WO2020037648A1 - Gène de colza résistant aux herbicides à base d'acide salicylique de pyrimidine et son utilisation - Google Patents

Gène de colza résistant aux herbicides à base d'acide salicylique de pyrimidine et son utilisation Download PDF

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WO2020037648A1
WO2020037648A1 PCT/CN2018/102232 CN2018102232W WO2020037648A1 WO 2020037648 A1 WO2020037648 A1 WO 2020037648A1 CN 2018102232 W CN2018102232 W CN 2018102232W WO 2020037648 A1 WO2020037648 A1 WO 2020037648A1
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seq
mutated
acetolactate synthase
plant
mutation
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Chinese (zh)
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胡茂龙
浦惠明
龙卫华
高建芹
张洁夫
陈松
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江苏省农业科学院
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Priority to DE112018006599.5T priority Critical patent/DE112018006599T5/de
Priority to CN201880072169.4A priority patent/CN112154207B/zh
Priority to PCT/CN2018/102232 priority patent/WO2020037648A1/fr
Priority to CA3087906A priority patent/CA3087906A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8274—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for herbicide resistance
    • C12N15/8278—Sulfonylurea
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A01H5/10—Seeds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1022—Transferases (2.) transferring aldehyde or ketonic groups (2.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y202/00—Transferases transferring aldehyde or ketonic groups (2.2)
    • C12Y202/01—Transketolases and transaldolases (2.2.1)
    • C12Y202/01006—Acetolactate synthase (2.2.1.6)

Definitions

  • the invention relates to the technical field of plant genetic engineering, in particular to rapeseed pyrimidine-salicylic acid herbicide gene and application thereof. More specifically, the present invention relates to a rape plant that is tolerant to pyrimidine salicylic acid herbicides and parts thereof, resistance genes, muteins, and applications thereof.
  • Rape (Brassica napus L.) is China's largest oilseed crop, providing edible oil sources for more than half of the population in China.
  • One of the important biological hazards in rapeseed production is farmland weeds. It not only competes with rapeseed for water and fertilizer, but also changes the microclimate of rapeseed fields. Some weeds are also intermediate hosts of rapeseed pests and diseases, and accelerate the spread of pests and diseases It severely affects the yield and quality of rapeseed crops.
  • manual weeding is time consuming and laborious, increasing production costs. Therefore, the application of herbicides to control field weeds has become an inevitable choice for people.
  • Herbicides mainly inhibit or disrupt plants by inhibiting or interfering with key metabolic processes in plants. Targeting key enzymes in the amino acid biosynthesis process is an important direction and hot spot in the development of new and efficient herbicides.
  • Herbicides developed with acetolactate synthase (ALS; EC2.2..16) as the target enzyme have become mainstream products of new high-efficiency herbicides.
  • ALS is an enzyme that catalyzes the first step in the biosynthesis of branched-chain amino acids (valine, leucine, and isoleucine).
  • ALS inhibitor herbicides can inhibit ALS enzyme activity in plant cells, hinder branch chain amino acids (valine, leucine, and isoleucine) biosynthesis, thereby inhibiting plant cell division and growth.
  • PB pyrimidyl-benzoates
  • thio pyrimidineoxy
  • Herbicides The first commercial variety of this class of herbicide was pyrithiobac-sodium. Subsequently, pyriminobac-methyl was developed in 1993, and bispyribac-sodium (Nongmeili) was developed in 1996.
  • ALS herbicides Since the application of ALS herbicides to agriculture, it has been observed that sensitive plant species, including naturally occurring weeds, occasionally show spontaneous tolerance to such herbicides. Substitution of a single base at a specific site in the ALS gene usually results in more or less resistance, and plants with mutated ALS alleles show different levels of tolerance to ALS herbicides, depending on The chemical structure of the ALS herbicide and the point mutation site of the ALS gene.
