CN106868027A - The application of japonica rice ALS mutated genes and its albumen in terms of antiweed - Google Patents

The application of japonica rice ALS mutated genes and its albumen in terms of antiweed Download PDF

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CN106868027A
CN106868027A CN201710120010.2A CN201710120010A CN106868027A CN 106868027 A CN106868027 A CN 106868027A CN 201710120010 A CN201710120010 A CN 201710120010A CN 106868027 A CN106868027 A CN 106868027A
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leu
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张保龙
陈天子
王金彦
凌溪铁
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Jiangsu Academy of Agricultural Sciences
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Abstract

The invention discloses a kind of ALS mutated genes of japonica rice, it is mutated into nucleotides T in the 536th nucleotides of the als gene sequence of japonica rice by C.The invention also discloses a kind of japonica rice ALS mutated genes coded by ALS mutains and its application.The mutain from the japonica rice mutant plants of anti-ALS inhibitor class herbicide, compared with the wild type ALS sequences of japonica rice, in Ala179 sites undergo mutation by its protein sequence.Green plants expresses the protein sequence can be resisted（It is resistance to）Inhibitor of acetolactate synthetase class herbicide, particularly imidazolinone herbicide.

Description

The application of japonica rice ALS mutated genes and its albumen in terms of antiweed
Technical field
The invention belongs to vegetable protein and plant antiweed field.Specifically, the present invention relates to the acetyl breast of japonica rice Acid enzyme (ALS) mutain, the mutain can assign plant especially Rice Resistance inhibitor of acetolactate synthetase class and remove The characteristic of careless agent.The invention discloses the sequence of the mutain, and their applications in plant antiweed field.
Background technology
Acetolactate synthestase (ALS) (also referred to as acetohydroxy acid synthetase, AHAS；EC 4.1.3.18), it is plant and micro- life Key enzyme in 3 kinds of amino acid bio building-up processes of thing.If the vigor of this enzyme is suppressed or loses, plant figured silk fabrics can be caused Propylhomoserin, leucine and isoleucine biosynthesis block, influence the synthesis of albumen, ultimately result in plant growth and are obstructed, until dead.Cause This, various highy potent herbicides with ALS as action target is developed, mainly including sulfonylurea (Sulfonylureas, SU), miaow Oxazoline ketone (Imidazolinones, IMI), triazolo pyrimidine class (Triazolopyrimidines, TP), pyrimidine oxygen (sulphur) benzene Formic acid class [Pyrimidinylthio (or oxy)-benzoates, PTB；pyrimidinyl-carboxyherbicides； PCs] and 13 classes such as sulfoamido carbonyltriazolinone (Sulfonylamino-carbonyltriazolinones, SCT) Compound.These herbicides are referred to as ALS inhibitor class herbicides or ALS class herbicides, and this kind of herbicide has that selectivity is strong, kills The features such as grass spectrum is wide, low toxicity is efficient, spread is used at present.But these herbicides belong to selective herbicide, killing While careless, also poisoning can be in itself produced to the general crops without anti-(resistance to) herbicidal properties, strongly limit it makes With the time and space is used, be just avoided that crops are subjected to medicine using herbicide for the previous period if desired in crop seeding Evil.Cultivating anti-(resistance to) herbicide crop varieties can reduce chemical injury of crops, widen the use scope of herbicide.
ALS belongs to the family for relying on thiamine pyrophosphatase, is had been observed that in vivo in plant, fungi and algae etc.. The enzyme is made up of catalytic subunit and regulation subunit, and catalytic subunit molecular weight is about 65kD, and regulation subunit is between 9-54kD.For The holoenzyme activity of ALS is realized, in addition to this 2 subunits, 3 essential confactors, ThDP, divalence gold is also relied on Category ion and flavine purine dinucleotides.Ripe ALS albumen is about made up of 670 amino acid, and its sequence is in different plant species Between it is highly conserved.ALS albumen is 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、 The amino acid sites such as Ser 653, Gly 654, Val 669 (the ALS amino acid positions of plant Arabidopsis thaliana are calculated in mode) occur Mutation can produce ALS inhibitor class Herbicid resistants, and this is in multiple kinds of crops (including corn, wheat, paddy rice, rape, Xiang Certain herbaceous plants with big flowers etc.), have been reported that in model plant arabidopsis and hundreds of weeds.And, the amino acid residue variation of different loci is produced Different types of ALS inhibitor class Herbicid resistant.
Wherein, anti-ALS inhibitor mutational site known to paddy rice include Gly 95, Ala 96, Ala 122, Trp 548, Ser 627, Ser 653 and Gly 654.ALS mutant antiweed levels are relevant with the position of ALS amino acid mutations, also with The number of amino acid classes and mutating acid after mutation is relevant.Some researchs are it is also shown that the ALS that amino acid variation causes dashes forward While becoming enzyme to inhibitor class herbicide generation resistance, the enzymatic property of some ALS mutant enzymes is also changed.
At present, the mechanism of action of ALS inhibitor class herbicide not yet determines, it is difficult to look-ahead ALS albumen other amino Whether the mutation in sour site can produce Herbicid resistant, rely only on long-term, the arduous practical exploration of scientific research personnel, and rely on Some fortune are only possible to find the Herbicid resistant site that ALS albumen is new.
