CN106755019B - Application of a wheat ALS mutant gene and its protein in herbicide resistance - Google Patents

Abstract

The invention discloses a kind of wheat ALS mutated genes, become nucleotide A by G in the 331st nucleotide of the als gene sequence of the 6BL chromosome of wheat 1 B gene group.The invention also discloses a kind of encoded wheat ALS mutain of wheat ALS mutated genes and its applications.The albumen source is in the Wheat Mutant plant of anti-ALS inhibitor class herbicide, and compared with wheat wild type ALS sequence, protein sequence is in Val111 site mutation.Green plants, which expresses the mutein sequence, can resist (resistance to) inhibitor of acetolactate synthetase class herbicide, especially imidazolinone herbicide.1 leaf of wheat, the 1 core seedling of wheat ALS mutain of the invention application hundred ridge 3mL it is logical/L water (9 times are recommended to use concentration) after, plant still normal growth and development and solid.

Description

Application of a wheat ALS mutant gene and its protein in herbicide resistance

technical field

The invention belongs to the fields of plant protein and plant herbicide resistance, and relates to the application of a wheat ALS mutant gene and its protein in herbicide resistance.

Background technique

Weeds in farmland are one of the main causes of crop yield reduction. Compared with the traditional methods of relying on cultivation measures, manual weeding and mechanical weeding, the use of chemical herbicides is recognized as the most efficient, simple and economical method of controlling weeds in farmland.

Acetolactate synthase (ALS) (also known as acetohydroxyacid synthase, AHAS; EC 4.1.3.18) inhibitor herbicides use ALS as a target to cause weed death, mainly including sulfonylureas (Sulfonylureas, SU), Imidazolinones (IMI), triazolopyrimidines (TP), pyrimidinylthio(or oxy)-benzoates [Pyrimidinylthio(or oxy)-benzoates, PTB; pyrimidinyl-carboxyherbicides; PCs] and sulfonamide carbonyl Thirteen types of compounds such as Sulfonylamino-carbonyltriazolinones (SCT). ALS inhibitor herbicides have the advantages of strong selectivity, broad herbicidal spectrum, low toxicity and high efficiency, and low toxicity to mammals, and have been widely used at present. However, these herbicides also cause phytotoxicity to crops that generally do not have the characteristics of resistance (tolerance) to herbicides, which greatly limits their use time and space. Harmful. Therefore, cultivating some herbicide-resistant (tolerant) crop varieties can reduce crop phytotoxicity and broaden the application range of herbicides.

The mature ALS protein consists of about 670 amino acids, and its sequence is highly conserved among different species. ALS protein at 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 , Ser 638, Ser 653, Gly654, Val 669 and other amino acid sites (calculated based on the ALS amino acid positions of the model plant Arabidopsis thaliana) can produce ALS inhibitor herbicide resistance, which is in a variety of crops (including corn, Wheat, wheat, rapeseed, sunflower, etc.), the model plant Arabidopsis and hundreds of weeds have been reported.

Common wheat, which is widely grown in production, is an allohexaploid, containing three genomes A, B and D. Known anti-ALS inhibitor mutation sites in wheat include Ala 179 and Ser 627 (authorized patent CN102559646B). Gene sequence comparison analysis found that the known mutations of Ala 179 and Ser 627 were located on chromosome 6BL of wheat B genome. The herbicide resistance level of ALS mutants is related to the position of ALS amino acid mutation, and also related to the type of amino acid after mutation and the number of mutated amino acids.

At present, the mechanism of action of ALS inhibitor herbicides has not yet been determined, and it is difficult to predict in advance whether mutations at other amino acid sites of the ALS protein will cause herbicide resistance. A new herbicide-resistance locus in the ALS protein may be discovered by luck.

Contents of the invention

Purpose of the invention: The first technical problem to be solved by the present invention is to provide a wheat ALS mutant gene.

