WO2020037642A1 - 油菜抗三唑嘧啶磺酰胺类除草剂基因及其应用 - Google Patents

油菜抗三唑嘧啶磺酰胺类除草剂基因及其应用 Download PDF

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WO2020037642A1
WO2020037642A1 PCT/CN2018/102175 CN2018102175W WO2020037642A1 WO 2020037642 A1 WO2020037642 A1 WO 2020037642A1 CN 2018102175 W CN2018102175 W CN 2018102175W WO 2020037642 A1 WO2020037642 A1 WO 2020037642A1
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seq
mutated
acetolactate synthase
plant
leucine
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PCT/CN2018/102175
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French (fr)
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胡茂龙
浦惠明
龙卫华
高建芹
张洁夫
陈松
程丽
彭琦
陈锋
周晓婴
张维
付三雄
王晓东
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江苏省农业科学院
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Priority to PCT/CN2018/102175 priority Critical patent/WO2020037642A1/zh
Priority to CN201880071918.1A priority patent/CN111373035B/zh
Priority to CA3084844A priority patent/CA3084844A1/en
Priority to DE112018006097.7T priority patent/DE112018006097T5/de
Publication of WO2020037642A1 publication Critical patent/WO2020037642A1/zh

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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
    • 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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8274—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for herbicide resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • 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 triazole pyrimidinesulfonamide herbicide genes and applications thereof. More specifically, the present invention relates to a rape plant tolerant to a triazolopyrimidine sulfonamide herbicide, a part thereof, a resistance gene, a mutein, and an application thereof.
  • Rapeseed is the oilseed crop with the largest area sown in China and the most widely distributed area. China is the country that produces the most rapeseed in the world.
  • 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. severely affected the yield and quality of rape 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.
  • the acetolactate synthase inhibitors that have been developed include herbicides such as sulfonylureas, imidazolinones, pyrimidinesalicylic acid, and triazolopyrimidines (
  • triazopyrimidine sulfonamide herbicides have developed very rapidly since they were successfully developed in the 1990s. Nearly 10 commercial varieties have been reported, mainly diflusulfuron, Flubensulfuron, mesosulfuril, penoxsulam and the like.
  • the mechanism of action of triazolopyrimidine sulfonamide herbicides is similar to that of sulfonylurea herbicides, and it is a typical acetolactate synthase inhibitor.
  • Triazole pyrimidine sulfonamide herbicides reduce the activity of ALS in plants, and the synthesis of valine, leucine and isoleucine is inhibited, affecting protein synthesis and causing plant growth to stop and die.
  • Plant roots and leaves absorb drugs, which are conducted throughout the body, accumulate in meristematic tissues, and inhibit cell division.
  • the typical symptoms of weed damage are: chlorosis of leaf leaves, fading of veins, whitening or purplening of leaves, shortening of internodes, death of terminal buds, and death of the whole plant to kill weeds.
  • 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 on rapeseed plants to triazopyrimidinesulfonamide herbicides, wherein the ALS polypeptide encoded by the ALS gene is at position 556 Contains an amino acid different from tryptophan and contains an amino acid different from proline at position 179.
  • the ALS polypeptide encoded by the ALS gene has a double mutation selected from the group consisting of: W556L and P179S; W556L and P179T; W556L and P179L; W556L and P179A.
  • the ALS polypeptide encoded by the ALS gene has the following mutations: W556L and P179S.
  • the invention provides an isolated nucleic acid encoding a mutant acetolactate synthase (ALS3), said mutant acetolactate synthase (ALS3) protein comprising the following mutations:
  • Proline (P) is mutated to serine (S), threonine (T), leucine (L) or alanine (A) at a position corresponding to position 179 of SEQ ID NO: 2;
  • 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 to produce a herbicide-resistant plant.
  • the plant is rapeseed.
  • the present invention provides a method for producing a herbicide-resistant plant, which is characterized by comprising 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 resistance to triazolopyrimidine sulfonamides.
  • 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 triazole pyrimidinesulfonamide herbicide, comprising an isolated nucleic acid encoding a mutant acetolactate synthase protein, said mutant acetolactate synthase protein Contains the following mutations:
  • Proline (P) is mutated to serine (S), threonine (T), leucine (L) or alanine (A) at the position corresponding to position 179 of SEQ ID NO: 2,
  • said plant is rape; wherein said parts are organs, tissues and cells of the plant, and preferably seeds;
  • the mutated acetolactate synthase protein comprises a tryptophan (W) mutation at a position corresponding to position 556 of SEQ ID NO: 2 to a leucine (L) and a SEQ ID NO: Proline (P) was mutated to serine (S) at position 179 of 2;
  • 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 rapeseed plants, the method comprising applying an effective amount of a triazopyrimidinesulfonamide herbicide to the weed and rapeseed plants Field, the rapeseed plant contains an isolated nucleic acid encoding a mutant acetolactate synthase protein, the mutant acetolactate synthase protein comprises the following mutations:
  • Proline (P) is mutated to serine (S), threonine (T), leucine (L) or alanine (A) at a position corresponding to position 179 of SEQ ID NO: 2;
  • the mutated acetolactate synthase protein comprises a tryptophan (W) mutation at a position corresponding to position 556 of SEQ ID NO: 2 to a leucine (L) and a SEQ ID NO: Proline (P) was mutated to serine (S) at position 179 of 2;
  • 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 wild type strain N131
  • ALS3 amino acid partial sequence of ALS3_EM28 resistant material EM28 ALS3 amino acid partial sequence of ALS3_DS6 resistant material DS6.
  • 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 diflusulfuron at different concentrations.
  • 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 proline P ⁇ serine S substitution occurred at position 179 of the polypeptide encoded by the endogenous ALS gene of the rape plant, which is due to the position 535 corresponding to the nucleotide sequence shown in SEQ ID NO: 1
  • the "C” nucleotide is mutated to the "T” nucleotide at the position.
  • the present invention provides a rapeseed plant, and the endogenous ALS gene of the rapeseed plant comprises (or consists of) the nucleotide sequence shown in SEQ ID NO: 3, which encodes SEQ ID mutated ALS 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 triazopyrimidinesulfonamide herbicides described herein (or, Agent is less sensitive). This is due to the point mutations described herein that lead to amino acid substitutions.
  • ALS tolerance to triazopyrimidinesulfonamide herbicides (or, less sensitivity to triazopyrimidinesulfonamide herbicides) can be obtained from ALS is obtained from cell extracts from plants containing mutant ALS sequences and plants without mutant ALS sequences and their activity is measured for comparison, such as the method described in Singh et al (1988) [J. Chromatogr., 444, 251-261].
  • triazolidinesulfonamide 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 “triazolopyrimidinesulfonamide herbicide” may be one herbicide or two, three, four, or more known in the art. Mixtures of herbicides, the herbicides are preferably those listed herein, such as, for example, diflufenacil, saflufenacil, mesosulfuril, penoxsulam and the like.
  • the present invention provides a rape plant with tolerance to a triazolopyrimidinesulfonamide herbicide having an endogenous acetolactate synthase (ALS) gene mutation.