And also the situation on ALS site mutations in japonica rice was reported in nobody's research both at home and abroad at present.
The content of the invention
First technical problem to be solved by this invention is to provide a kind of ALS mutated genes of japonica rice.
The technical problem also to be solved of the invention is to provide the ALS mutation coded by the ALS mutated genes of above-mentioned japonica rice Albumen.
The technical problem also to be solved of the invention there is provided the expression of the ALS mutated genes containing above-mentioned japonica rice Box, recombinant vector or cell.
The technical problem also to be solved of the invention there is provided ALS mutated genes, mutain, the expression of above-mentioned japonica rice The application of box, recombinant vector or cell in terms of green plants herbicide is prepared.
The technical problem also to be solved of the invention there is provided the method for obtaining the green plants with Herbicid resistant.
The method that the technical problem finally to be solved of the invention there is provided green plants of the identification with Herbicid resistant.
The present inventor by the plant of japonica rice variety town rice 18 carried out for a long time, the screening that constantly EMS is mutated, it was found that one Individual new ALS mutains, it is insensitive to ALS inhibitor class herbicides, so that plant has ALS inhibitor class weedings Agent resistance.Application of the present invention in plant breeding, can be used to cultivate the plant with Herbicid resistant, especially crops, The present invention also developed the application of these albumen and its encoding gene in transgenic paddy rice.
In order to solve the above technical problems, the technical solution adopted by the present invention is as follows：A kind of ALS mutated genes of japonica rice, It sports T in the 536th nucleotides of the als gene sequence of japonica rice by C.
Its nucleotide sequence of the ALS mutated genes of above-mentioned japonica rice such as SEQ ID No:Shown in 1.
ALS muteins coded by the ALS mutated genes of above-mentioned japonica rice.The mutagenic origin is in japonica rice variety town Rice 18, with wild rice japonica rice ALS (such as Genbank accession number XM_ to ALS inhibitor class herbicide sensitives 015770973.1) compare, its amino acid is in Ala179 site mutations.Show there is presently no report, from paddy rice japonica rice The Ala179 site mutations of ALS, amino acid Ala sports amino acid Val, can cause that japonica rice has ALS inhibitor class herbicides Resistance.
The ALS mutains of above-mentioned japonica rice, its amino acid sequence is as shown in SEQ ID NO.2.
Present invention also includes expression cassette, recombinant vector or cell, its ALS saltant type base for containing above-mentioned japonica rice Cause.
Present invention also the ALS mutated genes including above-mentioned japonica rice, albumen, expression cassette, recombinant vector or cell Application in terms of green plants antiweed.
Above-mentioned green plants is paddy rice etc..
Present invention also includes the method for obtaining the green plants with Herbicid resistant, comprises the following steps：
1) ALS mutated genes of the green plants comprising described japonica rice are made；Or
2) green plants is made to express the ALS muteins of described japonica rice.
Above-mentioned method, including transgenosis, hybridization, backcrossing or vegetative propagation step.
The method for identifying the green plants that the method for preceding claim is obtained, comprises the following steps：
1) the ALS the mutated genes whether green plants includes above-mentioned japonica rice are determined；Or,
2) the ALS the muteins whether green plants expresses above-mentioned japonica rice are determined.
Beneficial effect：Field spray ALS inhibitor classes herbicide " hundred ridges lead to " test result indicate that, containing of the invention After logical/L water (9 times recommend concentration) using the ridges of 3mL hundred, plant is still for the plant 3-4 leaves seedling of the ALS mutains of japonica rice Right normal growth development and solid, and wild type japonica rice 3-4 leaves seedling apply the ridges of 1mL hundred it is logical/3L water (1 times recommend it is dense Degree) that whole strain is shown as after 30 days is dead.
Brief description of the drawings
The resistant rice japonica rice mutant that the logical herbicide screening in the ridges of Fig. 1 hundred is obtained；
Fig. 2 PCR expand the als gene result figure of the mutant of town rice 18；1st swimming lane is Marker；Marker molecular weight from Top to bottm is followed successively by 8kb, 5kb, 3kb, 2kb, 1kb, 750bp, 500bp, 250bp, and the 2nd swimming lane is the wild rice of town rice 18 DNA, the 3rd swimming lane is the DNA of the mutant plant of antiweed town rice 18；Purpose fragment length 1935bp；
Fig. 3 is the logical herbicide in hundred ridges to wild type and the ALS inhibition of enzyme activity of antiweed mutant plant；
Logical suction of the herbicide to wild type and the ALS inhibition of enzyme activity of the mutant plants of antiweed town rice 18 in the ridges of Fig. 4 hundred Light value is determined；
The Overexpression vector BamHI/SacI double digestion proof diagrams of the als gene of Fig. 5 mutant plants；1st swimming lane is Marker；Marker molecular weight is followed successively by 8kb, 5kb, 3kb, 2kb, 1kb, 750bp, 500bp, 250bp, the 2nd swimming from top to bottom Road is the genetic fragment and plasmid fragments DNA that recombinant vector is produced after BamHI/SacI double digestions, and genetic fragment size meets It is expected, it was demonstrated that vector construction success；
Fig. 6 saltant type als gene paddy rice PCR detection figures；A total of 11 swimming lanes, the 1st swimming lane is Marker；Marker points Son amount is followed successively by 8kb, 5kb, 3kb, 2kb, 1kb, 750bp, 500bp, 250bp from top to bottom, and the 2nd swimming lane is DNA, as Positive control, the 3rd~11 swimming lane is the transgenosis DNA of mutant plant.
