CN106591334B

CN106591334B - A kind of wheat ALS mutated genes and its application in terms of antiweed

Info

Publication number: CN106591334B
Application number: CN201611179338.3A
Authority: CN
Inventors: 张保龙; 陈天子; 王金彦; 凌溪铁
Original assignee: Jiangsu Academy of Agricultural Sciences
Current assignee: Jiangsu Academy of Agricultural Sciences
Priority date: 2016-12-19
Filing date: 2016-12-19
Publication date: 2019-10-22
Anticipated expiration: 2036-12-19
Also published as: CN106591334A

Abstract

The invention discloses a kind of wheat ALS mutated genes, become nucleotide A by G in the 1180th 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 mutain derives from the Wheat Mutant plant of anti-ALS inhibitor class herbicide, and compared with wheat wild type ALS sequence, mutein sequence is in Ala394 site mutation.Green plants, which expresses the protein 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

A kind of wheat ALS mutated genes and its application in terms of antiweed

Technical field

The invention belongs to vegetable proteins and plant antiweed field, and in particular to a kind of wheat ALS mutated genes and Its application in terms of antiweed.

Background technique

Farmland weed is to lead to one of main reason of crop production reduction.The use of chemical herbicide is recognised by people Prevent and kill off the administering method that farmland weed is most efficient, easy and economic.

Acetolactate synthestase (ALS) (also referred to as acetohydroxy acid synthetase, AHAS；EC 4.1.3.18) inhibitor class weeding Agent causes weeds dead using ALS as target, mainly includes sulfonylurea (Sulfonylureas, SU), imidazolone type (Imidazolinones, IMI), triazolo pyrimidine class (Triazolopyrimidines, TP), pyrimidine oxygen (sulphur) benzoic acids [Pyrimidinylthio(or oxy)–benzoates,PTB；pyrimidinyl-carboxyherbicides；PCs] and sulphur The 13 class compound such as amide groups carbonyltriazolinone (Sulfonylamino-carbonyltriazolinones, SCT).ALS Inhibitor class herbicide has the advantages that strong selectivity, broad weed-killing spectrum, low toxicity are efficient, low etc. to mammalian toxicity, greatly at present Area is promoted the use of.But these herbicides also generate phytotoxicity to the general crops for not having anti-(resistance to) herbicidal properties itself, It is strongly limited using time and use space, such as needs just to be avoided that using herbicide for the previous period in crop seeding Crops are by phytotoxicity.Therefore, chemical injury of crops can be reduced, widen making for herbicide by cultivating some anti-(resistance to) herbicide crop varieties Use range.

Mature ALS albumen is about made of 670 amino acid, and sequence is highly conserved between different plant species.ALS albumen In Gly 95, Ala 96, Ala 122, Pro 171, Pro 196, Pro 197, Ala 205, Asp 376, Trp 537, Trp 548、Trp 552、Trp 557、Trp 563、Trp 574、Ser 621、Ser 627、Ser 638、Ser 653、Gly 654、 The amino acid sites such as Val 669 (the ALS amino acid position of plant Arabidopsis thaliana calculates in mode), which mutate, can produce ALS suppression Preparation class Herbicid resistant, this is quasi- in multiple kinds of crops (including corn, wheat, wheat, rape, sunflower etc.), model plant It is had been reported that in southern mustard and hundreds of weeds.

The common wheat planted extensively in production is a kind of allohexaploid, contains tri- genomes of A, B and D.Wheat is The anti-ALS inhibitor mutational site known includes Ala 179, Ser 627 (granted patent CN102559646B).Gene order compares Analysis finds that known Ala 179, the Ser 627 mutation are located on the 6BL chromosome of wheat 1 B gene group.ALS mutant is anti- Herbicide level is related with the position of ALS amino acid mutation, also the number with amino acid classes and mutating acid after mutation It is related.

Currently, the mechanism of action of ALS inhibitor class herbicide not yet determines, it is difficult the other amino of look-ahead ALS albumen Whether the mutation in sour site can generate Herbicid resistant, rely only on the practical exploration that scientific research personnel is long-term, arduous, and rely on Some fortune are likely to the new Herbicid resistant site of discovery ALS albumen.