The technical problem to be solved by the present invention is to provide the wheat ALS mutant protein encoded by the above wheat 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-mentioned wheat ALS mutant gene.

The technical problem to be solved by the present invention is to provide the application of the above-mentioned wheat ALS mutant gene, mutant protein, expression cassette, recombinant vector or cell in the preparation of green plant herbicide.

The technical problem to be solved by the present invention is to provide a method for obtaining green plants with herbicide resistance.

The final technical problem to be solved by the present invention is to provide a method for identifying green plants with herbicide resistance.

Through long-term and arduous research, the inventors have conducted long-term and continuous screening of the EMS mutant plants of Xumai wheat (purchased from the Agricultural Germplasm Resources Protection and Utilization Platform of Jiangsu Province), and found a series of ALS mutant proteins, including those mentioned above. Certain known ALS muteins and novel ALS muteins described above are insensitive to ALS inhibitor herbicides, thereby rendering plants resistant to ALS inhibitor herbicides. The application of the present invention in plant breeding can be used to breed plants with herbicide resistance, especially crops, and the application of these proteins and their coding genes in transgenic or non-transgenic crops such as wheat has also been developed.

Technical solution: In order to solve the above-mentioned technical problems, the technical solution adopted in the present invention is as follows: a wheat ALS mutant gene, whose 331st nucleotide in the 6BL chromosome ALS gene sequence of the wheat B genome is changed from G to nucleotide a.

Wherein, the nucleotide sequence of the aforementioned wheat ALS mutant gene is shown in SEQ ID NO:1.

Wherein, the wheat ALS mutant protein encoded by the above-mentioned wheat ALS mutant gene. The mutation is derived from the 6BL chromosome ALS gene of the wheat B genome. Compared with the wild-type wheat ALS that is sensitive to ALS inhibitor herbicides, its 331st nucleotide in the 6BL chromosome ALS gene sequence of the wheat B genome is represented by G Change into nucleotide A, causing the 111th amino acid of its corresponding coded amino acid sequence to change from valine to isoleucine. So far, no reports have shown that mutations at the Val111 site of the ALS protein on the 6BL chromosome from the wheat B genome will confer resistance to ALS inhibitor herbicides in wheat.

Wherein, the above-mentioned wheat ALS protein has an amino acid sequence as shown in SEQ ID NO:2.

The content of the present invention also includes expression cassettes, recombinant vectors or cells, which contain the above-mentioned wheat ALS mutant gene.

The content of the present invention also includes the application of the above-mentioned wheat ALS mutant gene, mutant protein, expression cassette, recombinant vector or cell in green plant herbicide resistance.

Wherein, the above-mentioned green plants are wheat, tobacco, Arabidopsis and the like.

The content of the present invention also includes a method for obtaining a green plant with herbicide resistance, comprising the steps of:

1) making green plants contain the wheat ALS mutant gene; or

2) Making green plants express the wheat ALS mutant protein.

The above method includes the steps of transgenic, hybrid, backcross or asexual reproduction.

Identify the method for the green plant that above-mentioned method obtains, comprise the following steps:

1) determining whether the green plant comprises the above-mentioned wheat ALS mutant gene; or,

2) Determining whether the green plant expresses the above-mentioned wheat ALS mutein.

The content of the present invention also includes a herbicide protectant, which is made from the above-mentioned wheat ALS mutant protein.