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 unreceipted specific in embodiment Condition person, the condition advised according to normal condition or manufacturer is carried out.Agents useful for same or the unreceipted production firm person of instrument, are Can by city available from conventional products.
Embodiment 1：The anti-imidazolinone herbicide mutant acquisition process of paddy rice japonica rice town rice 18 (hundred ridges lead to)
Japonica rice conventional rice town rice town rice 18 (this is M0, is soaked 2 hours with clear water) 150kg is divided 6 times and uses 0.5- 1.0% (w/w) ethyl methane sulfonate (EMS) soaks 6-9 hours at room temperature, and every 1 hour of period shook a seed；Discard EMS molten Liquid, running water stirs immersion seed 5 times, 5 minutes every time, then rinses seed overnight with running water, and next day carries out field sowing, And carry out conventional fertilizer water management (this is M1).After plant maturation, seed is mixed to be received, dries, preservation of passing the winter.Next year sows field.Treat During paddy rice (this is M2) seedling leaf phase to 3-4 long, spray for the ridges of 3mL hundred it is logical/(" hundred ridges lead to " is BASF Aktiengesellschaft's life to L water A kind of water aqua type imidazolinone herbicide is produced, it is logical/1.5~3L water in the ridges of 1mL hundred to recommend minimum concentration), after 30 days also The japonica rice mutant plant (Fig. 1) of anti-imidazolinone herbicide is in normal green plant.The M2 of antiweed is obtained altogether 421 plants of individual plant, these resistance individual plants carry out conventional rich water quality management, have 385 M2 individual plants normally solid, single after seed maturity Strain sowing, dry, preservation of passing the winter.
Embodiment 2：Anti- imidazolinone herbicide japonica rice mutant mutational site analysis
The antiweed japonica rice mutant plant for taking the acquisition of above-described embodiment 1 chooses the blade of mutant plant, and base is extracted respectively Because of a group DNA, serving Hai Han spaces bio tech ltd carries out gene order-checking.Sequencing result and japonica rice reference gene sequence (https://www.ncbi.nlm.nih.gov/nuccore/XM_015770973.1) compare, find above-mentioned antiweed round-grained rice Rice mutant there occurs 1 mutation in site on als gene, wherein the 536th in als gene of antiweed mutant plant Point base is undergone mutation, and T is become by C, causes the 179th site of the amino acid sequence of its corresponding encoded to be changed into figured silk fabrics ammonia from alanine Acid, the i.e. nucleotide sequence of the als gene of antiweed mutant plant as shown in SEQ ID NO.1, its coding ALS saltant types The amino acid sequence of albumen is as shown in SEQ ID NO.2.Its Classification And Nomenclature of mutant plant rice paddy seed zhen4 of the invention is water Rice zhen4 (Oryza sativa Japonica Group zhen 4), the plant in 2 months 2017 No. 12 be preserved in China typical culture collection center (CCTCC), collection (Wuhan University first of Wuhan City, Hubei Province Wuchang District Wuhan University Affiliated primary school opposite), postcode：430072, deposit number is CCTCC No：P201705.The rice paddy seed and light water of the preservation The condition of culture of rice is consistent, happiness high temperature, how wet, short-day, not tight to soil requirement, in 20-32 DEG C, damp soil All can preferably be grown under (pH 5.5-7.5), normal sunshine condition.After rice paddy seed is soaked with clear water, it is placed in incubated 25-30 DEG C of case is cultivated 48 hours, respectively changes a water sooner or later；Then rice paddy seed is placed in the plate for being lined with wet gauze, is covered Lid, is placed on 25-30 DEG C of culture of constant incubator, just can detect germination within about 3-4 days.
The anti-imidazolinone herbicide japonica rice mutant als gene clone of embodiment 3
The blade of above-mentioned antiweed japonica rice mutant zhen4 is taken, the genomic DNA of mutant zhen4 is extracted respectively.Root Designed according to the chromosome sequence where paddy rice japonica rice wild type als gene (such as Genbank accession number XM_015770973.1) and expanded Increase als gene special primer be：Forward primer F5 '-ATCCGAGCCACACATCGCCTCAC-3 ', reverse primer R5 '- CTCTTTATGGGTCATTCAGGTCAA-3’。
Using OneTaqTM Quick2 × Master Mix (are purchased from NEB companies, article No.：M0483L ALS) is expanded Gene 5 ' terminal sequence, 3 ' terminal sequences, its reaction system are as follows：
Pcr amplification reaction program uses two-step method, and annealing and extension are combined into together, using 68 degree.