  • the present invention addresses this need and provides mutant acetolactate synthase (ALS) nucleic acids and proteins encoded by these mutant nucleic acids.
  • the invention also relates to rapeseed plants, cells and seeds comprising these mutant nucleic acids and proteins, said mutations conferring tolerance to a pyrimidine salicylic acid herbicide, wherein the ALS polypeptide encoded by the ALS gene is at position 556 thereof Contains amino acids different from tryptophan and contains amino acids different from serine at position 635.
  • the ALS polypeptide encoded by the ALS gene has a double mutation selected from the group consisting of: W556L and S635N; W556L and S635T; W556L and S635I.
  • the ALS polypeptide encoded by the ALS gene has the following mutations: W556L and S635N.
  • the invention provides an isolated nucleic acid encoding a mutant acetolactate synthase (ALS3), said mutant acetolactate synthase (ALS3) protein comprising the following mutations:
  • S serine
  • N asparagine
  • T threonine
  • I isoleucine
  • nucleotide sequence of the isolated nucleic acid is as shown in SEQ ID NO: 3;
  • amino acid sequence of the mutated ALS3 protein is as shown in SEQ ID NO: 4.
  • the invention provides an expression cassette, vector or cell, which contains a nucleic acid according to the invention. Accordingly, the present invention provides the use of a nucleic acid, an expression cassette, a vector or a cell or a mutant acetolactate synthase (ALS3) protein of the present invention for producing a pyrimidine salicylic acid herbicide-resistant plant.
  • the plant For rapeseed.
  • the present invention provides a method for producing a plant having pyrimidine salicylic acid herbicide resistance, which comprises the following steps:
  • the nucleic acid according to the present invention is introduced into a plant, and preferably the nucleic acid according to the present invention is introduced into a plant through steps such as transgenic, hybridization, backcrossing or asexual propagation, wherein the plant expresses the mutant acetolactate synthase according to the present invention. (ALS3) protein and has pyrimidine salicylic acid herbicide resistance.
  • ALS3 mutant acetolactate synthase according to the present invention.
  • the present invention provides a non-transgenic plant or part thereof that is resistant to a pyrimidine salicylic acid herbicide, comprising an isolated nucleic acid encoding a mutant acetolactate synthase protein, the mutant acetolactate synthase protein comprising The following mutations:
  • S serine
  • N asparagine
  • T threonine
  • I isoleucine
  • said plant is rape; wherein said parts are organs, tissues and cells of the plant, and preferably seeds;
  • said protein comprises a mutation of tryptophan (W) to leucine (L) at a position corresponding to position 556 of SEQ ID NO: 2 and a position corresponding to position 635 of SEQ ID NO: 2 Mutation of serine (S) to asparagine (N);
  • amino acid sequence of the mutated ALS3 protein is shown in SEQ ID NO: 4.
  • the present invention provides a method for controlling weeds in a field containing rape plants, the method comprising applying an effective amount of a pyrimidine salicylic acid herbicide to the field containing the weeds and rape plants
  • the rapeseed plant contains an isolated nucleic acid encoding a mutant acetolactate synthase protein, and the mutant acetolactate synthase protein contains the following mutations:
  • S serine
  • N asparagine
  • T threonine
  • I isoleucine
  • said protein comprises a mutation of tryptophan (W) to leucine (L) at a position corresponding to position 556 of SEQ ID NO: 2 and a position corresponding to position 635 of SEQ ID NO: 2 Mutation of serine (S) to asparagine (N);
  • amino acid sequence of the mutated ALS3 protein is shown in SEQ ID NO: 4.
  • Figure 1 shows the results of amino acid partial sequence alignment of rapeseed ALS3 from different sources.