Beneficial effects: the experimental results of spraying the ALS inhibitor herbicide "Bailongtong" in the field show that the wheat 1 leaf and 1 core seedling containing the wheat ALS mutant protein of the present invention is applied 3mL Bailongtong/L water (9 After 1 times the recommended concentration), the plants still grow and develop normally and bear fruit. However, after the wild-type wheat seedlings are applied with 1mL Bailongtong/3L water (1 times the recommended concentration), the plant growth gradually stops, and the leaves gradually turn green and become lighter. Yellow, the plant cannot be jointed, and eventually the whole plant dies; further, the wheat ALS mutant gene of the present invention is transformed into tobacco leaf discs, and after the transgenic plants are harvested, when the offspring plants grow to the 3-4 leaf stage, they are positive by PCR Finally, spray 4mL Bailongtong/L water (12 times the recommended concentration), and after 30 days, it was found that the growth state of the transgenic tobacco was good, while the non-transgenic tobacco was all withered; further, the Arabidopsis plant transformed with the wheat ALS mutant gene After the thaliana matured and harvested, the seeds were sown immediately, and the unbolted Arabidopsis seedlings were sprayed with 3mL of Bailongtong/L water (9 times the recommended concentration). After 30 days, it was found that the non-transgenic Arabidopsis died, while the transgenic Arabidopsis grew. in good shape.

Description of drawings

The resistant wheat mutant of the present invention obtained by screening with Bailongtong herbicide in Fig. 1;

Figure 2 PCR amplification results of the full-length wheat ALS gene; the first lane is Marker; the molecular weight of Marker from top to bottom is 5kb, 3kb, 2kb, 1kb, 750bp, 500bp, and the second lane is the DNA of herbicide-resistant mutant plants; The target fragment length is 2104bp;

Fig. 3 BamHI and SacI double-digestion verification electrophoresis of recombinant plasmid pCAMBIA1301-ALS; lane 1 is Marker; marker molecular weight is 8kb, 5kb, 3kb, 2kb, 1kb, 750bp, 500bp, 250bp from top to bottom; lane 2 It is the digested fragment of the recombinant plasmid pCAMBIA1301-ALS.

Detailed ways

Embodiments of the present invention will be described in detail below in conjunction with examples, but those skilled in the art will understand that the following examples are only used to illustrate the present invention, and should not be considered as limiting the scope of the present invention. Those who do not indicate the specific conditions in the examples are carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used were not indicated by the manufacturer, and they were all commercially available conventional products.

Example 1: The process of obtaining wheat mutants resistant to imidazolinone herbicides (Bailongtong)

Xumai seeds (purchased from Jiangsu Provincial Agricultural Germplasm Resources Protection and Utilization Platform) (this is M0, soaked for 2 hours with clear water) 120kg were divided into 6 times with 0.4-0.6% (w/w) ethyl methanesulfonate (EMS ) at room temperature for 6-9 hours, during which the seeds were shaken every 1 hour; the EMS solution was discarded, and the seeds were stirred and soaked in tap water for 5 times, each time for 5 minutes, and then the seeds were rinsed with tap water overnight, and the field was sown the next day, and routine Fertilizer and water management (this is M1). After the plants are mature, the seeds are mixed, dried, and stored for the winter. Sow in the field the following year. When the wheat (this is M2) seedlings grow to the stage of 1 leaf and 1 core, spray 3mL of Bailongtong/L water ("Bailongtong" is a water-based imidazolinone herbicide produced by BASF in Germany, it is recommended The minimum concentration used is 1mL Bailongtong/1.5-3L water), and the plants that are still green and can joint normally after 3 months are the wheat mutant plants resistant to imidazolinone herbicides (Figure 1). A total of 141 herbicide-resistant M2 individual plants were obtained. These resistant individual plants were treated with conventional fertilizer and water management, and all of them could bear fruit normally. After the seeds matured, the individual plants were harvested, dried, and stored for winter. We harvested the 141 M2 obtained above, and planted M3 seeds, and further screened and identified Bailongtong herbicide. The screening concentration was the same as before, and found that the growth of the No. 75 mutant had no effect at all, while the wild-type The leaves are completely yellowed. We further measured the enzyme activities of the mutant and wild-type ALS proteins, and found that the enzyme activities of the two ALS proteins were between 1.2 and 1.4 when no herbicide was added, but after adding the herbicide Bailongtong, the mutant The ALS protease activity of ALS protein was significantly higher than that of wild-type ALS protein.