Program is as follows：Predegeneration：98℃3min；35 circulations：98 DEG C of 10sec of denaturation；Extend 68 DEG C of 1min；Insulation：72 ℃10min。
Take 2 μ l PCR primers to be detected through 1% agarose gel electrophoresis, discovery has after the fragment of expected size (Fig. 2), it is remaining PCR primer reclaimed through PCR cleaning agents box (be purchased from Axygen companies) cleaning after, be cloned into pMD19-T carriers and (be purchased from Takara companies), then convert Escherichia coli.Each random 12 Escherichia coli clones of picking of conversion enters performing PCR detection, takes 6 monoclonals that PCR results are positive, send Jin Sirui bio tech ltd to be sequenced, and obtain the als gene sequence of mutation.
The anti-imidazolinone herbicide of japonica rice M3 mutant of embodiment 4 (hundred ridges lead to)
The seed insemination and emergence (this is M3) that zhen4 is harvested, when the M3 japonica rice seedling leaf phase to 3-4 long, sprays 4mL hundred Ridge leads to/L water (recommending minimum concentration to lead to/3L water for the ridges of 1mL hundred, equivalent to 12 times of concentration).After herbicide spraying 15 days, resist Property seedling M3 in normal green, can continue up to 20-30cm long, and non-resistance seedling leaf withered and yellow, the plant that loses green or even part Do not grow tall, only 5-9cm.After 30 days, M3 resistant strains are in normal green plant, and spray the wild type round-grained rice of same concentration herbicidal agent Rice is all withered, shows that mutation japonica rice at least resists the logical herbicide in 12 times of hundred ridges of concentration.
The ALS enzyme activity determinations of the japonica rice M3 mutant of embodiment 5
In order to verify the Herbicid resistant of japonica rice mutant whether caused by above-mentioned ALS is mutated, present inventor has performed ALS enzyme assays.Method (Singh B.K., Stidham M.A., Shaner of the assay method with reference to Singh etc. D.L.Assay of acetohydroxyacid synthase.Analytical Biochemistry,1988,171:173- 179.).Specifically, taking the M3 plant leaf 0.2g of wild type japonica rice and zhen4 respectively, crushed with liquid nitrogen grinding in mortar, Add 2mL extract solutions (100mM K2HPO4, pH 7.5,10mM Sodium Pyruvates, 5mM EDTA, 1mM valine, 1mM leucines, 10mM cysteines, 0.1mM flavin adenine dinucleotide (FAD)s, 5mM magnesium chlorides, 10% (V/V) glycerine, 1% (w/v) polyethylene pyrrole Pyrrolidone), lyolysis to be extracted continues to grind 1min or so after freezing.12000rpm, 4 DEG C of centrifugation 30min, Aspirate supernatant are added Ammonium sulfate makes up to 50% saturation degree, and in placing on ice half an hour, 12000rpm, 4 DEG C of centrifugation 30min abandon supernatant, will precipitate It is dissolved in 0.2mL reaction buffers (100mM K2HPO4, pH 7.0,1mM EDTA, 10mM magnesium chloride, 100mM Sodium Pyruvates, 1mM diphosphothiamines, 0.1mM flavin adenine dinucleotide (FAD)s), respectively each plant ALS extract solutions.
10 μ L herbicides " hundred ridges lead to " (aqua, active ingredient 240g/L) are separately added into the ALS extract solutions for obtaining, are mixed It is even, 37 DEG C of incubation 1h, plus 0.1ml 3M sulfuric acid terminating reactions, reactant mixture is incubated in 60 DEG C of reactions is easy to decarboxylation in 30 minutes, Plus 0.4mL nitrite ions (0.09g/L 1- naphthols and 0.009g/L creatines, with 2.5M NaOH dissolve) obtain mixing thing liquid then. Mixed liquor is developed the color for 30 minutes in 37 DEG C of incubations, and (ALS catalysis pyruvic acid forms acetolactic acid, and acetolactic acid decarboxylation forms 3- hydroxyls Base butanone, then pink compound is formed with creatine and 1- naphthols, the compound has obtained the maximum absorption at 530nm), then survey The absorbance of fixed its 530nm, ALS activity represents that the height of A530 light absorption values reflects the height of ALS activity with A530 light absorption values. Experiment is control with water, and wild type, zhen4 strains all respectively survey 5 individual plants.
A530 light absorption values measurement result finds, leads to when not having the ridge of ALS inhibitor hundred in wild type, the ALS extract solutions of zhen4 When, their A530 light absorption values show the ALS enzymatic activitys there was no significant difference (figure of wild type and mutant between 1.3-1.4 3,4)；And after adding the ridge of ALS inhibitor hundred logical, the A530 light absorption values of wild type are only 0.28, and the A530 light absorption values of zhen4 are The ALS enzymatic activitys of 1.15, i.e. wild type are only the 25.4% of control, and the ALS enzymatic activitys of zhen4 still have 83% (Fig. 3,4), are dashed forward The ALS enzymatic activitys of variant are wild types more than 3 times, show that the mutation ALS of zhen4 leads to insensitive to hundred ridges, anti-so as to impart Property.