  • ALS acetolactate synthase
  • the term "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.
  • the seed of EM28 plant was deposited on June 19, 2017 at the Common Microbiological Center (CGMCC) of the China Microbial Strain Collection Management Committee, Address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, 100101, deposit number CGMCC No .14299, the taxonomic name of this strain: Brassica napus.
  • CGMCC Common Microbiological Center
  • EMS mutagenesis was the same as before.
  • rapeseed seedlings were almost all dead, and only more than 20 rapeseed seedlings survived and grew normally.
  • the vegetable seedlings grew to the 5-6 leaf stage more than 20 rapeseed suspected to be triazole pyrimidinesulfonamide herbicides were transferred to rapeseed breeding fields, and M3 seeds were harvested by self-bagging during flowering.
  • M3 seeds were harvested by self-bagging during flowering.
  • 3.75 g of aiha -1 diflusulfuron was sprayed at the seedling stage of M3 seeds for resistance effect identification. From 1 week after spraying, observe the phytotoxicity reaction every day.
  • strain number DS6 showed strong resistance, no symptoms of phytotoxicity, and normal growth, while other lines and controls had a phytotoxicity reaction one week after spraying, and the heart leaves of vegetable seedlings began to change. Yellow, and gradually decayed, eventually dying. So far, we have obtained a new Brassica napus germplasm DS6, which is resistant to triazole pyrimidinesulfonamide herbicides, and is temporarily named RT-1. Later, through classical genetics research, it was found that the resistance ratio of RT-1 resistance traits between the live and dead strains in the F 2 generation population was 3: 1, which is consistent with the genetic rule of single dominant genes. That is, the mutation trait locus is controlled by a dominant nuclear gene.
  • Example 2 Molecular cloning of resistance genes in new germplasms of Brassica napus with resistance to triazole pyrimidinesulfonamide herbicides
  • Triazole pyrimidinesulfonamide herbicides are ALS inhibitor herbicides, and the target of the herbicide is acetolactate synthase.
  • the target of the 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 genomic DNA from leaves of resistant strains RT-1 and N131 and EM28, respectively, and the wild-type and mutant ALS1, ALS2, and ALS3 genes were cloned by PCR. 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. Sequencing alignment revealed that point mutations were detected at two sites on the ALS3 gene in the resistant strain RT-1.
  • a point mutation occurs at position +535, and the nucleotide changes from C to T, which results in the mutation of proline (P) to serine (S) at position 179 of the corresponding encoded protein; the point +1667 occurs at the ALS3 gene Mutation, the nucleotide changes from G to T, resulting in mutation of tryptophan (W) to leucine (L) at position 556 of the corresponding encoded protein; ( Figure 1). Therefore, compared with the mutant EM28, the ALS3 gene in the resistant strain has a new mutation site (P179S), 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 (P179S and W556L) increased the resistance of resistant mutants to triazolopyrimidinesulfonamide herbicides.
  • the original material EM28 is resistant to sulfonylurea and imidazolinone herbicides and sensitive to triazopyrimidinesulfonamide herbicides. Therefore, N131 and EM28 were used as control materials to identify and evaluate the resistance effect of resistance mutant RT-1 to sulfonylureas, imidazolinones, and triazolidinesulfonamide herbicides.
  • the identification method is carried out by two methods: field identification and greenhouse pot test.
  • 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.
  • the triazolopyrimidine sulfonamide herbicide diflusulfachlor was sprayed separately [chemical name: 2 ', 6'-difluoro-5-methoxy-8- Fluoro [1,2,4] triazole [1,5-c] pyrimidine-2-sulfonylanilide, SU-type herbicide bensulfuron (2- [N- (4-methoxy-6-methyl- 1,3,5-triazin-2-yl) -N-methylcarbamylsulfonyl] benzoic acid methyl ester) and IMI herbicide is imidazolenicotinic acid ((RS) -5-ethyl-2- ( 4-isopropyl-4-methyl-5-oxo-1H-imidazolin-2-yl) nicotinic acid].
  • 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
  • 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 RT-1 shows strong resistance to the TP herbicide diflubenzulam, because compared with N131 and EM28, with the increase of diflusulfuron concentration, N131 and The ALS enzyme activity in EM28 decreased rapidly and showed the same change trend, while the mutant enzyme activity in RT-1 was less inhibited by herbicides. Even under high concentration (250 ⁇ mol L-1) diflusulfuron, RT The mutant enzyme activity in -1 was about 47.3% of the control. However, at this time, the ALS mutant enzymes in N131 and EM28 were basically inactive, only 8.98% and 19.6% of the control, respectively.
  • the sensitivity of the ALS enzyme in the mutant RT-1 to the TP herbicide disulfachlor is significantly lower than that of the ALS enzymes in N131 and EM28, which further illustrates that the RT-1 ALS3 gene has a double mutation site.
  • the resistance of RT-1 to the TP herbicide disulfachlor was shown.
  • 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 primer 3 5'CGC GGTACC CTCTCTCTCTCTCATCTAACCAT3 'and ALS3 primer 4: 5'CGC ACTAGT CTCTCAGTACTTAGTGCGACC3', 5 'of which were added to 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. After 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.
  • R represents that the plant growth is good after herbicide treatment, and there is no phytotoxicity
  • S represents that the plant growth is severely suppressed after herbicide treatment, showing obvious phytotoxicity, and eventually the vegetable seedlings die.
  • the acetolactate synthase mutation gene of the nucleotide in RT-1 as shown in SEQ ID NO: 3 was cloned into the plant expression vector pCAMBIA1390 plasmid (purchased from CAMBI, Australia).
  • the positive clones were selected to transform Agrobacterium EHA105, and the conventional Agrobacterium-mediated transformation method was used to transform the B. benthamiana leaf discs.
  • 3.75 g aiha -1 was sprayed at the seedling stage of the T3 generation transgenic tobacco. Sulfuramide was identified for resistance.
  • Example 7 Functional verification of resistance genes in common rapeseed
  • RT-1 acetolactate synthase mutation gene of nucleotides in RT-1 as shown in SEQ ID NO: 3 into other common rapeseed varieties or lines that were not resistant to TP herbicides.
  • RT-1 and 3075R Pu Huiming et al., 2002, Jiangsu Agricultural Science, 4: 33-3
  • 3018R Pu Huiming et al., 1999, Jiangsu
  • Agricultural Science, 6: 32-33) Prepare hybrid combinations, harvest 2 F1 seeds in the rape vernalization culture room for additional planting in the same year, select a single plant with consistent growth and self-bagging during flowering, and harvest F2 seeds at Jiangsu Academy of Agricultural Sciences Seeds were planted in the Huangshui Plant Science Base. Each F2 population was sown 20 rows. A single leaf of the F2 population was taken at the seedling stage, DNA was extracted, and the ALS3 mutant gene was amplified by PCR. The product was purified, recovered, and sequenced according to Example 2. According to the sequencing results, a homozygous F2 single strain with an acetolactate synthase mutation gene shown in SEQ ID NO: 1 in RT-1 was screened.