  • ALS3, reference sequence on Genbank accession number: Z11526
  • ALS3 amino acid partial sequence of ALS3_N131 wild type strain N131 ALS3_EM28 resistant EM3 amino acid partial sequence of EM28; ALS3_Sh4 resistant material Sh4; ALS3 amino acid partial sequence of Sh5; ALS3_Sh6 resistant material Sh6; ALS3 amino acid partial sequence of ALS3_Sh7 resistant material Sh7.
  • Figure 2 shows the in vitro inhibition of wild-type and mutant ALS enzyme activity by bensulfuron-methyl at different concentrations.
  • Figure 3 shows the in vitro inhibition of ALS enzyme activity of wild-type and mutants by different concentrations of imidazolenic acid.
  • Figure 4 shows the in vitro inhibition of ALS enzyme activity of wild-type and mutants by bisamprol at different concentrations.
  • Figure 5 shows the resistance performance of herbicide-resistant Arabidopsis thaliana and tobacco after spraying with herbicide (Col, wild-type Arabidopsis, 3A-1 and 3A-2 Arabidopsis; Tob, wild-type tobacco, Y3A-1 and Y3A-2 transgenic tobacco-resistant tobacco). + Indicates spraying with 60 g of aiha -1 bispyrisol,-indicates no treatment with herbicide.
  • non-transgenic means that the individual genes have not been introduced by an appropriate biological vector or by any other physical means. However, the mutated gene can be passed on by pollination (naturally or by breeding methods) to produce another non-transgenic plant containing the specific gene.
  • endogenous gene is meant a gene in a plant that is not introduced into said plant by genetic engineering techniques.
  • nucleotide sequence refers to a polymerized unbranched form of nucleotides of any length , Ribonucleotides or deoxyribonucleotides, or a combination of both.
  • Nucleic acid sequences include DNA, cDNA, genomic DNA, RNA, including synthetic forms, and mixed polymers, including the sense and antisense strands, or may contain unnatural or derived nucleotide bases, as will be understood by those skilled in the art this point.
  • polypeptide or "protein” (these two terms are used interchangeably herein) means a peptide, protein or polypeptide comprising an amino acid chain of a given length, wherein the amino acid residue is Covalent peptide bonds.
  • the present invention also includes peptide mimics of the protein / polypeptide (in which amino acids and / or peptide bonds have been replaced by functional analogs), and amino acids other than the amino acids encoded by the 20 genes, such as selenohalides Cystine.
  • Peptides, oligopeptides, and proteins can be referred to as polypeptides.
  • polypeptide also refers to (but does not exclude) modification of the polypeptide, such as glycosylation, acetylation, phosphorylation, and the like. This modification is well documented in the basic literature and in more detail in monographs and research literature.
  • Amino acid substitutions include amino acid changes in which amino acids are replaced by different naturally occurring amino acid residues. Such substitutions can be classified as “conservative” in which the amino acid residues contained in the wild-type ALS protein are replaced by another naturally occurring and similarly characteristic amino acid. For example, or the substitutions included in the present invention may also be “non-conservative" ", Where the amino acid residues present in the wild-type ALS protein are replaced with amino acids having different properties, such as naturally occurring amino acids from different groups (such as the replacement of charged or hydrophobic amino acids with alanine).
  • similar amino acids refers to amino acids with similar amino acid side chains, ie, amino acids with polar, non-polar, or near neutral side chains.
  • dipolar amino acids refer to amino acids with different amino acid side chains, for example, amino acids with polar side chains are not similar to amino acids with non-polar side chains.
  • Polar side chains often tend to be present on the surface of proteins, where they can interact with the water environment present in the cell (“hydrophilic” amino acids).
  • non-polar amino acids tend to be located in the center of the protein, where they can interact with similar non-polar neighboring molecules (“hydrophobic” amino acids).
  • amino acids with polar side chains are arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, histidine, lysine, serine, and threonine (All are hydrophilic amino acids except cysteine is hydrophobic).
  • amino acids with non-polar side chains are alanine, glycine, isoleucine, leucine, methionine, phenylalanine, proline and tryptophan (all are hydrophobic, Except glycine is neutral).