The ridge of 6 transgenosis ALS Rice Resistances of embodiment hundred leads to
Design special primer 5 '-CGCGGATCCATCCGAGCCACACATCGCCTC-3 ' and 5 '- TCCCCGCGGCCTACGGAAAACAACACAC-3 ', it 5 ' is separately added into BamHI and SacI digestion decorating sites.Reference implementation The method of example 3, mutation als gene is amplified by PCR from the genomic DNA of the mutant zhen4 of above-mentioned japonica rice 18, and sequencing is just After really, distinguish double digestion mutation als gene fragment with BamHI and SacI and plant expression vector pCAMBIA1301 plasmids (are purchased from Pcambia companies), digestion products T4-DNA enzymes (being purchased from TaKaRa companies) connection, connection product conversion Escherichia coli.Restructuring Plasmid extraction DNA, is verified with BamHI and SacI double digestions, can produce big plasmid fragments and small genetic fragment (Fig. 5), Prove that the als gene by nucleotide sequence as shown in SEQ ID NO.1 is cloned into plant expression vector pCAMBIA1301 plasmids In (being purchased from pcambia companies).The plasmid vector conversion Agrobacterium EHA105 that will be built, cultivates thalline.Using conventional agriculture Bacillus mediated method conversion Japonica rice Nipponbare (is purchased from Jiangsu Province's agricultural plasm resource protection and utilizes platform), obtains transgenosis After plant sowing, during Progeny plants leaf phase to 3-4 long, PCR detects transfer-gen plant (Fig. 6).PCR detection primers are forward primer - the ATGGTTAGAGAGGCTTACGC-3 ' of 35SF 5 ', reverse primer 5R 5 '-AGCAACAGGTCAGCCTTATCCAC-3 ', amplification Fragment includes 5 ' terminal sequences of CaMV35S promoters and als gene, size about 2kb.Pcr amplification reaction system reference implementation example 3, pcr amplification reaction program uses two-step method, and annealing and extension are combined into together, using 68 degree.Amplification program is as follows：Predegeneration： 98℃3min；30 circulations：98 DEG C of 10sec of denaturation；Extend 68 DEG C of 2min；Insulation：72℃10min.It is positive through PCR identifications Afterwards, the ridges of 4mL hundred are sprayed and leads to/L water (12 times recommend concentration), after 7 days, ALS enzyme activity is determined with reference to the methods described of embodiment 6 Property, it is found that the ALS enzymatic activitys of transgenic paddy rice are significantly higher than the ALS enzyme activity 64% of wild type town rice 18；Find to turn base after 30 days Because paddy growth is in good condition, and non-transgenic wild type town rice 18 is then all withered.
Although specific embodiment of the invention has been described in detail, it will be understood to those of skill in the art that.According to Those details can be carried out various modifications and replacement by disclosed all teachings, and these are in protection scope of the present invention It is interior.Four corner of the invention is given by extremely any equivalent of appended patent requirements.
SEQUENCE LISTING
<110>Jiangsu Province Agriculture Science Institute
<120>Application of the ALS mutated genes and its albumen of japonica rice in terms of antiweed
<130> SG20170221002
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 1935
<212> DNA
<213>The ALS mutated genes of town rice 18
<220>
<221> CDS
<222> (1)..(1935)
<400> 1
atg gct acg acc gcc gcg gcc gcg gcc gcc gcc ctg tcc gcc gcc gcg 48
Met Ala Thr Thr Ala Ala Ala Ala Ala Ala Ala Leu Ser Ala Ala Ala
1 5 10 15
acg gcc aag acc ggc cgt aag aac cac cag cga cac cac gtc ctt ccc 96
Thr Ala Lys Thr Gly Arg Lys Asn His Gln Arg His His Val Leu Pro
20 25 30