Example 2: Analysis of mutation sites in herbicide-resistant wheat mutants

Among the herbicide-resistant wheat mutant plants obtained in Example 1 above, the leaves of the mutant plant No. 75 and the leaves of the wild-type plant were selected, and genomic DNA was extracted and sent to Shanghai Hanyu Biotechnology Co., Ltd. for genome sequencing. Sequencing results revealed that, compared with the wild-type plant, the above-mentioned herbicide-resistant wheat mutant No. 75 had a single-base mutation at the 331st nucleotide of the 6BL chromosome ALS gene sequence of its B genome, changing from G to A , causing the 111th amino acid of its corresponding encoded amino acid sequence to be changed from valine to isoleucine, that is, the nucleotide sequence of the 6BL chromosome ALS gene of the B genome of the No. 75 wheat herbicide-resistant mutant plant such as SEQ ID NO. 1, the amino acid sequence of the encoded ALS protein is shown in SEQ ID NO.2. The No. 75 wheat mutant plant of the present invention is classified as common wheat seed Xumai M3 (Triticumaetivum L.Xumai-M3), and the bacterial strain has been preserved in the China Type Culture Collection Center (CCTCC) on September 27, 2016, address : Wuhan University Preservation Center (opposite to the First Affiliated Primary School of Wuhan University), Wuchang District, Wuhan City, Hubei Province, Zip Code: 430072, and the deposit number is CCTCC No: P201622.

Example 3 Cloning of ALS gene of wheat mutant resistant to imidazolinone herbicides

The leaves of the above-mentioned herbicide-resistant wheat mutant No. 75 were taken to extract genomic DNA. The specific primers for amplifying the full-length ALS gene were designed according to the sequencing sequence: forward primer 5'-CCATCACCCCTCCCCAATTCC-3', reverse primer 5'-CACTTGTAGGTCTTGTAGGTCG-3'. Using Takara PrimerSTAR Max DNA Polymerase polymerase (10U/μl) (purchased from Takara Company) to amplify the full-length sequence of the ALS gene, the reaction system is as follows:

The PCR amplification reaction program adopts a two-step method, annealing and extension are combined together, and 68 ° C is used.

The program is as follows: pre-denaturation: 98°C for 3min; 35 cycles; denaturation at 98°C for 10sec; extension at 68°C for 3min; incubation: 72°C for 10min.

Take 2 μl of the PCR product and detect it by 1% agarose gel electrophoresis. After a fragment of the expected size is found (Figure 2), the remaining PCR product is cleaned and recovered by a PCR cleaning kit (purchased from Axygen), and cloned into pMD19-T Vector (purchased from Takara Company), and then transformed into Escherichia coli. For each transformation, 12 single clones of Escherichia coli were randomly selected for PCR detection, and 6 single clones with positive PCR results were sent to GenScript Biotechnology Co., Ltd. for sequencing to obtain the mutant ALS gene sequence.

Example 4 Determination of ALS Enzyme Activity of Wheat Mutants Resistant to Bailongtong Herbicide

In order to verify whether the herbicide resistance of the wheat mutants is caused by the ALS mutation, the present inventors performed an ALS enzyme activity assay. The determination method refers to the method of Singh et al. (Singh BK, Stidham MA, Shaner DLAssayofacetohydroxyacid synthase. Analytical Biochemistry, 1988, 171: 173-179.). Specifically, 0.2 g leaves of wild-type Xumai wheat and wheat mutant No. 75 were taken, ground and pulverized with liquid nitrogen in a mortar, and 2 mL of extract (100 mM K ₂ HPO ₄ , pH 7.5, 10 mM sodium pyruvate, 5mM EDTA, 1mM valine, 1mM leucine, 10mM cysteine, 0.1mM flavin adenine dinucleotide, 5mM magnesium chloride, 10% (V/V) glycerol, 1% (w/v) poly Vinylpyrrolidone), continue to grind for about 1 min after the extract is thawed. Centrifuge at 12000rpm, 4°C for 30min, absorb the supernatant, add ammonium sulfate to make it reach 50% saturation, place on ice for half an hour, centrifuge at 12000rpm, 4°C for 30min, discard the supernatant, dissolve the precipitate in 0.2mL reaction buffer (100mM K ₂ HPO ₄ , pH 7.0, 1mM EDTA, 10mM magnesium chloride, 100mM sodium pyruvate, 1mM thiamine pyrophosphate, 0.1mM flavin adenine dinucleotide), and obtain ALS extracts from each plant.