Summary of the invention

Goal of the invention: to achieve the goals above, first technical problem to be solved by this invention is to provide a kind of small Wheat ALS mutated genes.

It is prominent that the present invention also technical problems to be solved are to provide the encoded wheat ALS of above-mentioned wheat ALS mutated genes Bleach.

The present invention also technical problems to be solved there is provided containing above-mentioned wheat ALS mutated genes expression cassette, Recombinant vector or cell.

Also there is provided above-mentioned wheat ALS mutated genes, mutain, expression for technical problems to be solved by the present invention The application of box, recombinant vector or cell in terms of preparing green plants herbicide.

There is provided obtain the method with the green plants of Herbicid resistant for the present invention also technical problems to be solved.

The method of green plants of the last technical problems to be solved of the present invention there is provided identification with Herbicid resistant.

The present inventor (is purchased from Jiangsu Province's agricultural kind by long-term, arduous research, to the EMS mutant plant of Xu's wheat wheat Matter resources protection and utilization platform) for a long time, constantly screen, it was found that a series of ALS mutains, including it is described previously Certain known ALS mutains and new ALS mutain, it is insensitive to ALS inhibitor class herbicide so that plant With ALS inhibitor class Herbicid resistant.Application of the present invention in plant breeding, can be used for cultivating has Herbicid resistant Plant, especially crops also developed these albumen and its encoding gene and make in transgenosis or non-transgenic such as wheat etc. Application in object.

Technical solution: in order to solve the above technical problems, The technical solution adopted by the invention is as follows: a kind of wheat ALS is mutated Type gene becomes nucleotide A by G in the 1180th nucleotide of the 6BL chromosome als gene sequence of wheat 1 B gene group.

Wherein, its nucleotide sequence of above-mentioned wheat ALS mutated genes is as shown in SEQ ID NO:1.

Wherein, the above-mentioned encoded wheat ALS mutain of wheat ALS mutated genes.The mutagenic origin is in wheat B The 6BL chromosome als gene of genome, compared with the wild-type wheat ALS to ALS inhibitor class herbicide sensitive, small 1180th nucleotide of the 6BL chromosome als gene sequence of wheat 1 B gene group becomes nucleotide A by G, leads to its corresponding encoded The 394th amino acids of amino acid sequence threonine is become from alanine.There is presently no reports to show from wheat B The Ala394 site mutation of the 6BL chromosome ALS albumen of genome can make wheat have ALS inhibitor class Herbicid resistant.

Wherein, above-mentioned wheat ALS mutain, amino acid sequence is as shown in SEQ ID NO:2.

The content of present invention further includes expression cassette, recombinant vector or cell, contains above-mentioned wheat ALS mutated genes.

The content of present invention further includes above-mentioned wheat ALS mutated genes, mutain, expression cassette, recombinant vector or thin Application of the born of the same parents in terms of green plants antiweed.

Wherein, above-mentioned green plants is wheat, tobacco, arabidopsis etc..

The content of present invention further includes the method for obtaining the green plants with Herbicid resistant, is included the following steps:

1) making green plants includes the wheat ALS mutated genes；Or

2) the wheat ALS mutain for making green plants expression described.

Above-mentioned method, including transgenosis, hybridization, backcrossing or vegetative propagation step.

The method for the green plants that the method for identifying preceding claim obtains, comprising the following steps:

1) measure whether the green plants includes above-mentioned wheat ALS mutated genes；Or,

2) measure whether the green plants expresses above-mentioned wheat ALS mutain.

The utility model has the advantages that the present invention field spray ALS inhibitor class herbicide " hundred ridges logical " the experimental results showed that, contain 1 leaf of wheat, the 1 core seedling of wheat ALS mutain of the invention leads to/L water on application hundred ridge 3mL (9 times are recommended to use concentration) Afterwards, plant still normal growth and development and solid, and wild-type wheat seedling application hundred ridge 1mL it is logical/(1 times of recommendation makes 3L water With concentration) after, plant strain growth tapers off, blade gradually chlorosis, shoal yellow, plant is unable to jointing, and final whole strain is dead；Into One step, wheat ALS mutated gene of the invention converts Ben Shi cigarette leaf dish, and after obtaining transgenic plant sowing, Progeny plants are long When to the 3-4 leaf phase, after PCR identification is positive, spray hundred ridge 4mL it is logical/L water (12 times are recommended to use concentration), found after 30 days Transgene tobacco growth conditions are good, and non-transgenic tobacco is then all withered；Further, wheat ALS of the invention mutation It after the arabidopsis maturation sowing of genetic transformation, sows immediately, hundred ridge 3mL is sprayed to the Arabidopsis thaliana Seedlings of non-bolting and leads to/(9 times of L water It is recommended to use concentration), find that non-transgenic arabidopsis is withered after 30 days, and transgenic arabidopsis growth conditions are good.