  • each selected F2 single plant was self-bagging, and F3 seeds were harvested.
  • all selected rapeseed seedlings with resistance genes introduced were in good growth condition, but all rapeseed seedlings without resistance genes were yellowed and died, indicating that the nucleotides in RT-1 are shown in SEQ ID NO: 3
  • the acetolactate synthase mutant gene expressed in rapeseed also has anti-TP herbicide function (Table 3).
  • R represents that the plant growth is good after herbicide treatment, and there is no phytotoxicity
  • S represents that the plant growth is severely suppressed after herbicide treatment, showing obvious phytotoxicity, and eventually the vegetable seedlings die.
  • the 535th nucleotide of the ALS3 gene changed from C to A, and the + 1667th nucleotide changed from G to T, which resulted in the 179th position of the corresponding encoded protein.
  • Example 5 the above three mutant sequences were constructed into the plant expression vector pCAMBIA1390 plasmid (purchased from Australian CAMBI company), transformed into Arabidopsis thaliana, and after obtaining a positive seedling, 3.75 g of aiha -1 was sprayed at the seedling stage. Diflubenzulam was identified for resistance. After 3 weeks of spraying treatment, all transgenic Arabidopsis seedlings grew well, while all non-transgenic Arabidopsis seedlings yellowed and died (Table 4), indicating that the sequence of the above three amino acid mutation combinations had Anti-TP herbicide function.

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Abstract

一种耐受三哇嚓咤磺酞胺类除草剂的油菜植物及其部分、抗性基因、突变蛋白及其应用。

Description

油菜抗三唑嘧啶磺酰胺类除草剂基因及其应用 技术领域
本发明涉及植物基因工程技术领域，具体地，涉及油菜抗三唑嘧啶磺酰胺类除草剂基因及其应用。更具体地，本发明涉及耐受三唑嘧啶磺酰胺类除草剂的油菜植物及其部分、抗性基因、突变蛋白及其应用。
背景技术
油菜是我国播种面积最大，地区分布最广的油料作物。我国是世界上生产油菜籽最多的国家。油菜生产过程中一类重要的生物危害是农田杂草，其不但与油菜作物争水争肥争光，而且改变油菜作物田间小气候，甚至有些杂草还是油菜作物病虫害的中间寄主，加快病虫害的蔓延，严重影响了油菜作物产量和品质。然而，人工除草费时、费力，增加生产成本。因此，应用除草剂防治田间杂草成为人们的必然选择。
除草剂主要是通过抑制或干扰植物关键的代谢过程而抑制植物生长或杀死植物。以氨基酸生物合成过程中的关键酶为靶标，是研发新型高效除草剂的一个重要方向和热点。以乙酰乳酸合酶(acetolactate synthase，ALS；EC2.2..16)为靶酶开发的除草剂，己经成为新型高效除草剂的主流产品。ALS是催化支链氨基酸(缬氨酸、亮氨酸和异亮氨酸)生物合成第一步的酶。ALS抑制剂类除草剂能抑制植物细胞内的ALS酶活性，阻碍支链氨基酸(缬氨酸、亮氨酸和异亮氨酸)生物合成，从而抑制植物细胞的分裂和生长。已研制开发的乙酰乳酸合酶抑制剂主要有磺酰脲类、咪唑啉酮类、嘧啶水杨酸类、三唑嘧啶磺酰胺类(triazolopyrimidines，TP)等除草剂。
在众多的乙酰乳酸合酶抑制剂中，三唑嘧啶磺酰胺类除草剂自20世纪90年代开发成功以来发展非常迅速，已报道了近10个商品化的品种，主要有双氟磺草胺、唑嘧磺草胺、双氯磺草胺、五氟磺草胺等。三唑嘧啶磺酰胺类除草剂的作用机制与磺酰脲类除草剂类似，是典型的乙酰乳酸合酶抑制剂。三唑嘧啶磺酰胺类除草剂使植物体内ALS活力降低，缬氨酸、亮氨酸与异亮氨酸的合成受到抑制，影响蛋白质的合成从而导致植物生长停止而死亡。植物根与叶均吸收药剂，在体内周身传导，积累于分生组织，抑制细胞分裂。杂草受害的典型症状是：叶片中脉失绿，叶脉褪色，叶片白化或紫化，节间变短，顶芽死亡，最终全株死亡而起到杀死杂草的目的。
研究发现，ALS上氨基酸替换发生的位点及其位点处替换氨基酸不同产生的抗性功能存在显著差异(Yu Q,Han HP,Martin M,Vila-Aiub,Powles SB.AHAS herbicide resistance endowing mutations:effect on AHAS functionality and plant growth.J Exp Botany,2010,61:3925-3934)。不同位点氨基酸替换产生的ALS抑制剂除草剂抗性效应存在显著差异，同时不同位点突变对其它ALS抑制剂除草剂存在比较复杂的交互抗性关系。