Add 10 μL of the herbicide “Bailongtong” (aqua, active ingredient 240g/L) to the obtained ALS extract, mix well, incubate at 37°C for 1 hour, add 0.1ml of 3M sulfuric acid to terminate the reaction, and incubate the reaction mixture at 60°C 30 minutes facilitates decarboxylation. Then add 0.4mL color developing solution (0.09g/L 1-naphthol and 0.009g/L creatine, dissolved with 2.5M NaOH). The mixture was incubated at 37°C for 30 minutes for color development (ALS protein catalyzes pyruvate to form acetolactate, acetolactate decarboxylates to form 3-hydroxybutanone, and then forms a pink complex with creatine and 1-naphthol. There is a maximum absorbance value at 530nm), and then measure its absorbance at 530nm, and the ALS protein activity is represented by the A530 absorbance value, and the level of A530 absorbance value reflects the level of ALS protein activity. Water was used as the control in the experiment, and three repetitions were set.

The results of A530 absorbance measurement showed that when the ALS extracts of the wild-type and No. 75 wheat mutants did not contain Biotron, their A530 absorbance values were between 1.2-1.5, indicating that the ALS enzyme activity of the wild-type and mutants There was no significant difference (Table 1); after adding Bailongtong, the A530 absorbance value of the wild-type plant was only 0.31, and the A530 absorbance value of the mutant wheat plant was about 0.85, that is, the ALS enzyme activity of the wild-type plant was only 25.6% of the mutant wheat plants, while the ALS enzyme activity of the mutant wheat plants was still 61.6% (Table 1), indicating that the mutant ALS of No. 75 wheat mutant was not sensitive to Bailongtong, thus imparting resistance.

Table 1

Embodiment 5 transgenic tobacco obtains

Specific primers 5'-CGCGGATCCCCATCACCCCTCCCAATTCC-3' and 5'-TCCCCGCGGCACTTGTAGGTCTTGTAGGTCG-3' were designed, and BamHI and SacI restriction modification sites were added to the 5' respectively. The mutated ALS gene was amplified from the genomic DNA of the above-mentioned No. 75 wheat mutant by PCR. After the sequencing was correct, the mutated ALS gene fragment and the plant expression vector pCAMBIA1301 plasmid (purchased from pcambia company) were double-digested with BamHI and SacI respectively. The excised product was ligated with T4-DNase (purchased from TaKaRa Company), and the ligated product was transformed into Escherichia coli to obtain the recombinant plasmid pCAMBIA1301-ALS. DNA was extracted from the recombinant plasmid pCAMBIA1301-ALS, verified by double digestion with BamHI and SacI, large plasmid fragments and small gene fragments (Fig. 3) could be produced, proving that the nucleotide sequence of ALS as shown in SEQ ID NO.1 The gene was cloned into the plant expression vector pCAMBIA1301 plasmid (purchased from pcambia company). Transform the constructed plasmid vector into Agrobacterium EHA105, select positive clones, and culture the cells. The conventional Agrobacterium-mediated method was used to transform tobacco leaf discs to obtain transgenic plants. After harvesting, when the progeny plants grew to the 3-4 leaf stage and were positive by PCR, they were sprayed with 4mL Bailongtong/L water (12 times the recommended use concentration), after 7 days, the ALS enzyme activity was measured with reference to the method described in Example 4, and it was found that the ALS enzyme activity of transgenic tobacco was significantly higher than that of non-transgenic tobacco; after 30 days, it was found that the growth state of transgenic tobacco was good, while that of non-transgenic tobacco was all dead.