Detailed description of the invention

Hundred ridge Fig. 1 leads to the resistant wheat mutant that herbicide screening obtains；

Fig. 2 PCR amplification wheat als gene overall length result figure；1st swimming lane is Marker；Marker molecular weight is from top to bottom It is followed successively by 5kb, 3kb, 2kb, 1kb, 750bp, 500bp, the 2nd swimming lane is the DNA of antiweed mutant plant；Target fragment length 2104bp；

BamHI the and SacI double digestion of Fig. 3 recombinant plasmid pCAMBIA1301-ALS verifies electrophoretogram；Swimming lane 1 be for Marker；Marker molecular weight is followed successively by 8kb, 5kb, 3kb, 2kb, 1kb, 750bp, 500bp, 250bp from top to bottom；Swimming lane 2 For the segment after recombinant plasmid pCAMBIA1301-ALS digestion.

Specific embodiment

Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will Understand, the following example is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.It is not specified in embodiment specific Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is It can be with conventional products that are commercially available.

Embodiment 1: the anti-imidazolinone herbicide mutant acquisition process of wheat (hundred ridges are logical)

By Xu's Wheat Seeds (purchased from Jiangsu Province's agricultural plasm resource protection and utilizing platform), (this is M0, and it is small to impregnate 2 with clear water When) is impregnated at room temperature 6-9 hours with 0.4-0.6% (w/w) ethyl methane sulfonate (EMS) for 120kg points 6 times, shake in during which every 1 hour Seed；EMS solution is discarded, tap water stirs immersion seed 5 times, 5 minutes every time, then rinses seed mistake with tap water At night, next day carries out field sowing, and carries out conventional fertilizer water management (this is M1).After plant is mature, seed is mixed to be received, dries, and is passed the winter It saves.Next year sows field.When wheat (this is M2) seedling length to the 1 core phase of 1 leaf, sprays and lead to/L water (" hundred ridges for hundred ridge 3mL It is logical " it is that BASF Aktiengesellschaft produces a kind of water aqua type imidazolinone herbicide, recommendation is minimum to use concentration for hundred ridge 1mL Logical/1.5~3L water), the plant for after 3 months being also in the normal jointing of normal green and energy is anti-imidazolinone herbicide Wheat mutant plant (Fig. 1).Total 141 plants of M2 single plant for obtaining antiweed, these resistance single plants carry out conventional fertilizer and water management, Can be normal solid, after seed is mature, single plant sowing is dried, preservation of passing the winter.We carry out 141 plants of M2 of above-mentioned acquisition single Strain harvest, and plantation M3 seed is carried out, further hundred ridge leads to herbicide screening identification, and screening concentration finds No. 38 as before The growth of mutant absolutely not influences, and the then complete yellow of the blade of WT lines.We further to the mutant with The ALS albumen of wild type carries out enzyme activity determination, and when hair is there are currently no herbicide is added, the enzyme activity of two kinds of ALS albumen is in 1.2-1.4 Between, but be added hundred ridge of herbicide it is logical after, the ALS protease activity of mutant is significantly higher than wild type ALS albumen.