本领域需要获得相对于强生命力的杂草具有生长优势的油菜植物，需要获得可耐受三唑嘧啶磺酰胺类除草剂的非转基因油菜植物。
发明概述
本发明解决了这种需要，并提供了突变的乙酰乳酸合酶(ALS)核酸和这些突变的核酸编码的蛋白。本发明还涉及包含这些突变核酸和蛋白的油菜植物、细胞和种子，所述突变赋予油菜植物对于三唑嘧啶磺酰胺类除草剂的耐受，其中所述ALS基因编码的ALS多肽在其位置556处含有不同于色氨酸的氨基酸并且在其位置179处含有不同于脯氨酸的氨基酸。在优选的实施方式中，所述ALS基因编码的ALS多肽具有选自下列的双重突变：W556L和P179S；W556L和P179T；W556L和P179L；W556L和P179A。在最优选的实施方式中，所述ALS基因编码的ALS多肽具有下列突变：W556L和P179S。
在一个实施方式中，本发明提供了编码突变的乙酰乳酸合酶(ALS3)的分离的核酸，所述突变的乙酰乳酸合酶(ALS3)蛋白包含如下的突变：
在对应于SEQ ID NO:2的位置556的位置处色氨酸(W)突变为亮氨酸(L)；和
在对应于SEQ ID NO:2的位置179的位置处脯氨酸(P)突变为丝氨酸(S)、苏氨酸(T)、亮氨酸(L)或丙氨酸(A)；
优选地，所述的分离的核酸的核苷酸序列如SEQ ID NO：3所示；
优选地，其中所述突变的ALS3蛋白的氨基酸序列如SEQ ID NO:4所示。
在一个方面，本发明提供了表达盒、载体或细胞，其含有本发明所述的核酸。相应地，本发明提供了本发明的核酸、表达盒、载体或细胞或突变的乙酰乳酸合酶(ALS3)蛋白用于产生抗除草剂植物的用途，优选地，所述植物为油菜。
在另一个方面，本发明提供了产生具有除草剂抗性的植物的方法，其特征在于，包括如下步骤：
将本发明所述的核酸导入植物，优选地通过转基因、杂交、回交或无性繁殖等步骤将本发明所述的核酸导入植物，其中所述植物表达本发明所述的突变的乙酰乳酸合酶(ALS3)蛋白并具有三唑嘧啶磺酰胺类除草剂抗性。
在又一个方面，本发明提供了抗三唑嘧啶磺酰胺类除草剂的非转基因植物或其部分，其包含编码突变的乙酰乳酸合酶蛋白的分离的核酸，所述突变的乙酰乳酸合酶蛋白包含如下的突变：
在对应于SEQ ID NO:2的位置556的位置处色氨酸(W)突变为亮氨酸(L)；和
在对应于SEQ ID NO:2的位置179的位置处脯氨酸(P)突变为丝氨酸(S)、苏氨酸(T)、亮氨酸(L)或丙氨酸(A)，
优选地，其中所述植物是油菜；其中所述部分为植物的器官、组织和细胞，并且优选种子；
优选地，其中所述突变的乙酰乳酸合酶蛋白包含在对应于SEQ ID NO:2的位置556的位置处色氨酸(W)突变为亮氨酸(L)和在对应于SEQ ID NO:2的位置179的位置处脯氨酸(P)突变为丝氨酸(S)；
更优选地，其中所述突变的ALS3蛋白的氨基酸序列如SEQ ID NO:4所示。
在另一个方面，本发明提供了在含有油菜植物的田地中控制杂草的方法，所述方法包括施用有效量的三唑嘧啶磺酰胺类除草剂至含有所述杂草和油菜植物的所述田地，所述油菜植物包含编码突变的乙酰乳酸合酶蛋白的分离的核酸，所述突变的乙酰乳酸合酶蛋白包含如下的突变：
在对应于SEQ ID NO:2的位置556的位置处色氨酸(W)突变为亮氨酸(L)；和
在对应于SEQ ID NO:2的位置179的位置处脯氨酸(P)突变为丝氨酸(S)、苏氨酸(T)、亮氨酸(L)或丙氨酸(A)；
优选地，其中所述突变的乙酰乳酸合酶蛋白包含在对应于SEQ ID NO:2的位置556的位置处色氨酸(W)突变为亮氨酸(L)和在对应于SEQ ID NO:2的位置179的位置处脯氨酸(P)突变为丝氨酸(S)；
更优选地，其中所述突变的ALS3蛋白的氨基酸序列如SEQ ID NO:4所示。
附图说明
图1显示了不同来源的油菜ALS3氨基酸部分序列比对结果。
ALS3，Genbank上参考序列(登录号：Z11526)；ALS3_N131野生型品系N131的ALS3氨基酸部分序列；ALS3_EM28抗性材料EM28的ALS3氨基酸部分序列；ALS3_DS6抗性材料DS6的ALS3氨基酸部分序列。箭头表示突变氨基酸。
图2显示了不同浓度的苯磺隆对野生型和突变体的ALS酶活性体外抑制。
图3显示了不同浓度的咪唑乙烟酸对野生型和突变体的ALS酶活性体外抑制。
图4显示了不同浓度的双氟磺草胺对野生型和突变体的ALS酶活性体外抑制。
发明详述
通过参考在附图中描述和/或说明的并且在下面说明书中详细说明的非限制性实施方案以及实例将更全面地说明本发明的实施方案以及它们的不同特征以及有利的细节。应当注意的是在附图中所述的特征不必按比例绘制，并且当本领域技术人员可以认可时，一个实施方案的特征可以与其他实施方案一起使用，尽管在此没有清楚地说明。
定义
除非另外说明，权利要求以及说明书中所使用的术语是如下面列出定义的。
术语“非转基因”是指没有通过适当的生物载体或通过任何其他物理方式引入各个基因。但是，突变的基因可通过授粉(自然地或通过育种方法)被传递，以产生另一种含有该特定基因的非转基因植物。
“内源”基因意指植物中不是通过基因工程技术引入到所述植物中的基因。
术语“核苷酸序列”、“多核苷酸”、“核酸序列”、“核酸”、“核酸分子”在本文中可互换使用，其是指任意长度的聚合无支链形式的核苷酸，核糖核苷酸或者脱氧核糖核苷酸或其二者组合。核酸序列包括DNA、cDNA、基因组DNA、RNA，包括合成形式以及混合的聚合物，包括正义链和反义链，或可以含有非天然的或衍生的核苷酸碱基，本领域技术人员可理解这点。
当用于本文时，术语“多肽”或“蛋白质”(本文中这两个术语可互换地使用)意指包含给定长度的氨基酸链的肽、蛋白质或多肽，其中所述氨基酸残基通过共价的肽键连接。但是，本发明也包括所述蛋白质/多肽的肽模拟物(其中氨基酸和/或肽键已经被功能性类似物替换)，以及除了所述20种基因编码的氨基酸之外的氨基酸例如硒代半胱氨酸。肽、寡肽和蛋白质可被称为多肽。所述术语多肽还指(不排除)多肽的修饰，例如糖基化、乙酰化、磷酸化等。这种修饰很好地记载于基础文献中，并更详细地记载于专著以及研究文 献中。