  • ALS ALS
  • ALSL AHAS
  • AHASL AHASL
  • the term “gene” refers to a polymerized form of nucleotides (ribonucleotides or deoxyribonucleosides) of any length. This term includes double- and single-stranded DNA and RNA. It also includes known Types of modification, such as methylation, "capping," replacing one or more naturally occurring nucleotides with analogs.
  • the gene includes a coding sequence that encodes a polypeptide as defined herein.
  • the "coding sequence” is when A nucleotide sequence that is placed or under the control of appropriate regulatory sequences that can be transcribed into mRNA and / or translated into a polypeptide.
  • the boundaries of the coding sequence are the translation initiation codon at the 5 'end and the 3' end
  • the translation stop codon is determined.
  • the coding sequence can include, but is not limited to, mRNA, cDNA, recombinant nucleic acid sequence or genomic DNA, but introns may be present in some cases.
  • Brainssica napus may be abbreviated as “B. napus”.
  • rapeseed is used herein. The three terms are used interchangeably and should be understood to completely include rape in cultivated form.
  • Alabidopsis thaliana can be abbreviated as “A. thaliana”. These two terms are used interchangeably herein.
  • position means the position of an amino acid in an amino acid sequence described herein or the position of a nucleotide in a nucleotide sequence described herein, such as the wild type shown in SEQ ID NO: 1
  • corresponding as used herein also includes positions determined not only by the aforementioned nucleotide / amino acid numbering.
  • nucleotides at other positions in the ALS 5 'untranslated region (UTR) including promoters and / or any other regulatory sequences) or genes (including exons and introns)
  • UTR untranslated region
  • genes including exons and introns
  • the position of a given nucleotide that can be substituted in the can be different.
  • the positions of a given amino acid that can be replaced in the present invention may differ. Therefore, "corresponding positions" in the present invention should be understood as the nucleotides / amino acids at the indicated numbers may be different, but still may have similar adjacent nucleotides / amino acids.
  • nucleotides / amino acids which can be exchanged, deleted or inserted are also included by the term "corresponding position".
  • the art Skilled artisans can use tools and methods well known in the art, such as comparisons manually or by using computer programs, such as BLAST (Altschul et al. (1990), Journal of Molecular Biology, 215, 403-410) (which represents a substantial local Alignment Search Tool) or ClustalW (Thompson et al. (1994), Nucleic Acid Res., 22, 4673-4680) or any other suitable program suitable for generating sequence alignments.
  • the present invention provides a rapeseed plant in which a tryptophan W ⁇ leucine L substitution occurs at a position 556 of a polypeptide encoded by the endogenous ALS gene of the rapeseed plant, which is due to corresponding to SEQ ID ID NO:
  • the "G” nucleotide is mutated to the "T” nucleotide at the position 1667 of the nucleotide sequence shown in 1.
  • the present invention provides a rapeseed plant, and the endogenous ALS3 gene of the rapeseed plant comprises (or consists of) the nucleotide sequence shown in SEQ ID NO: 3, which encodes SEQ ID mutated ALS3 polypeptide shown by NO: 4.
  • ALS activity can be measured according to the assay described in Singh (1991), Proc. Natl. Acad. Sci. 88: 4572-4576.
  • the ALS nucleotide sequences encoding ALS polypeptides referred to herein preferably confer resistance to one or more pyrimidinesalicylic acid herbicides described herein (alternatively, Low sensitivity). This is due to the point mutations described herein that lead to amino acid substitutions. Therefore, tolerance to pyrimidine salicylic acid herbicides (or, less sensitive to pyrimidine salicylic acid herbicides) can be derived from the presence of mutations in the presence of pyrimidine salicylic acid herbicides.
  • ALS is obtained from cell extracts of plants of the ALS sequence and plants without a mutant ALS sequence and their activity is measured for comparison, for example the method described in Singh et al (1988) [J. Chromatogr., 444, 251-261].