gct cga ggc cgg gtg ggg gcg gcg gcg gtc agg tgc tcg gcg gtg tcc 144
Ala Arg Gly Arg Val Gly Ala Ala Ala Val Arg Cys Ser Ala Val Ser
35 40 45
ccg gtc acc ccg ccg tcc ccg gcg ccg ccg gcc acg ccg ctc cgg ccg 192
Pro Val Thr Pro Pro Ser Pro Ala Pro Pro Ala Thr Pro Leu Arg Pro
50 55 60
tgg ggg ccg gcc gag ccc cgc aag ggc gcg gac atc ctc gtg gag gcg 240
Trp Gly Pro Ala Glu Pro Arg Lys Gly Ala Asp Ile Leu Val Glu Ala
65 70 75 80
ctg gag cgg tgc ggc gtc agc gac gtg ttc gcc tac ccg ggc ggc gcg 288
Leu Glu Arg Cys Gly Val Ser Asp Val Phe Ala Tyr Pro Gly Gly Ala
85 90 95
tcc atg gag atc cac cag gcg ctg acg cgc tcc ccg gtc atc acc aac 336
Ser Met Glu Ile His Gln Ala Leu Thr Arg Ser Pro Val Ile Thr Asn
100 105 110
cac ctc ttc cgc cac gag cag ggc gag gcg ttc gcg gcg tcc ggg tac 384
His Leu Phe Arg His Glu Gln Gly Glu Ala Phe Ala Ala Ser Gly Tyr
115 120 125
gcg cgc gcg tcc ggc cgc gtc ggg gtc tgc gtc gcc acc tcc ggc ccc 432
Ala Arg Ala Ser Gly Arg Val Gly Val Cys Val Ala Thr Ser Gly Pro
130 135 140
ggg gca acc aac ctc gtg tcc gcg ctc gcc gac gcg ctg ctc gac tcc 480
Gly Ala Thr Asn Leu Val Ser Ala Leu Ala Asp Ala Leu Leu Asp Ser
145 150 155 160
gtc ccg atg gtc gcc atc acg ggc cag gtc ccc cgc cgc atg atc ggc 528
Val Pro Met Val Ala Ile Thr Gly Gln Val Pro Arg Arg Met Ile Gly
165 170 175
acc gac gtc ttc cag gag acg ccc ata gtc gag gtc acc cgc tcc atc 576
Thr Asp Val Phe Gln Glu Thr Pro Ile Val Glu Val Thr Arg Ser Ile
180 185 190
acc aag cac aat tac ctt gtc ctt gat gtg gag gac atc ccc cgc gtc 624
Thr Lys His Asn Tyr Leu Val Leu Asp Val Glu Asp Ile Pro Arg Val
195 200 205
ata cag gaa gcc ttc ttc ctc gcg tcc tcg ggc cgt cct ggc ccg gtg 672
Ile Gln Glu Ala Phe Phe Leu Ala Ser Ser Gly Arg Pro Gly Pro Val
210 215 220
ctg gtc gac atc ccc aag gac atc cag cag cag atg gcc gtg ccg gtc 720
Leu Val Asp Ile Pro Lys Asp Ile Gln Gln Gln Met Ala Val Pro Val
225 230 235 240
tgg gac acc tcg atg aat cta cca ggg tac atc gca cgc ctg ccc aag 768
Trp Asp Thr Ser Met Asn Leu Pro Gly Tyr Ile Ala Arg Leu Pro Lys
245 250 255
cca ccc gcg aca gaa ttg ctt gag cag gtc ttg cgt ctg gtt ggc gag 816
Pro Pro Ala Thr Glu Leu Leu Glu Gln Val Leu Arg Leu Val Gly Glu
260 265 270
tca cgg cgc ccg att ctc tat gtc ggt ggt ggc tgc tct gca tct ggt 864
Ser Arg Arg Pro Ile Leu Tyr Val Gly Gly Gly Cys Ser Ala Ser Gly
275 280 285
gac gaa ttg cgc tgg ttt gtt gag ctg act ggt atc cca gtt aca acc 912
Asp Glu Leu Arg Trp Phe Val Glu Leu Thr Gly Ile Pro Val Thr Thr
290 295 300
act ctg atg ggc ctc ggc aat ttc ccc agt gac gac ccg ttg tcc ctg 960
Thr Leu Met Gly Leu Gly Asn Phe Pro Ser Asp Asp Pro Leu Ser Leu
305 310 315 320
cgc atg ctt ggg atg cat ggc acg gtg tac gca aat tat gcc gtg gat 1008
Arg Met Leu Gly Met His Gly Thr Val Tyr Ala Asn Tyr Ala Val Asp
325 330 335
aag gct gac ctg ttg ctt gcg ttt ggt gtg cgg ttt gat gat cgt gtg 1056
Lys Ala Asp Leu Leu Leu Ala Phe Gly Val Arg Phe Asp Asp Arg Val
340 345 350
aca ggg aaa att gag gct ttt gca agc agg gcc aag att gtg cac att 1104
Thr Gly Lys Ile Glu Ala Phe Ala Ser Arg Ala Lys Ile Val His Ile
355 360 365
gac att gat cca gca gag att gga aag aac aag caa cca cat gtg tca 1152
Asp Ile Asp Pro Ala Glu Ile Gly Lys Asn Lys Gln Pro His Val Ser
370 375 380
att tgc gca gat gtt aag ctt gct tta cag ggc ttg aat gct ctg cta 1200
Ile Cys Ala Asp Val Lys Leu Ala Leu Gln Gly Leu Asn Ala Leu Leu
385 390 395 400
caa cag agc aca aca aag aca agt tct gat ttt agt gca tgg cac aat 1248
Gln Gln Ser Thr Thr Lys Thr Ser Ser Asp Phe Ser Ala Trp His Asn
405 410 415
gag ttg gac cag cag aag agg gag ttt cct ctg ggg tac aaa act ttt 1296
Glu Leu Asp Gln Gln Lys Arg Glu Phe Pro Leu Gly Tyr Lys Thr Phe
420 425 430
ggt gaa gag atc cca ccg caa tat gcc att cag gtg ctg gat gag ctg 1344
Gly Glu Glu Ile Pro Pro Gln Tyr Ala Ile Gln Val Leu Asp Glu Leu