Example 6 Transgenic Arabidopsis Obtained

Specific primers 5'-CGCGGATCCCCATCACCCCTCCCAATTCC-3' and 5'-TCCCCGCGGCACTTGTAGGTCTTGTAGGTCG-3' were designed, and BamHI and SacI restriction modification sites were added to the 5' respectively. Amplify the mutated ALS gene from the genomic DNA of the above-mentioned No. 75 wheat mutant by PCR. After the sequencing is correct, the ALS gene whose nucleotide sequence is shown in SEQ ID NO.1 is cloned to the plant for expression according to the method in Example 5 In the vector pCAMBIA2301 plasmid (purchased from pcambia company), select positive clones to transform Agrobacterium EHA105, culture the cells, and transform Arabidopsis thaliana through the Agrobacterium-mediated method (Clough S, Bent A. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant Journal, 1998, 16(6): 735-743.), after the transformed Arabidopsis thaliana matured and harvested seeds, they were sown immediately, and 3mL Bailongtong/L was sprayed on the unbolted Arabidopsis seedlings Water (9 times the recommended concentration), after 30 days, it was found that the non-transgenic Arabidopsis was dead, while the transgenic Arabidopsis was growing well.

While specific embodiments of the present invention have been described in detail, those skilled in the art will understand. Based on all the teachings that have been disclosed, various modifications and substitutions can be made to those details, which are all within the scope of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