  • plants preferably in the presence of multiple concentrations of pyrimidinesalicylic acid herbicides, more preferably in the presence of multiple concentrations of pyrimidinesalicylic acid herbicide "bisoxafen,” in the wild type ALS activity in cell extracts or leaf extracts of rapeseed and rapeseed cell extracts or leaf extracts of the obtained mutants.
  • the sensitivity is lower, and vice versa, can be considered as “resistant "Higher tolerance” or “higher resistance.”
  • “higher tolerance” or “higher resistance” can be considered “less sensitive.”
  • pyrimidinesalicylic acid herbicide is not intended to be limited to a single herbicide that can interfere with the enzyme activity of ALS. Therefore, unless otherwise stated or apparent from the context, a “pyrimidinesalicylic acid herbicide” may be one herbicide or two, three, four, or more herbicides known in the art. Mixtures of agents, the herbicides are preferably those listed herein, such as, for example, sulfamethoxazole, sulfachlor, ciprofen, bispyribac, sulfamethoxam, sulfamethoxam, and the like.
  • the present invention provides a rapeseed plant that is tolerant to pyrimidine salicylic acid herbicides and has an endogenous acetolactate synthase (ALS) gene mutation.
  • plant means a plant at any stage of development, unless explicitly stated otherwise. A part of the plant may be connected to the whole plant or may be separated from the whole plant. Parts of such plants include, but are not limited to, the organs, tissues and cells of the plant, preferably seeds.
  • the rapeseed plant of the present invention is non-transgenic with respect to the endogenous ALS gene. Of course, the exogenous gene can be transferred into the plant by genetic engineering or by conventional methods such as crossing.
  • CGMCC Common Microbiological Center
  • the recommended concentration of spraying the bispyribenzide herbicide weed control on M3 seeds at seedling stage was used to identify the resistance effect. From 1 week after spraying, observe the phytotoxicity reaction every day. It was found that the six strains numbered Sh1, Sh2, Sh3, Sh8, Sh9, and Sh10 had a phytotoxicity reaction one week after spraying. The heart leaves of the vegetable seedlings began to turn yellow, and gradually rot, and finally died. These seedlings should be caused by leakage of pesticides under high-density planting conditions; the four strains numbered Sh4, Sh5, Sh6, and Sh7 showed strong resistance without any symptoms of phytotoxicity and could grow normally.
  • Example 2 Molecular cloning of a resistance gene in a new germplasm of Brassica napus resistant to pyrimidine salicylic acid herbicides
  • Pyrimidine salicylic acid herbicides belong to the class of ALS inhibitor herbicides.
  • the target of this type of herbicide is acetolactate synthase.
  • ALS1 primer 1 GTGGATCTAACTGTTCTTGA
  • primer 2 AGAGATGAAGCTGGTGATC.
  • ALS2 primer 1 GAGTGTTGCGAGAAATTGCTT and primer 2: TTGATTATTCTATGCTCTCTTCTG.
  • ALS3 primer 1 AGGGTTAGATGAGAGAGAGAG and primer 2: GGTCGCACTAAGTACTGAGAG.
  • the CTAB method was used to extract leaf genomic DNA from resistant strains Sh4, Sh5, Sh6, Sh7 and non-resistant strains Sh1, Sh2, Sh3, Sh8, Sh9, Sh10, and N131 and EM28.
  • PCR wild-type and mutant ALS1 were cloned.
  • ALS2 and ALS3 genes 50 ⁇ L PCR reaction system was prepared according to the instructions of Toyobo (Shanghai) Biotechnology Co., Ltd. high-fidelity DNA polymerase KOD-Plus kit.
  • Amplification was performed on a MJ Research PTC-200 PCR instrument.