氨基酸取代包括氨基酸改变，其中氨基酸被不同的天然存在的氨基酸残基代替。这种取代可被分类为“保守的”，其中野生型ALS蛋白质中含有的氨基酸残基被另外的天然存在且特性相似的氨基酸替换，例如或本发明中包括的取代还可能是“非保守的”，其中存在于野生型ALS蛋白质中的氨基酸残基被具有不同性质的氨基酸取代，例如来自于不同组的天然存在的氨基酸(例如用丙氨酸取代带电荷的或疏水的氨基酸)。本文使用的“相似的氨基酸”是指具有相似的氨基酸侧链的氨基酸，即具有极性、非极性或接近中性的侧链的氨基酸。本文使用的“不相似氨基酸”是指具有不同氨基酸侧链的氨基酸，例如具有极性侧链的氨基酸与具有非极性侧链的氨基酸是不相似的。极性侧链通常趋向存在于蛋白质的表面，在此它们可以与细胞中存在的水环境相互作用(“亲水性”氨基酸)。另一方面，“非极性”氨基酸趋向于位于蛋白质内的中心，在此它们可以与相似的非极性相邻分子相互作用(“疏水性”氨基酸)。具有极性侧链的氨基酸的实例为精氨酸、天冬酰胺、天冬氨酸、半胱氨酸、谷氨酰胺、谷氨酸、组氨酸、赖氨酸、丝氨酸和苏氨酸(全部为亲水性氨基酸，除了半胱氨酸为疏水性的)。具有非极性侧链的氨基酸的实例为丙氨酸、甘氨酸、异亮氨酸、亮氨酸、甲硫氨酸、苯丙氨酸、脯氨酸和色氨酸(全部为疏水性的，除了甘氨酸为中性的)。
通常，本领域技术人员根据其通常的常识和使用术语ALS、ALSL、AHAS或AHASL的上下文即可知晓分别是指所述核苷酸序列或核酸，或者是指所述氨基酸序列或多肽。
当用于本文时，术语“基因”是指任何长度的核苷酸(核糖核苷酸或脱氧核糖核苷的聚合形式。该术语包括双链和单链的DNA和RNA。其还包括已知类型的修饰，例如甲基化、“加帽”、用类似物取代一个或多个天然存在的核苷酸。优选地，基因包括编码本文所定义的多肽的编码序列。“编码序列”是当被置于或在适当调节序列的控制下可被转录为mRNA和/或被翻译为多肽的核苷酸序列。所述编码序列的界限是由5'末端的翻译起始密码子和3'末端的翻译终止密码子确定的。编码序列可以包括但不限于mRNA、cDNA、重组核酸序列或基因组DNA，但是某些情况下也可能存在内含子。
当用于本文时，术语“甘蓝型油菜(Brassica napus)”可缩写为“油菜(B.napus)”。此外，本文中使用了术语“油菜”。所述三个术语可互换使用并且应被理解为完全包括栽培形式的油菜。相似地，例如术语“拟南芥(Arabidopsis thaliana)”可缩写为“拟南芥(A.thaliana)”。在本文中这两个术语可互换地使用。
当用于本发明时，术语“位置”意指氨基酸在本文描述的氨基酸序列中的位置或核苷酸在本文描述的核苷酸序列中的位置，例如SEQ ID NO:1所示的野生型油菜ALS3蛋白的编码序列或SEQ ID NO:2所示的野生型油菜ALS3蛋白的氨基酸序列中的位置或其对应位置。本文使用的术语“对应的”还包括不仅由前述核苷酸/氨基酸的编号所确定的位置。由于在ALS 5'非翻译区(UTR)(包括启动子和/或任何其他的调节序列)或基因(包括外显子和内含子)中其他位置处核苷酸的缺失或插入，本发明中可被取代的给定核苷酸的位置可以不同。相似地，由于在ALS多肽中其他位置氨基酸的缺失或插入，本发明中可被替换的给定氨基酸的位置可以不同。因此，本发明中“对应位置”应被理解为在所指示编号处的核苷酸/氨基酸可以不同，但仍然可具有相似的相邻核苷酸/氨基酸。所述可被交换、缺失或插入的核苷酸/氨基酸也被术语“对应位置”所包括。为了确定在给定的ALS核苷酸/氨基酸序列中核苷酸残基或氨基酸残基是否对应于核苷酸序列SEQ ID NO:1或氨基酸序列SEQ ID NO:2中的某些位置，本领域技术人员可使用本领域中公知的工具和方法，例如人工地或通过使用计算机程序的比对，例如BLAST(Altschul et al.(1990),Journal of Molecular Biology,215,403-410)(其代表基本局部比对搜索工具)或ClustalW(Thompson et al.(1994),Nucleic Acid Res.,22,4673-4680)或任何其他适合于产生序列比对的适合程序。
具体地，本发明提供了一种油菜植物，在所述油菜植物的内源ALS基因编码的多肽位置556处发生色氨酸W→亮氨酸L取代，这是由于在对应于SEQ ID NO:1中示出的核苷酸序列位置1667的位置处"G"核苷酸突变为"T"核苷酸。并且，在所述油菜植物的内源ALS基因编码的多肽位置179处发生脯氨酸P→丝氨酸S取代，这是由于在对应于SEQ ID NO:1中示出的核苷酸序列位置535的位置处"C"核苷酸突变为"T"核苷酸。在最优选的实施方案中，本发明提供了一种油菜植物，在所述油菜植物的内源ALS基因包含SEQ ID NO:3所示的核苷酸序列(或由其组成)，其编码SEQ ID NO:4所示的突变的ALS多肽。
可根据Singh(1991),Proc.Natl.Acad.Sci.88:4572-4576中描述的测定法来测量ALS活性。本文提到的编码ALS多肽的ALS核苷酸序列优选地可赋予对本文所述的一种或多种三唑嘧啶磺酰胺类除草剂的耐受性(或者，对三唑嘧啶磺酰胺类除草剂更低的敏感度)。这是由于本文所述的导致氨基酸取代的点突变。因此，对三唑嘧啶磺酰胺类除草剂的耐受性(或者，对三唑嘧啶磺酰胺类除草剂更低的敏感度)可通过在存在三唑嘧啶磺酰胺 类除草剂的情况下，从来自含有突变ALS序列的植物和没有突变ALS序列的植物的细胞提取物中获得ALS并比较其活性来测量，例如Singh et al(1988)[J.Chromatogr.,444,251-261]中描述的方法。当使用植物时，优选地在存在多种浓度的三唑嘧啶磺酰胺类除草剂的情况下，更优选在存在多种浓度的三唑嘧啶磺酰胺类除草剂"双氟磺草胺的情况下，在野生型的细胞提取物或叶提取物以及在所获得突变体的油菜细胞提取物或叶提取物中测定ALS活性。相似地，“耐受性更高”或“抗性更高”，反之亦然，可被看作是“敏感度更低”。
术语“三唑嘧啶磺酰胺类除草剂”不意图受限于可干扰ALS酶活性的单一除草剂。因此，除非另有说明或可从上下文中明显看出，否则“三唑嘧啶磺酰胺类除草剂”可以是本领域中已知的一种除草剂或者两种、三种、四种或多种除草剂的混合物，所述除草剂优选为本文所列出的那些，例如双氟磺草胺、唑嘧磺草胺、双氯磺草胺、五氟磺草胺等。
本发明提供了具有内源性乙酰乳酸合酶(ALS)基因突变的可耐受三唑嘧啶磺酰胺类除草剂的油菜植物。用于本文时，除非另有明确说明，否则术语“植物”意指处在任何发育阶段的植物。植物的部分可被连接到整个完整植物或者可从整个完整植物分离。这样的植物的部分包括但不限于植物的器官、组织和细胞，优选种子。本发明的油菜植物在内源ALS基因方面是非转基因的。当然，可通过基因工程或通过常规方法例如杂交来将外源基因转移到所述植物中。
以下基于实施例对本发明进行描述，但是本发明并不仅仅限于这些实施例。