SEQUENCE LISTING

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195 200 205

Arg Val Ile Gln Glu Ala Phe Phe Leu Ala Ser Ser Ser Gly Arg Pro Gly

210 215 220

Pro Val Leu Val Asp Ile Pro Lys Asp Ile Gln Gln Gln Met Ala Val

225 230 235 240

Pro Val Trp Asp Thr Pro Met Ser Leu Pro Gly Tyr Ile Ala Arg Leu

245 250 255

Pro Lys Pro Pro Ser Thr Glu Ser Leu Glu Gln Val Leu Arg Leu Val

260 265 270

Gly Glu Ser Arg Arg Pro Ile Leu Tyr Val Gly Gly Gly Cys Ala Ala

275 280 285

Ser Gly Glu Glu Leu Arg Arg Phe Val Glu Leu Thr Gly Ile Pro Val

290 295 300

Thr Thr Thr Leu Met Gly Leu Gly Asn Phe Pro Ser Asp Asp Pro Leu

305 310 315 320

Ser Leu Arg Met Leu Gly Met His Gly Thr Val Tyr Ala Asn Tyr Ala

325 330 335

Val Asp Lys Ala Asp Leu Leu Leu Ala Phe Gly Val Arg Phe Asp Asp

340 345 350

Arg Val Thr Gly Lys Ile Glu Ala Phe Ala Ser Arg Ser Lys Ile Val

355 360 365

His Ile Asp Ile Asp Pro Ala Glu Ile Gly Lys Asn Lys Gln Pro His

370 375 380

Val Ser Ile Cys Ala Asp Val Lys Leu Ala Leu Gln Gly Leu Asn Ala

385 390 395 400

Leu Leu Asn Gly Ser Lys Ala Gln Gln Gly Leu Asp Phe Gly Pro Trp

405 410 415

His Lys Glu Leu Asp Gln Gln Lys Arg Glu Phe Pro Leu Gly Phe Lys

420 425 430

Thr Phe Gly Glu Ala Ile Pro Pro Gln Tyr Ala Ile Gln Val Leu Asp

435 440 445

Glu Leu Thr Lys Gly Glu Ala Ile Ile Ala Thr Gly Val Gly Gln His

450 455 460

Gln Met Trp Ala Ala Gln Tyr Tyr Thr Tyr Lys Arg Pro Arg Gln Trp

465 470 475 480

Leu Ser Ser Ser Ser Gly Leu Gly Ala Met Gly Phe Gly Leu Pro Ala Ala

485 490 495

Ala Gly Ala Ala Val Ala Asn Pro Gly Val Thr Val Val Asp Ile Asp

500 505 510

Gly Asp Gly Ser Phe Leu Met Asn Ile Gln Glu Leu Ala Leu Ile Arg

515 520 525

Ile Glu Asn Leu Pro Val Lys Val Met Ile Leu Asn Asn Gln His Leu

530 535 540

Gly Met Val Val Gln Trp Glu Asp Arg Phe Tyr Lys Ala Asn Arg Ala

545 550 555 560

His Thr Tyr Leu Gly Asn Pro Glu Asn Glu Ser Glu Ile Tyr Pro Asp

565 570 575

Phe Val Thr Ile Ala Lys Gly Phe Asn Val Pro Ala Val Arg Val Thr

580 585 590

Lys Lys Ser Glu Val Thr Ala Ala Ile Lys Lys Met Leu Glu Thr Pro

595 600 605

Gly Pro Tyr Leu Leu Asp Ile Ile Val Pro His Gln Glu His Val Leu

610 615 620

Pro Met Ile Pro Ser Gly Gly Ala Phe Lys Asp Met Ile Met Glu Gly

625 630 635 640

Asp Gly Arg Thr Ser Tyr

645

<210> 3

<211> 21

<212>DNA

<213> forward primer

<400> 3

ccatcacccc tccccaattc c 21

<210> 4

<211> 22

<212>DNA

<213> reverse primer

<400> 4

cacttgtaggt cg 22

<210> 5

<211> 30

<212>DNA

<213> specific upstream primer

<400> 5

cgcggatccc catcacccct ccccaattcc 30

<210> 6

<211> 31

<212>DNA

<213> specific downstream primer

<400> 6

tccccgcggc acttgtaggt cttgtaggtc g 31

Claims (8)

1. A wheat ALS mutant gene, which changes from G to nucleotide A at the 331st nucleotide in the ALS gene sequence of wheat, and its nucleotide sequence of the ALS mutant gene is as SEQ ID No:1 shown. 2. the wheat ALS mutein encoded by the wheat ALS mutant gene of claim 1. 3. ALS mutein according to claim 2, characterized in that its amino acid sequence is as shown in SEQ ID No:2. 4. An expression cassette, a recombinant vector or a cell, which contains the wheat ALS mutant gene of claim 1. 5. the wheat ALS mutant gene described in claim 1, the wheat ALS mutant protein described in claim 2 or 3, or the application of the expression cassette, recombinant vector or cell described in claim 4 in green plant herbicide resistance , the green plant is wheat, tobacco or Arabidopsis, and the herbicide is an imidazolinone herbicide. 6. the method for obtaining the green plant with herbicide resistance, is characterized in that, comprises the steps: 1) making green plants contain the wheat ALS mutant gene according to claim 1; or 2) making green plants express the wheat ALS mutein according to any one of claims 2 or 3; The green plant is wheat, tobacco or Arabidopsis, and the herbicide is an imidazolinone herbicide. 7. The method according to claim 6, characterized in that it comprises a transgenic or hybridization step. 8. identify the method for the green plant that the method described in claim 6 or 7 obtains, is characterized in that, comprises the following steps: 1) determining whether said green plant comprises the wheat ALS mutant gene of claim 1; or, 2) determining whether said green plant expresses the wheat ALS mutein described in any one of claim 2 or 3; said green plant is wheat, tobacco or Arabidopsis, and said herbicide is an imidazolinone herbicide.