
CN107267656A (zh) * 2017-08-15 2017-10-20 江苏省农业科学院 用于抗咪草烟除草剂基因型检测的功能标记as‑als及其应用

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005020673A1 (fr) * 2003-08-29 2005-03-10 Instituto Nacional De Technologia Agropecuaria Plants de riz presentant une tolerance accrue aux herbicides imidazolinone
CN102586215A (zh) * 2011-09-06 2012-07-18 深圳兴旺生物种业有限公司 水稻抗除草剂蛋白及其在植物育种中的应用
CN102936591A (zh) * 2012-11-22 2013-02-20 北京兴博雅生物技术有限公司 乙酰乳酸合成酶突变体及其应用
CN105695493A (zh) * 2016-04-12 2016-06-22 江苏省农业科学院 一种als突变型基因在抗除草剂方面的应用
CN105755024A (zh) * 2016-04-12 2016-07-13 江苏省农业科学院 Als突变型基因及其蛋白和应用
CN105779479A (zh) * 2016-04-12 2016-07-20 江苏省农业科学院 一种als突变型基因及其在抗除草剂方面的应用

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101691583A (zh) * 2009-09-25 2010-04-07 南京农业大学 一种农杆菌介导的不依赖于组织培养的棉花转基因方法
CA2814591C (fr) * 2010-10-15 2021-08-31 Kws Saat Ag Mutants beta vulgaris tolerants a un herbicide inhibiteur d'als
MA38173B2 (fr) * 2012-12-13 2017-12-29 Bayer Cropscience Ag Utilisation d'herbicides inhibiteurs de l'als pour la lutte contre une végétation indésirable chez des plantes de type betterave fourragère tolérantes aux herbicides inhibiteurs de l'als

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005020673A1 (fr) * 2003-08-29 2005-03-10 Instituto Nacional De Technologia Agropecuaria Plants de riz presentant une tolerance accrue aux herbicides imidazolinone
CN102586215A (zh) * 2011-09-06 2012-07-18 深圳兴旺生物种业有限公司 水稻抗除草剂蛋白及其在植物育种中的应用
CN102936591A (zh) * 2012-11-22 2013-02-20 北京兴博雅生物技术有限公司 乙酰乳酸合成酶突变体及其应用
CN105695493A (zh) * 2016-04-12 2016-06-22 江苏省农业科学院 一种als突变型基因在抗除草剂方面的应用
CN105755024A (zh) * 2016-04-12 2016-07-13 江苏省农业科学院 Als突变型基因及其蛋白和应用
CN105779479A (zh) * 2016-04-12 2016-07-20 江苏省农业科学院 一种als突变型基因及其在抗除草剂方面的应用

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
A. OKUZAKI ET AL.: "A novel mutated acetolactate synthase gene conferring specific resistance to pyrimidinyl carboxy herbicides in rice", PLANT MOL BIOL, vol. 64, 3 March 2007 (2007-03-03), pages 219 - 224, XP019507416, ISSN: 0167-4412 *
DATABASE Nucleotide [O] 1 July 2008 (2008-07-01), SALES, M. A. ET AL.: "Oryza sativa Indica Group biotype Lin-3 acetolactate synthase (ALS) gene, complete cds", XP055431058, Database accession no. EU548083.1 *
SONG, GUISHENG ET AL.: "Isolation and Functional Analysis of Rice Acetolactate-synthase (ALS)", JOURNAL OF AGRICULTURAL SCIENCE AND TECHNOLOGY, vol. 9, no. 3, 15 June 2007 (2007-06-15), pages 66 - 72, XP055431062, ISSN: 1008-0864 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113717957A (zh) * 2021-11-04 2021-11-30 科稷达隆(北京)生物技术有限公司 一种als突变蛋白及其应用

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