实施例1
在之前申请的专利(胡茂龙等，中国专利：CN 107245480 A，具有除草剂抗性的乙酰乳酸合酶突变蛋白及其应用)中，通过对野生型油菜品系N131(公知公用，见浦惠明等，江苏农业学报，2010，26(6)：1432-1434)进行甲磺酸乙酯(EMS)诱变处理，在诱变的M2代，我们筛选并鉴定获得了抗磺酰脲类除草剂突变体EM28。EM28植株种子于2017年06月19日保藏于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，地址：北京市朝阳区北辰西路1号院3号，邮编：100101，保藏编号为CGMCC No.14299，该菌株的分类命名为：甘蓝型油菜(Brassica napus)。为期望获得抗三唑嘧啶磺酰胺类除草剂油菜种质或资源满足抗除草剂油菜品种选育的需求，我们再次对EM28种子进行 EMS诱变处理，EMS诱变方法同前。待M2代菜苗长至3-4叶期时，喷施三唑嘧啶磺酰胺类除草剂双氟磺草胺[化学名称：2',6'-二氟-5-甲氧基-8-氟[1,2,4]三唑[1,5-c]嘧啶-2-磺酰苯胺。分子式：C12H8F3N5O3S。CAS号：145701-23-1]，喷施杂草防治推荐使用浓度3.75g a.i.ha –1的双氟磺草胺，进行抗三唑嘧啶磺酰胺类除草剂种质的筛选。处理3周后，油菜幼苗几乎全部接近死亡，只有20多株菜苗存活并正常生长。待菜苗生长至5-6叶期后，将20多株疑似为抗三唑嘧啶磺酰胺类除草剂油菜移至油菜育种大田，当年花期套袋自交收获得到M3种子。在光照培养室内，对M3种子苗期喷施3.75g a.i.ha –1的双氟磺草胺，进行抗性效应鉴定。从喷药1周开始，每天观察药害反应。结果发现，其中编号为DS6的株系表现出较强抗性，无任何药害症状，能正常生长，而其它株系和对照在喷药后1周就有药害反应，菜苗心叶开始变黄，并渐渐腐烂，最后死亡。至此，我们获得了抗三唑嘧啶磺酰胺类除草剂甘蓝型油菜新种质DS6，暂命名为RT-1。后期通过经典遗传学研究发现，RT-1的抗性性状在F 2代群体中的成活株和死亡株的分离比例为3：1，符合单显性基因的遗传规律。也就是说，突变性状位由1个显性核基因控制。
实施例2：抗三唑嘧啶磺酰胺类除草剂甘蓝型油菜新种质中抗性基因的分子克隆
三唑嘧啶磺酰胺类除草剂属于ALS抑制剂类除草剂，除草剂的靶标是乙酰乳酸合酶。在甘蓝型油菜基因组内共有3个具有功能的乙酰乳酸合酶基因，分别是位于A基因组ALS2和ALS3(Genebank登录号：Z11525和Z11526)，C基因组的ALS1(Genebank登录号：Z11524)。根据这3个ALS基因序列，分别设计3对PCR引物。ALS1引物1：GTGGATCTAACTGTTCTTGA和引物2：AGAGATGAAGCTGGTGATC。ALS2引物1：GAGTGTTGCGAGAAATTGCTT和引物2：TTGATTATTCTATGCTCTCTTCTG。ALS3引物1：ATGGTTAGATGAGAGAGAGAGAG和引物2：GGTCGCACTAAGTACTGAGAG。采用CTAB法分别提取抗性株系RT-1和N131、EM28的叶片基因组DNA，PCR克隆野生型与突变体ALS1、ALS2和ALS3基因。按东洋纺(上海)生物科技有限公司高保真性DNA聚合酶KOD-Plus试剂盒说明书配制50μL PCR反应体系。在MJ Research PTC-200型PCR仪上进行扩增，反应程序为94℃预变性5min；94℃变性30s，55℃退火30s，72℃延伸2.5min，共35个循环。产物经平末端加A后，在1.2％(V/W)琼脂糖凝胶电泳分离后，用北京Tiangen公司生产的琼脂糖凝胶DNA回收试剂盒(目录号：DP209)纯化回收，纯化的PCR产物委托南京金斯瑞生物有限公司测序。测序比对发现，抗性株系RT-1在ALS3基因上检测到两个位点发生了点突变。即，第+535处发生点突变，核苷酸由C变为T，导致相应编码 蛋白的第179位由脯氨酸(P)突变为丝氨酸(S)；ALS3基因的第+1667处发生点突变，核苷酸由G变为T，导致相应编码蛋白的第556位由色氨酸(W)突变为亮氨酸(L)；(图1)。因此，与突变体EM28相比，抗性株系中的ALS3基因增加了1个新的突变位点(P179S)，其核苷酸如SEQ ID NO：3所示，氨基酸序列如SEQ ID NO：4所示。ALS3基因的双位点突变(P179S和W556L)增加了抗性突变体对三唑嘧啶磺酰胺类除草剂的抗性。
实施例3：抗性株系的除草剂抗性效应评价与鉴定
原始材料EM28对磺酰脲类、咪唑啉酮类除草剂具有抗性而对三唑嘧啶磺酰胺类除草剂敏感。因此以N131和EM28作为对照材料，鉴定评价抗性突变体RT-1对磺酰脲类、咪唑啉酮类和三唑嘧啶磺酰胺类除草剂的抗性效应。鉴定方法采用田间鉴定和温室盆栽试验两种方法进行。油菜田间鉴定试验在江苏省农业科学院油菜隔离繁殖区进行，温室盆栽试验在恒温光照培养室中进行。待所有处理材料播种出苗生长至3-4叶苗龄，分别喷施三唑嘧啶磺酰胺类除草剂双氟磺草胺[化学名称：2',6'-二氟-5-甲氧基-8-氟[1,2,4]三唑[1,5-c]嘧啶-2-磺酰苯胺、SU类除草剂苯磺隆(2-[N-(4-甲氧基-6-甲基-1,3,5-三嗪-2-基)-N-甲基氨基甲酰胺基磺酰基]苯甲酸甲酯)、IMI类除草剂为咪唑乙烟酸[(RS)-5-乙基-2-(4-异丙基-4-甲基-5-氧代-1H-咪唑啉-2-基)烟酸]。喷药3周后，根据菜苗的生长表现确定它们的在不同用药浓度下的抗性效应，结果如表1所示。从表1可以看出，突变抗性材料RT-1增加了对三唑嘧啶磺酰胺类除草剂的抗性。
表1不同浓度的ALS抑制剂除草剂处理3个油菜后的抗性表现
Figure PCTCN2018102175-appb-000001
表注：R代表除草剂处理后油菜植株生长良好，无药害表现；S代表除草剂处理后油菜植株生长受到严重抑制，表现明显药害，最终菜苗死亡(以下同)。
实施例4：除草剂对ALS酶活性的抑制性试验