  • the reaction procedure was pre-denaturation at 94 ° C for 5 minutes; denaturation at 94 ° C for 30s, annealing at 55 ° C for 30s, and extension at 72 ° C for 2.5 minutes, a total of 35 cycles.
  • the product was separated by 1.2% (V / W) agarose gel electrophoresis, and then purified and recovered using an agarose gel DNA recovery kit (catalog number: DP209) produced by Beijing Tiangen Company.
  • the purified PCR The products were commissioned by Nanjing Kingsray Biological Co., Ltd. for sequencing.
  • the ALS3 gene in the resistant strain has a new mutation site (S635N), the nucleotides of which are shown in SEQ ID NO: 3 and the amino acid sequence of which is shown in SEQ ID NO: 4 shown.
  • a two-site mutation of the ALS3 gene (W556L and S635N) increased the resistance of resistant mutants to pyrimidine salicylic acid herbicides.
  • RP-1 Sh7 with strong growth potential and good plant type was tentatively named RP-1.
  • N131 and EM28 were used as control materials.
  • the rape field identification test was conducted in the rape isolation breeding area of the Jiangsu Academy of Agricultural Sciences, and the greenhouse pot experiment was performed in a constant temperature light culture room.
  • ALS inhibitor herbicides widely used in China are sprayed, among which the pyrimidine salicylic acid herbicide is bispyrither [2,6-bis (4 , 6-dimethoxypyrimidin-2-oxy) sodium benzoate], SU-type herbicide is besulfuron-methyl (2- [N- (4-methoxy-6-methyl-1,3,5 -Triazin-2-yl) -N-methylcarbamidosulfonyl] benzoic acid methyl ester) and IMI herbicides are imidazolenicotinic acid ((RS) -5-ethyl-2- (4-isopropyl) 4-methyl-5-oxo-1H-imidazolin-2-yl) nicotinic acid].
  • the pyrimidine salicylic acid herbicide is bispyrither [2,6-bis (4 , 6-dimethoxypyrimidin-2-oxy) sodium benzoate]
  • SU-type herbicide is besulfuron-methyl (2- [N- (4-methoxy
  • R represents that the rapeseed plants grow well after herbicide treatment, and there is no phytotoxicity
  • S represents that the growth of rapeseed plants is severely inhibited after herbicide treatment, showing obvious phytotoxicity, and the final vegetable seedlings die (the same below).
  • Example 4 Inhibition test of ALS enzyme activity by herbicide
  • an in vitro enzyme activity test was performed to compare the ALS enzyme in RP-1, EM28 and the original wild-type N131 with three types of herbicides bispyribenzol (PB), benzsulfuron (SU type) and imidazolidinic acid (IMI type), compared the difference between the three materials.
  • PB bispyribenzol
  • SU type benzsulfuron
  • II type imidazolidinic acid
  • ALS enzyme activity refer to the method of Singh et al. (Singh BK, et al., Analytical Biochemistry, 1988, 171: 173-179). Specifically, 0.2 g of leaf samples were taken and ground and pulverized with liquid nitrogen in a mortar.
  • the ground sample was added to a 4.5 ml primary enzyme extract [100 mM K2HPO4, 0.5 mM MgCl2, 0.5 mM thiamine pyrophosphate ( TPP), 10 ⁇ M flavin adenine dinucleotide (FAD), 10 mM sodium pyruvate, 10% (v / v) glycerol, 1 mM dithiothreitol, 1 mM benzylsulfonyl fluoride (PMSF), 0.5 % (W / v) polyvinylpyrrolidone], centrifuged at 4 ° C and 12000 rpm for 20 min.