Embodiment 2: antiweed Wheat Mutant mutational site analysis

In the antiweed wheat mutant plant that above-described embodiment 1 obtains, the blade of No. 38 mutant plants and wild is chosen The blade of type plant extracts genomic DNA, serves Hai Hanyu Biotechnology Co., Ltd and carries out gene order-checking.Sequencing result It was found that: compared with WT lines, the 6BL chromosome ALS base of No. 38 Wheat Mutants of above-mentioned antiweed in its 1 B gene group Because single base mutation has occurred in the 1180th nucleotide of sequence, A is become by G, leads to the amino acid sequence of its corresponding encoded 394th amino acid becomes threonine, i.e., the 6BL chromosome of the 1 B gene group of No. 38 endowing wheat with herbicide resistance mutant plants from alanine The nucleotide sequence of als gene is as shown in SEQ ID NO.1, the amino acid sequence such as SEQ ID of the ALS mutain of coding Shown in NO.2.No. 38 its classification naming of wheat mutant plant of the invention are common wheat seed Xu wheat M2 (Triticum Aestivum L.Xumai-M2), which was preserved in China typical culture collection center on September 27th, 2016 (CCTCC), address: Wuhan City, Hubei Province Wuchang District Wuhan University collection (the first affiliated primary school, Wuhan University opposite), postal It compiles: 430072, deposit number is CCTCC No:P201621.

The anti-imidazolinone herbicide Wheat Mutant als gene of embodiment 3 clone

The blade of No. 38 Wheat Mutants of above-mentioned antiweed is taken, genomic DNA is extracted.It is designed and is expanded according to sequencing sequence Increase the special primer of als gene full-length gene are as follows: forward primer 5 '-CCATCACCCCTCCCCAATTCC-3 ', reverse primer 5 '- CACTTGTAGGTCTTGTAGGTCG-3'.Using Takara PrimerSTAR Max DNA Polymerase polymerase (10U/ μ l) (be purchased from Takara company) amplification als gene full length sequence, reaction system is as follows:

Pcr amplification reaction program uses two-step method, and annealing and extension are combined into together, using 68 DEG C.

Program is as follows: initial denaturation: 98 DEG C of 3min；35 circulations；It is denaturalized 98 DEG C of 10sec；Extend 68 DEG C of 3min；Heat preservation: 72 ℃10min。

2 μ l PCR products are taken to detect through 1% agarose gel electrophoresis, discovery has after the segment of expected size (Fig. 2), remaining PCR product after PCR cleaning agents box (be purchased from Axygen company) cleaning recycling, be cloned into pMD19-T carrier and (be purchased from Takara company), then convert Escherichia coli.Each random 12 Escherichia coli clones of picking of conversion carry out PCR detection, take 6 monoclonals that PCR result is positive send Jin Sirui Biotechnology Co., Ltd to be sequenced, and obtain mutation als gene sequence.

Embodiment 4 resists the ALS enzyme activity determination of the logical herbicide Wheat Mutant in hundred ridges

In order to verify the Herbicid resistant of Wheat Mutant whether caused by ALS is mutated, present inventor has performed ALS enzyme activity Property measurement.Method (Singh B.K., Stidham M.A., Shaner D.L.Assay of of the measuring method referring to Singh etc. acetohydroxyacid synthase.Analytical Biochemistry,1988,171:173-179.).Specifically, The blade 0.2g for taking wild type Xu wheat wheat and No. 38 Wheat Mutants respectively, is crushed in mortar with liquid nitrogen grinding, and 2mL is added Extracting solution (100mM K₂HPO₄, pH 7.5,10mM Sodium Pyruvate, 5mM EDTA, 1mM valine, 1mM leucine, half Guang of 10mM Propylhomoserin, 0.1mM flavin adenine dinucleotide (FAD), 5mM magnesium chloride, 10% (V/V) glycerol, 1% (w/v) polyvinylpyrrolidone), Lyolysis to be extracted continues to grind 1min or so after freezing.12000rpm, 4 DEG C of centrifugation 30min, Aspirate supernatant, ammonium sulfate, which is added, to be made Reach 50% saturation degree, in placing on ice half an hour, 12000rpm, 4 DEG C of centrifugation 30min abandon supernatant, will be precipitated and dissolved in 0.2mL reaction buffer (100mM K₂HPO₄, pH 7.0,1mM EDTA, 10mM magnesium chloride, 100mM Sodium Pyruvate, 1mM coke phosphorus Allithiamine element, 0.1mM flavin adenine dinucleotide (FAD)), respectively each plant ALS extracting solution.