根据抗性表型鉴定结果，在体外进行酶活性离体测定试验，比较RT-1、EM28和野生型N131的中ALS酶被3种类型除草剂双氟磺草胺(TP类)、苯磺隆(SU类)和咪唑乙烟酸(IMI类)的抑制影响，比较3个材料间的差异。ALS酶活性测定参照Singh等的方法(Singh BK,et al.,Analytical Biochemistry,1988,171:173-179)。具体的，分别 取0.2g叶片样品，在研钵中用液氮研磨粉碎，将磨好的样品加入含有4.5ml的初酶提取液[100mM K2HPO4、0.5mM MgCl2、0.5mM硫胺素焦磷酸(TPP)、10μM黄素腺嘌呤双核苷酸(FAD)、10mM丙酮酸钠、10％(v/v)丙三醇、1mM二硫苏糖醇、1mM苯甲基磺酰氟(PMSF)、0.5％(w/v)聚乙烯基吡咯烷酮]中，于4℃、12000rpm离心20min。取上清液，加入等体积的饱和(NH4)2SO4，于冰上放置30min后，4℃、12000rpm离心20min，弃上清液，加入1mL初酶提取液，振荡溶解，获得每个样品的ALS酶液。取200μL提取好的ALS酶液，分别加入360μL 50mMHepes-NaOH(PH＝7.5)酶反应缓冲液、80μL 20mM TPP、80μL200μMFAD、80μL2M丙酮酸钠+200mM MgCl2和不同浓度的ALS类除草剂，混匀、放入37℃反应1h后，加入160μL 3M H2SO4终止反应，60℃脱羧15min。然后加入780μL5.5％α-萘酚溶液和780μL0.55％肌酸，65℃显色15min，在530nm比色，读取吸光值，根据标准曲线计算酶活性。将未加入除草剂对照的ALS酶活性分别记为100％，计算双氟磺草胺(TP类)、苯磺隆(SU类)和咪唑乙烟酸(IMI类)除草剂对RT-1、EM28和原始野生型N131的ALS酶活性的影响。
从图2和3可以看出，随着SU类除草剂苯磺隆和IMI类除草剂咪唑乙烟酸浓度的增加，野生型N131、EM28和RT-1的ALS酶活性均受到抑制，但EM28和RT-1中的突变酶都表现对除草剂具有一定抗性，因为与野生型N131相比，随苯磺隆和咪唑乙烟酸浓度的增加，EM28和RT-1中的ALS酶活性抑制下降趋势较缓，表明EM28和RT-1中的ALS突变酶对这2种除草剂具有抗性。同时，EM28和RT-1的下降变化趋势基本相同，表明RT-1和EM28对SU类、IMI类除草剂抗性效应基本相当，与表型鉴定结果一致。
从图4可以看出，RT-1中的突变酶表现对TP类除草剂双氟磺草胺具有较强抗性，因为与N131和EM28相比，随双氟磺草胺浓度增加，N131和EM28中的ALS酶活性快速下降，且变化趋势相同，而RT-1中的突变酶活性受到除草剂抑制程度较轻，即使在高浓度(250μmol L-1)双氟磺草胺条件下，RT-1中的突变酶活性是对照的47.3％左右。然而此时N131和EM28中的ALS突变酶基本没有了活性，分别仅为对照的8.98％和19.6％。综上，突变体RT-1中的ALS酶对TP类除草剂双氟磺草胺的敏感性显著低于N131和EM28中ALS酶，从而进一步说明，RT-1的ALS3基因双突变位点赋予了RT-1对TP类除草剂双氟磺草胺抗性。
实施例5：抗性基因在拟南芥中的功能验证
构建植物表达载体，通过常规的农杆菌介导法将抗性基因转入拟南芥植株，在转基 因后代中PCR筛选阳性纯合转基因株系进行除草剂表型鉴定。过程简言之，根据ALS3基因序列设计特异引物，ALS3引物3：5'CGC GGTACCCTCTCTCTCTCTCATCTAACCAT3'和ALS3引物4：5'CGC ACTAGTCTCTCAGTACTTAGTGCGACC3'，其5'分别加入KpnI和SpeI酶切修饰位点，下划线序列为酶切位点。以突变体RT-1的基因组DNA为模板，PCR扩增获得抗性基因，其核苷酸如SEQ ID NO：3所示，氨基酸序列如SEQ ID NO：4所示。PCR产物按实施例2方法经回收、克隆、测序，获得带有突变酶编码基因的重组T载体。用KpnI和SpeI双酶切T载体获得含目的基因的片段回收、连接到同样经双酶切的pCAMBIA1390载体上(购自澳大利亚CAMBI公司)，得到重组的植物表达载体。将构建好的重组载体转化大肠杆菌DH5α，提取质粒用于酶切和测序检测。将检测表明正确的含有目的基因的重组载体转化农杆菌EHA105菌株，提取质粒进行PCR和酶切鉴定。培养获得的重组菌株，利用农杆菌侵染花序法(flower dipping)转化拟南芥。T0代中在培养基上经抗生素筛选后，获得T1代植株移栽到盆钵中，置于人工培养箱中生长，PCR筛选、扩繁获得T3代的纯合转基因株系。在T3代转基因苗期，喷施3.75g a.i.ha –1双氟磺草胺进行抗性鉴定。喷药处理3周后，所有转基因拟南芥幼苗生长状态良好，而野生型拟南芥(Col)幼苗全部黄化死亡(表2)，表明RT-1中核苷酸如SEQ ID NO：3所示的乙酰乳酸合酶突变基因在拟南芥中表达具有抗TP类除草剂的功能。
表2抗性基因在拟南芥和烟草中表达的抗性表现
Figure PCTCN2018102175-appb-000002
表注：R代表除草剂处理后植株生长良好，无药害表现；S代表除草剂处理后植株生长受到严重抑制，表现明显药害，最终菜苗死亡。
实施例6：抗性基因在烟草中的功能验证
按照实施例5的方法，将RT-1中核苷酸如SEQ ID NO：3所示的乙酰乳酸合酶突变基因克隆至植物表达载体pCAMBIA1390质粒(购自澳大利亚CAMBI公司)中。挑选阳性克隆转化农杆菌EHA105，采用常规的农杆菌介导法转化本氏烟叶盘，获得转基因植株烟 草收种后，经PCR鉴定，在T3代转基因烟草苗期，喷施3.75g a.i.ha –1双氟磺草胺进行抗性鉴定。喷药处理3周后，所有转基因烟草幼苗生长状态良好，而野生型烟草(Tob)幼苗全部黄化死亡(表2)，表明RT-1中核苷酸如SEQ ID NO：3所示的乙酰乳酸合酶突变基因在烟草中表达也具有抗TP类除草剂的功能。
实施例7：抗性基因在普通油菜中的功能验证
采用杂交转育方法将RT-1中核苷酸如SEQ ID NO：3所示的乙酰乳酸合酶突变基因导入其它对TP类除草剂无抗性的普通油菜品种或品系。过程简言之，用RT-1分别与无抗性的普通油菜品种恢复系3075R(浦惠明等，2002，江苏农业科学，4：33-34)和3018R(浦惠明等，1999，江苏农业科学，6：32-33)配制杂交组合，当年收获2个F1种子在油菜春化培养室进行加代种植，花期选生长一致的单株套袋自交，收获F2种子在江苏省农科院溧水植物科学基地播种，每个F2群体播种20行，苗期取F2群体单株叶片，提取DNA，PCR扩增ALS3突变基因，产物按实例2步骤纯化、回收、测序。根据测序结果，筛选具有RT-1中核苷酸如SEQ ID NO：1所示的乙酰乳酸合酶突变基因的纯合型F2单株。于油菜开花期对每个入选的F2单株套袋自交，收获F3种子。在F3代苗期，喷施3.75ga.i.ha –1双氟磺草胺进行抗性鉴定。喷药处理3周后，所有入选的导入抗性基因的油菜幼苗生长状态良好，而未含抗性基因的油菜幼苗全部黄化死亡，表明RT-1中核苷酸如SEQ ID NO：3所示的乙酰乳酸合酶突变基因在油菜中表达也具有抗TP类除草剂的功能(表3)。