  • a primary enzyme extract [100 mM K2HPO4, 0.5 mM MgCl2, 0.5 mM thiamine pyrophosphate ( TPP), 10 ⁇ M flavin adenine dinucleotide (FAD), 10 mM sodium pyruvate, 10% (v / v
  • the mutant enzyme in RP-1 shows strong resistance to the pyrimidine salicylic acid herbicide dioxadi because, as compared with N131 and EM28, N131 and EM28 increase with the increase of the dioxadi concentration
  • the ALS enzyme activity in RP-1 decreased rapidly and showed the same trend, while the mutant enzyme activity in RP-1 was lightly inhibited by herbicides. Even under high concentration (250 ⁇ mol L-1) bispyrisol, The mutant enzyme activity was about 51% of the control.
  • the inhibition rates of N131 and EM28 enzyme activity were close to 100%, that is, the ALS in N131 and EM28 had basically no activity, only 4% and 10% of the control, respectively.
  • the sensitivity of the ALS enzyme in the mutant RP-1 to the pyrimidine salicylate herbicide dioxadi is significantly lower than that of the ALS enzymes in N131 and EM28, which further illustrates that the two-site mutation of the ALS3 gene (W556L and S635N) confers resistance to pyrimidine salicylic acid herbicides.
  • a plant expression vector was constructed, and the resistance gene was transferred into an Arabidopsis thaliana plant by a conventional Agrobacterium-mediated method, and positive homozygous transgenic lines were screened by PCR in the transgenic offspring to identify the herbicide phenotype.
  • specific primers were designed based on the ALS3 gene sequence.
  • ALS3 primers 3 5'CGC GGTACC CTCTCTCTCTCTCATCTAACCAT3 'and ALS3 primers 4: 5'CGC ACTAGT CTCTCAGTACTTAGTGCGACC3', 5 ′ of which were added with KpnI and SpeI restriction sites, underlined The sequence is a restriction site.
  • the resistance gene was obtained by PCR amplification.
  • the nucleotides are shown in SEQ ID NO: 3 and the amino acid sequence is shown in SEQ ID NO: 4.
  • the PCR product was recovered, cloned, and sequenced according to the method of Example 2 to obtain a recombinant T vector carrying a gene encoding a mutant enzyme.
  • a KpnI and SpeI double-digested T vector was used to obtain a fragment containing the gene of interest, which was recovered and ligated to the same double-digested pCAMBIA1390 vector (purchased from Australian CAMBI) to obtain a recombinant plant expression vector.
  • the constructed recombinant vector was transformed into E.
  • Agrobacterium tumefaciens EHA105 strain was transformed with the recombinant vector containing the correct target gene, and the plasmid was extracted for identification by PCR and enzyme digestion.
  • the obtained recombinant strain was cultured, and the Arabidopsis thaliana was transformed by Agrobacterium flower dipping.
  • the T0 generation was screened with antibiotics on the medium, the T1 generation plants were transplanted into pots, grown in artificial incubators, and screened and expanded to obtain homozygous transgenic lines of the T3 generation.
  • 60g of aiha -1 bisoxafen was sprayed for resistance identification.
  • the acetolactate synthase mutation gene of the nucleotide in RP-1 as shown in SEQ ID NO: 3 was cloned into the plant expression vector pCAMBIA1390 plasmid (purchased from Australian CAMBI company).
  • the positive clones were selected to transform Agrobacterium EHA105.
  • the conventional Agrobacterium-mediated transformation method was used to transform the tobacco leaf disc of Benn's. After the transgenic plants were harvested, they were identified by PCR. At the seedling stage of T3 generation transgenic tobacco, 60g aiha -1 was sprayed. Ether was identified for resistance.
  • Example 7 Functional verification of resistance genes in common rapeseed
  • acetolactate synthase mutation gene of nucleotides in RP-1 as shown in SEQ ID NO: 3 is introduced into other common rapeseed varieties or strains that are not resistant to pyrimidine salicylic acid herbicides by hybridization.