It is separately added into 10 μ L herbicides " hundred ridges are logical " (aqua, effective component 240g/L) in obtaining ALS extracting solution, mixes Even, 37 DEG C of incubation 1h add 0.1ml 3M sulfuric acid to terminate reaction, and reaction mixture is incubated for 30 minutes in 60 DEG C of reactions is convenient for decarboxylation. Plus 0.4mL developing solution (0.09g/L 1- naphthols and 0.009g/L creatine, with 2.5M NaOH dissolve) then.Mixed liquor is at 37 DEG C It is incubated for developed the color within 30 minutes (ALS mutain catalysis pyruvic acid formation acetolactic acid, acetolactic acid decarboxylation formation 3- hydroxyl fourth Ketone, then pink compound is formed with creatine and 1- naphthols, which has obtained the maximum absorption at 530nm), then measure it The absorbance of 530nm, ALS protein active indicate that the height of A530 light absorption value reflects the height of ALS protein active with A530 light absorption value It is low.Experiment is control with water, if 3 repetitions.

A530 light absorption value measurement result discovery, when not having hundred ridges in the ALS extracting solution of wild type and No. 38 Wheat Mutants When logical, their A530 light absorption value shows that the ALS enzymatic activity of wild type and mutant is poor without conspicuousness between 1.2-1.5 Different (table 1)；And after hundred ridges of addition are logical, the A530 light absorption value of WT lines is only 0.31, and the A530 of mutant wheat plant inhales Light value is 0.88 or so, i.e., the ALS enzymatic activity of WT lines is only 25.6% compareed, and the ALS of mutant wheat plant Enzymatic activity still has 61.5% (table 1), it is insensitive to show that the mutation ALS of No. 38 Wheat Mutants leads to hundred ridges, to impart anti- Property.

Table 1

5 transgene tobacco of embodiment obtains

Design special primer 5 '-CGCGGATCCCCATCACCCCTCCCCAATTCC-3 ' and 5 '- TCCCCGCGGCACTTGTAGGTCTTGTAGGTCG-3 ', 5 ' are separately added into BamHI and SacI digestion decorating site.Pass through PCR amplifies mutation als gene from the genomic DNA of above-mentioned No. 38 Wheat Mutants, after sequencing is correct, with BamHI and SacI distinguishes double digestion mutation als gene segment and plant expression vector pCAMBIA1301 plasmid (being purchased from pcambia company), Digestion products T4-DNA enzyme (being purchased from TaKaRa company) connection, connection product conversion Escherichia coli obtain recombinant plasmid pCAMBIA1301-ALS.Recombinant plasmid pCAMBIA1301-ALS extracts DNA, is verified, can be produced with BamHI and SacI double digestion Raw big plasmid fragments and small genetic fragment (Fig. 3), it was demonstrated that by nucleotide sequence ALS base as shown in SEQ ID NO.1 Because being cloned into plant expression vector pCAMBIA1301 plasmid (purchased from pcambia company).The plasmid vector built is converted Agrobacterium EHA105 selects positive colony, cultivates thallus.Using conventional Agrobacterium-mediated transformation Ben Shi cigarette leaf dish, obtain After transgenic plant sowing, Progeny plants it is long to the 3-4 leaf phase when, after PCR identification is positive, spray hundred ridge 4mL it is logical/L water (12 It is recommended to use concentration again), after 7 days, ALS enzymatic activity is measured referring to 4 the method for embodiment, finds the ALS enzyme of transgene tobacco Activity is significantly higher than non-transgenic tobacco (table 2)；Find that transgene tobacco growth conditions are good after 30 days, and non-transgenic tobacco It is then all withered.