表3抗性基因转育到普通油菜中的抗性表现
Figure PCTCN2018102175-appb-000003
表注：R代表除草剂处理后植株生长良好，无药害表现；S代表除草剂处理后植株生长受到严重抑制，表现明显药害，最终菜苗死亡。
实施例8：抗性突变位点不同氨基酸替换的抗性功能研究
为明确本发明中ALS3在Pro179和Trp556的两个位点突变成其他氨基酸后产生的抗性功能，我们通过查阅大量相关文献，设计了3种氨基酸突变组合(表4)，通过人工引入点突变位点并构建其植物表达载体，转化拟南芥验证其抗性功能。过程简言之，以突变体RT-1基因组为模板，利用PCR技术进行定点突变操作，实验委托南京钟鼎生物技术有限公司完成。结果获得了3个突变基因分别为：4T，ALS3基因的第+535处核苷酸由C变为A，第+1667处核苷酸由G变为T，分别导致相应编码蛋白的第179位由脯氨酸(P)突变为苏氨酸(T)，第556位由色氨酸(W)突变为亮氨酸(L)；4L，ALS3基因的第+536处核苷酸由C变为T，第+1667处核苷酸由G变为T，分别导致相应编码蛋白的第179位由脯氨酸(P)突变为亮氨酸(L)，第556位由色氨酸(W)突变为亮氨酸(L)；4A，ALS3基因的第+535处核苷酸由C变为G，第+1667处核苷酸由G变为T，分别导致相应编码蛋白的第179位由脯氨酸(P)突变为丙氨酸(A)，第556位由色氨酸(W)突变为亮氨酸(L)(表4)。
按照实施例5的方法，将上述3个突变序列构建至植物表达载体pCAMBIA1390质粒(购自澳大利亚CAMBI公司)中，转化拟南芥，获得阳性苗后，在苗期喷施3.75g a.i.ha –1的双氟磺草胺进行抗性鉴定。喷药处理3周后，所有转基因拟南芥幼苗生长状态良好，而未转基因的拟南芥幼苗全部黄化死亡(表4)，表明以上3种氨基酸突变组合的序列在拟南芥中表达具有抗TP类除草剂的功能。
表4不同氨基酸突变组合序列在拟南芥中的抗性表现
Figure PCTCN2018102175-appb-000004
注：斜体加粗字母表示突变的碱基，R代表除草剂处理后，转基因植株生长良好，无药害表现。
虽然以上描述了本发明的具体实施方式，但是本领域的技术人员应当理解，这些仅是举例说明，本发明的保护范围是由所附权利要求书限定的。本领域的技术人员在不背离本发明的原理和实质的前提下，可以对这些实施方式作出多种变更或修改，但这些变 更和修改均落入本发明的保护范围。

Claims (7)

  1. 编码突变的乙酰乳酸合酶蛋白的分离的核酸，所述突变的乙酰乳酸合酶蛋白包含如下的突变：
    在对应于SEQ ID NO:2的位置556的位置处色氨酸(W)突变为亮氨酸(L)；和
    在对应于SEQ ID NO:2的位置179的位置处脯氨酸(P)突变为丝氨酸(S)、苏氨酸(T)、亮氨酸(L)或丙氨酸(A)；
    优选地，所述的分离的核酸的核苷酸序列如SEQ ID NO：3所示；
    优选地，其中所述突变的乙酰乳酸合酶蛋白的氨基酸序列如SEQ ID NO:4所示。
  2. 表达盒、载体或细胞，其包含权利要求1所述的核酸。
  3. 突变的乙酰乳酸合酶蛋白，其包含如下的突变：
    在对应于SEQ ID NO:2的位置556的位置处色氨酸(W)突变为亮氨酸(L)；和
    在对应于SEQ ID NO:2的位置179的位置处脯氨酸(P)突变为丝氨酸(S)、苏氨酸(T)、亮氨酸(L)或丙氨酸(A)；
    优选地，其中所述突变的乙酰乳酸合酶蛋白包含在对应于SEQ ID NO:2的位置556的位置处色氨酸(W)突变为亮氨酸(L)和在对应于SEQ ID NO:2的位置179的位置处脯氨酸(P)突变为丝氨酸(S)；
    更优选地，其中所述突变的乙酰乳酸合酶蛋白的氨基酸序列如SEQ ID NO:4所示。
  4. 权利要求1所述的核酸或权利要求2所述的表达盒、载体或细胞或权利要求3所述的突变的乙酰乳酸合酶蛋白用于产生抗三唑嘧啶磺酰胺类除草剂植物的用途，优选地，所述植物为油菜。
  5. 产生具有三唑嘧啶磺酰胺类除草剂抗性的植物的方法，其特征在于，包括如下步骤：
    将权利要求1所述的核酸导入植物，优选地通过转基因、杂交、回交或无性繁殖等步骤将权利要求1所述的核酸导入植物，其中所述植物表达权利要求3所述的突变的乙酰乳酸合酶蛋白并具有三唑嘧啶磺酰胺类除草剂抗性。
  6. 抗三唑嘧啶磺酰胺类除草剂的非转基因植物或其部分，其包含编码突变的乙酰乳酸合酶蛋白的分离的核酸，所述突变的乙酰乳酸合酶蛋白包含如下的突变：
    在对应于SEQ ID NO:2的位置556的位置处色氨酸(W)突变为亮氨酸(L)；和
    在对应于SEQ ID NO:2的位置179的位置处脯氨酸(P)突变为丝氨酸(S)、苏氨酸(T)、亮氨酸(L)或丙氨酸(A)，
    优选地，其中所述植物是油菜；其中所述部分为植物的器官、组织和细胞，并且优选种子；
    优选地，其中所述突变的乙酰乳酸合酶蛋白包含在对应于SEQ ID NO:2的位置556的位置处色氨酸(W)突变为亮氨酸(L)和在对应于SEQ ID NO:2的位置179的位置处脯氨酸(P)突变为丝氨酸(S)；
    更优选地，其中所述突变的乙酰乳酸合酶蛋白的氨基酸序列如SEQ ID NO:4所示。
  7. 在含有油菜植物的田地中控制杂草的方法，所述方法包括施用有效量的三唑嘧啶磺酰胺类除草剂至含有所述杂草和油菜植物的所述田地，所述油菜植物包含编码突变的乙酰乳酸合酶蛋白的分离的核酸，所述突变的乙酰乳酸合酶蛋白包含如下的突变：
    在对应于SEQ ID NO:2的位置556的位置处色氨酸(W)突变为亮氨酸(L)；和
    在对应于SEQ ID NO:2的位置179的位置处脯氨酸(P)突变为丝氨酸(S)、苏氨酸(T)、亮氨酸(L)或丙氨酸(A)；
    优选地，其中所述突变的乙酰乳酸合酶蛋白包含在对应于SEQ ID NO:2的位置556的位置处色氨酸(W)突变为亮氨酸(L)和在对应于SEQ ID NO:2的位置179的位置处脯氨酸(P)突变为丝氨酸(S)；
    更优选地，其中所述突变的乙酰乳酸合酶蛋白的氨基酸序列如SEQ ID NO:4所示。
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