435 440 445
acg aaa ggt gag gca atc atc gct act ggt gtt ggg cag cac cag atg 1392
Thr Lys Gly Glu Ala Ile Ile Ala Thr Gly Val Gly Gln His Gln Met
450 455 460
tgg gcg gca caa tat tac acc tac aag cgg cca cgg cag tgg ctg tct 1440
Trp Ala Ala Gln Tyr Tyr Thr Tyr Lys Arg Pro Arg Gln Trp Leu Ser
465 470 475 480
tcg gct ggt ctg ggc gca atg gga ttt ggg ctg cct gct gca gct ggt 1488
Ser Ala Gly Leu Gly Ala Met Gly Phe Gly Leu Pro Ala Ala Ala Gly
485 490 495
gct tct gtg gct aac cca ggt gtc aca gtt gtt gat att gat ggg gat 1536
Ala Ser Val Ala Asn Pro Gly Val Thr Val Val Asp Ile Asp Gly Asp
500 505 510
ggt agc ttc ctc atg aac att cag gag ctg gca ttg atc cgc att gag 1584
Gly Ser Phe Leu Met Asn Ile Gln Glu Leu Ala Leu Ile Arg Ile Glu
515 520 525
aac ctc cct gtg aag gtg atg gtg ttg aac aac caa cat ttg ggt atg 1632
Asn Leu Pro Val Lys Val Met Val Leu Asn Asn Gln His Leu Gly Met
530 535 540
gtg gtg caa tgg gag gat agg ttt tac aag gcg aat agg gcg cat aca 1680
Val Val Gln Trp Glu Asp Arg Phe Tyr Lys Ala Asn Arg Ala His Thr
545 550 555 560
tac ttg ggc aac ccg gaa tgt gag agc gag ata tat cca gat ttt gtg 1728
Tyr Leu Gly Asn Pro Glu Cys Glu Ser Glu Ile Tyr Pro Asp Phe Val
565 570 575
act att gct aag ggg ttc aat att cct gca gtc cgt gta aca aag aag 1776
Thr Ile Ala Lys Gly Phe Asn Ile Pro Ala Val Arg Val Thr Lys Lys
580 585 590
agt gaa gtc cgt gcc gcc atc aag aag atg ctc gag act cca ggg cca 1824
Ser Glu Val Arg Ala Ala Ile Lys Lys Met Leu Glu Thr Pro Gly Pro
595 600 605
tac ttg ttg gat atc atc gtc ccg cac cag gag cat gtg ctg cct atg 1872
Tyr Leu Leu Asp Ile Ile Val Pro His Gln Glu His Val Leu Pro Met
610 615 620
atc cca agt ggg ggc gca ttc aag gac atg atc ctg gat ggt gat ggc 1920
Ile Pro Ser Gly Gly Ala Phe Lys Asp Met Ile Leu Asp Gly Asp Gly
625 630 635 640
agg act gtg tat taa 1935
Arg Thr Val Tyr
<210> 2
<211> 644
<212> PRT
<213>The ALS mutated genes of town rice 18
<400> 2
Met Ala Thr Thr Ala Ala Ala Ala Ala Ala Ala Leu Ser Ala Ala Ala
1 5 10 15
Thr Ala Lys Thr Gly Arg Lys Asn His Gln Arg His His Val Leu Pro
20 25 30
Ala Arg Gly Arg Val Gly Ala Ala Ala Val Arg Cys Ser Ala Val Ser
35 40 45
Pro Val Thr Pro Pro Ser Pro Ala Pro Pro Ala Thr Pro Leu Arg Pro
50 55 60
Trp Gly Pro Ala Glu Pro Arg Lys Gly Ala Asp Ile Leu Val Glu Ala
65 70 75 80
Leu Glu Arg Cys Gly Val Ser Asp Val Phe Ala Tyr Pro Gly Gly Ala
85 90 95
Ser Met Glu Ile His Gln Ala Leu Thr Arg Ser Pro Val Ile Thr Asn
100 105 110
His Leu Phe Arg His Glu Gln Gly Glu Ala Phe Ala Ala Ser Gly Tyr
115 120 125
Ala Arg Ala Ser Gly Arg Val Gly Val Cys Val Ala Thr Ser Gly Pro
130 135 140
Gly Ala Thr Asn Leu Val Ser Ala Leu Ala Asp Ala Leu Leu Asp Ser
145 150 155 160
Val Pro Met Val Ala Ile Thr Gly Gln Val Pro Arg Arg Met Ile Gly
165 170 175
Thr Asp Val Phe Gln Glu Thr Pro Ile Val Glu Val Thr Arg Ser Ile
180 185 190
Thr Lys His Asn Tyr Leu Val Leu Asp Val Glu Asp Ile Pro Arg Val
195 200 205
Ile Gln Glu Ala Phe Phe Leu Ala Ser Ser Gly Arg Pro Gly Pro Val
210 215 220
Leu Val Asp Ile Pro Lys Asp Ile Gln Gln Gln Met Ala Val Pro Val
225 230 235 240
Trp Asp Thr Ser Met Asn Leu Pro Gly Tyr Ile Ala Arg Leu Pro Lys
245 250 255
Pro Pro Ala Thr Glu Leu Leu Glu Gln Val Leu Arg Leu Val Gly Glu
260 265 270
Ser Arg Arg Pro Ile Leu Tyr Val Gly Gly Gly Cys Ser Ala Ser Gly
275 280 285
Asp Glu Leu Arg Trp Phe Val Glu Leu Thr Gly Ile Pro Val Thr Thr
290 295 300
Thr Leu Met Gly Leu Gly Asn Phe Pro Ser Asp Asp Pro Leu Ser Leu
305 310 315 320
Arg Met Leu Gly Met His Gly Thr Val Tyr Ala Asn Tyr Ala Val Asp
325 330 335
Lys Ala Asp Leu Leu Leu Ala Phe Gly Val Arg Phe Asp Asp Arg Val
340 345 350
Thr Gly Lys Ile Glu Ala Phe Ala Ser Arg Ala Lys Ile Val His Ile
355 360 365
Asp Ile Asp Pro Ala Glu Ile Gly Lys Asn Lys Gln Pro His Val Ser
370 375 380