  • RP-1 and 3075R Pan Huiming et al., 2002, Jiangsu Agricultural Science, 4: 33-3
  • 3018R Pu Huiming et al., 1999, Jiangsu
  • Agricultural Sciences, 6: 32-33) Prepare hybrid combinations, harvest two F1 seeds in the rape vernalization culture room for additional planting in the same year, select a single plant with self-bagging and self-consistent growth during flowering, and harvest F2 seeds in Nanjing Jiangsu province. The plant was seeded at the Yuanshui Water Plant Science Base. Each F2 population was sown 20 rows. A single leaf of the F2 population was taken at seedling stage, DNA was extracted, and the ALS3 gene was amplified by PCR. The product was purified, recovered, and sequenced according to Example 2.
  • Tryptophan (W) is mutated to leucine (L), serine (S) is mutated to threonine (T) at position 635;
  • LI the nucleotide at +1667 of ALS3 gene is changed from G to T and The nucleotide at +1904 changed from G to T, which resulted in mutation of tryptophan (W) to leucine (L) at position 556 and mutation of serine (S) to isoleucine at position 635 ( I);
  • GN the + 1666th nucleotide at +1666 changed from T to G and the + 1904th nucleotide changed from G to A, resulting in mutation of tryptophan (W) at position 556 of the corresponding encoded protein Glycine (G) and 635 positions were mutated from serine (S) to asparaginic acid (N);
  • GT ALS3 genes changed from T to G at +1666 and from G to +1904 C, resulting in mutation of tryptophan
  • Example 5 the above five mutant sequences were constructed into the plant expression vector pCAMBIA1390 plasmid (purchased from Australian CAMBI company), and transformed into Arabidopsis thaliana. After obtaining a positive transgenic seedling, 60 g of aiha -1 was sprayed at the seedling stage. Glyoxysin was tested for resistance.

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Abstract

L'invention concerne un gène de colza qui est résistant aux herbicides à base d'acide salicylique de pyrimidine et son utilisation, et concerne en outre une plante de colza et des parties de celle-ci qui peuvent résister à des herbicides à base d'acide salicylique de pyrimidine.
PCT/CN2018/102232 2018-08-24 2018-08-24 Gène de colza résistant aux herbicides à base d'acide salicylique de pyrimidine et son utilisation WO2020037648A1 (fr)

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DE112018006599.5T DE112018006599T5 (de) 2018-08-24 2018-08-24 Gegen Pyrimidinylsalicylat-Herbizid resistentes Rapsgen und dessen Verwendung
CN201880072169.4A CN112154207B (zh) 2018-08-24 2018-08-24 油菜抗嘧啶水杨酸类除草剂基因及其应用
PCT/CN2018/102232 WO2020037648A1 (fr) 2018-08-24 2018-08-24 Gène de colza résistant aux herbicides à base d'acide salicylique de pyrimidine et son utilisation
CA3087906A CA3087906A1 (fr) 2018-08-24 2018-08-24 Gene de colza resistant aux herbicides a base d'acide salicylique de pyrimidine et son utilisation

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CN108330116A (zh) * 2018-02-07 2018-07-27 北京大北农生物技术有限公司 除草剂耐受性蛋白质、其编码基因及用途
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CN108330116A (zh) * 2018-02-07 2018-07-27 北京大北农生物技术有限公司 除草剂耐受性蛋白质、其编码基因及用途

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HU , MAOLONG ET AL.: "Enzymatic Characteristics of Acetolactate Synthase Mutant S638N in Brassica Napus and Its Resistance to ALS Inhibitor Herbicides", ACTA AGRONOMICA SINICA, vol. 41, no. 9, 30 September 2015 (2015-09-30), pages 1353 - 1360, ISSN: 0496-3490 *
SIBONY, M. ET AL.: "Molecular basis for multiple resistance to acetolactate synthase-inhibiting herbicides and atrazine in Amaranthus blitoides (prostrate pigweed", PLANTA, vol. 216, 18 December 2002 (2002-12-18), pages 1022 - 1027, XP002631640, ISSN: 1432-2048 *

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