Table 2

6 transgenic arabidopsis of embodiment obtains

Design special primer 5 '-CGCGGATCCCCATCACCCCTCCCCAATTCC-3 ' and 5 '- TCCCCGCGGCACTTGTAGGTCTTGTAGGTCG-3 ', 5 ' are separately added into BamHI and SacI digestion decorating site.Pass through PCR amplifies mutation als gene from the genomic DNA of above-mentioned No. 38 Wheat Mutants, after sequencing is correct, referring to embodiment 5 Method nucleotide sequence als gene as shown in SEQ ID NO.1 is cloned into plant expression vector pCAMBIA2301 plasmid In (being purchased from pcambia company), positive colony conversion Agrobacterium EHA105 is selected, thallus is cultivated, passes through mediated by agriculture bacillus method Arabidopsis thaliana transformation (Clough S, Bent A.Floral dip:a simplified method for Agrobacterium- mediated transformation of Arabidopsis thaliana.Plant Journal,1998,16(6):735- 743.) it, after the arabidopsis maturation sowing of conversion, sows immediately, hundred ridge 3mL is sprayed to the Arabidopsis thaliana Seedlings of non-bolting and leads to/L water (9 It is recommended to use concentration again), find that non-transgenic arabidopsis is withered after 30 days, and transgenic arabidopsis growth conditions are good.

Although a specific embodiment of the invention has been described in detail, it will be understood to those of skill in the art that.According to All introductions having disclosed can be carry out various modifications and be replaced to those details, these are in protection scope of the present invention It is interior.Full scope of the invention is provided by extremely any equivalent of appended patent requirements.

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Arg Pro Trp Gly Pro Ser Glu Pro Arg Lys Gly Ala Asp Ile Leu Val

65 70 75 80

Glu Ala Leu Glu Arg Cys Gly Ile Val Asp Val Phe Ala Tyr Pro Gly

85 90 95

Gly Ala Ser Met Glu Ile His Gln Ala Leu Thr Arg Ser Pro Val Ile

100 105 110

Thr Asn His Leu Phe Arg His Glu Gln Gly Glu Ala Phe Ala Ala Ser

115 120 125

Gly Tyr Ala Arg Ala Ser Gly Arg Val Gly Val Cys Val Ala Thr Ser

130 135 140

Gly Pro Gly Ala Thr Asn Leu Val Ser Ala Leu Ala Asp Ala Leu Leu

145 150 155 160

Asp Ser Ile Pro Met Val Ala Ile Thr Gly Gln Val Pro Arg Arg Met

165 170 175

Ile Gly Thr Asp Ala Phe Gln Glu Thr Pro Ile Val Glu Val Thr Arg

180 185 190

Ser Ile Thr Lys His Asn Tyr Leu Val Leu Asp Val Glu Asp Ile Pro

195 200 205

Arg Val Ile Gln Glu Ala Phe Phe Leu Ala 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 Thr 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 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

cacttgtagg tcttgtaggt cg 22

<210> 5

<211> 30

<212> DNA

<213>special upstream primer

<400> 5

cgcggatccc catcacccct ccccaattcc 30

<210> 6

<211> 31

<212> DNA

<213>special downstream primer

<400> 6

tccccgcggc acttgtaggt cttgtaggtc g 31

Claims

1. a kind of wheat ALS mutated genes become nucleosides by G in the 1180th nucleotide of the als gene sequence of wheat Sour A, described its nucleotide sequence of ALS mutated genes is as shown in SEQ ID No:1.

2. the encoded wheat ALS mutain of wheat ALS mutated genes described in claim 1, amino acid sequence is such as Shown in SEQ ID NO. 2.

3. expression cassette or recombinant vector contain wheat ALS mutated genes described in claim 1.

4. wheat ALS mutated genes described in claim 1, wheat ALS mutain as claimed in claim 2, right are wanted The application of expression cassette described in asking 3 or recombinant vector in terms of green plants antiweed.

5. application according to claim 4, which is characterized in that the green plants is wheat, tobacco or arabidopsis.

6. obtaining the method with the green plants of Herbicid resistant, which comprises the steps of:

1) making green plants includes wheat ALS mutated genes described in claim 1；Or

2) green plants is made to express wheat ALS mutain as claimed in claim 2.

7. according to the method described in claim 6, it is characterized in that, it includes transgenosis, hybridization, backcrossing or vegetative propagation step Suddenly.

8. the method for identifying the green plants that method described in claim 6 or 7 obtains, which comprises the following steps:

1) measure whether the green plants includes wheat ALS mutated genes described in claim 1；Or,

2) measure whether the green plants expresses wheat ALS mutain as claimed in claim 2.

9. a kind of protective agent with Herbicid resistant, which is characterized in that the protective agent is by wheat ALS as claimed in claim 2 Mutain is made.