Ile Cys Ala Asp Val Lys Leu Ala Leu Gln Gly Leu Asn Ala Leu Leu
385 390 395 400
Gln Gln Ser Thr Thr Lys Thr Ser Ser Asp Phe Ser Ala Trp His Asn
405 410 415
Glu Leu Asp Gln Gln Lys Arg Glu Phe Pro Leu Gly Tyr Lys Thr Phe
420 425 430
Gly Glu Glu Ile Pro Pro Gln Tyr Ala Ile Gln Val Leu Asp Glu Leu
435 440 445
Thr Lys Gly Glu Ala Ile Ile Ala Thr Gly Val Gly Gln His Gln Met
450 455 460
Trp Ala Ala Gln Tyr Tyr Thr Tyr Lys Arg Pro Arg Gln Trp Leu Ser
465 470 475 480
Ser Ala Gly Leu Gly Ala Met Gly Phe Gly Leu Pro Ala Ala Ala Gly
485 490 495
Ala Ser Val Ala Asn Pro Gly Val Thr Val Val Asp Ile Asp Gly Asp
500 505 510
Gly Ser Phe Leu Met Asn Ile Gln Glu Leu Ala Leu Ile Arg Ile Glu
515 520 525
Asn Leu Pro Val Lys Val Met Val Leu Asn Asn Gln His Leu Gly Met
530 535 540
Val Val Gln Trp Glu Asp Arg Phe Tyr Lys Ala Asn Arg Ala His Thr
545 550 555 560
Tyr Leu Gly Asn Pro Glu Cys Glu Ser Glu Ile Tyr Pro Asp Phe Val
565 570 575
Thr Ile Ala Lys Gly Phe Asn Ile Pro Ala Val Arg Val Thr Lys Lys
580 585 590
Ser Glu Val Arg Ala Ala Ile Lys Lys Met Leu Glu Thr Pro Gly Pro
595 600 605
Tyr Leu Leu Asp Ile Ile Val Pro His Gln Glu His Val Leu Pro Met
610 615 620
Ile Pro Ser Gly Gly Ala Phe Lys Asp Met Ile Leu Asp Gly Asp Gly
625 630 635 640
Arg Thr Val Tyr
<210> 3
<211> 23
<212> DNA
<213>Expand the special primer forward primer of als gene
<400> 3
atccgagcca cacatcgcct cac 23
<210> 4
<211> 24
<212> DNA
<213>Expand the special primer reverse primer of als gene
<400> 4
ctctttatgg gtcattcagg tcaa 24
<210> 5
<211> 20
<212> DNA
<213>PCR detection primer forward primers 35SF
<400> 5
atggttagag aggcttacgc 20
<210> 6
<211> 23
<212> DNA
<213>PCR detection primer reverse primers 5R
<400> 6
agcaacaggt cagccttatc cac 23

Claims (11)

1. ALS mutated genes of a kind of japonica rice, it sports T in the 536th nucleotides of the als gene sequence of japonica rice by C.
2. ALS mutated genes of japonica rice according to claim 1, it is characterised in that described its core of ALS mutated genes Nucleotide sequence such as SEQ ID No:Shown in 1.
3. ALS muteins coded by the ALS mutated genes of the japonica rice described in claim 1 or 2.
4. ALS muteins of japonica rice according to claim 3, it is characterised in that its amino acid sequence such as SEQ ID NO. shown in 2.
5. expression cassette, recombinant vector or cell, it contains the ALS mutated genes described in claim 1 or 2.
6. the ALS mutated genes described in claim 1 or 2, the ALS muteins described in claim 3 or 4, right will Ask the application of expression cassette, recombinant vector or cell described in 5 in terms of green plants antiweed.
7. application according to claim 6, it is characterised in that the green plants is paddy rice.
8. the method for obtaining the green plants with Herbicid resistant, it is characterised in that comprise the following steps：
1）Green plants is set to include the ALS mutated genes described in claim 1 or 2；Or
2）Green plants is set to express any described ALS muteins of claim 3 or 4.
9. method according to claim 8, it is characterised in that it includes transgenosis, hybridization, backcrossing or vegetative propagation step.
10. the method for identifying the green plants that the method described in claim 8 or 9 is obtained, it is characterised in that including following step Suddenly：
1) whether the green plants is determined comprising the ALS mutated genes described in claim 1 or 2；Or,
2) any described ALS the muteins whether green plants expresses claim 3 or 4 are determined.
11. a kind of protective agents of herbicide, it is characterised in that the protective agent is dashed forward by any described ALS of claim 3 or 4 Modification albumen is made.
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