CN105695493A - Application of ALS mutant type genes in aspect of herbicide resistance - Google Patents
Application of ALS mutant type genes in aspect of herbicide resistance Download PDFInfo
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Abstract
The invention discloses ALS mutant type genes. The 75th nucleotide of the ALS mutant type genes in an ALS gene sequence of rice mutates from G to nucleotide C, the 339th nucleotide of the ALS mutant type genes in the ALS gene sequence of the rice mutates from A to nucleotide C, and the 710th nucleotide of the ALS mutant type genes in the ALS gene sequence mutates from C to nucleotide T. The invention further discloses application of ALS protein coded by the ALS mutant type genes in the aspect of herbicide resistance. The protein comes from a rice mutant plant resisting ALS inhibitor herbicide. Compared with the rice wild type ALS sequence, the protein sequence of the ALS mutant type genes mutates at Gln25, Gln113 and Ala237 sites. Green plants express the protein sequence, and acetolactate synthetase resistant inhibitor herbicide, particularly imidazolone and sulfonylurea herbicide, can be resisted.
Description
Technical field
The invention belongs to vegetable protein and Genes For Plant Tolerance herbicide field。Specifically, the present invention relates to the acetolactate synthestase of Oryza sativa L. (ALS) mutain, this albumen can give the characteristic of plant especially Rice Resistance inhibitor of acetolactate synthetase class herbicide。The invention discloses the sequence of this albumen and they application in Genes For Plant Tolerance herbicide field。
Background technology
Weeds are the unfavorable factors of restriction agricultural production high and stable yields。Relying on tradition compared with the methods such as cultivation step, artificial weeding and weeding by machine, the use of chemical herbicide is a kind of efficient, easy and the improvement weeds method of economy。
Acetolactate synthestase (ALS) is (also referred to as acetohydroxy acid synthetase, AHAS；EC4.1.3.18) inhibitor class herbicide causes that using ALS as target weeds are dead, mainly include sulfonylurea (Sulfonylureas, SU), imidazolone type (Imidazolinones, IMI), triazolo pyrimidine class (Triazolopyrimidines, TP), pyrimidine oxygen (sulfur) benzoic acids [Pyrimidinylthio (oroxy) benzoates, PTB；Pyrimidinyl-carboxyherbicides；PCs] and 13 compounds such as sulfoamido carbonyltriazolinone (Sulfonylamino-carbonyltriazolinones, SCT)。ALS inhibitor class herbicide has the features such as selectivity is strong, broad weed-killing spectrum, low toxicity are efficient, and spread uses at present。But the crops itself generally without anti-(resistance to) herbicidal properties are also produced poisoning by these herbicides, strongly limit its use time and use space, if desired for using herbicide to be just avoided that for the previous period in crop seeding, crops suffer poisoning。Cultivate anti-(resistance to) herbicide crop varieties to reduce chemical injury of crops, widen the use scope of herbicide。
Ripe ALS albumen is about made up of 670 aminoacid, its sequence high conservative between different plant species。ALS albumen is at Gly95, Ala96, Ala122, Pro171, Pro196, Pro197, Ala205, Asp376, Trp537, Trp548, Trp552, Trp557, Trp563, Trp574, Ser621, Ser627, Ser638, Ser653, Gly654, undergo mutation to produce ALS inhibitor class Herbicid resistant to the amino acid sites such as Val669 (the ALS amino acid position of plant Arabidopsis thaliana calculates in mode), this (includes Semen Maydis in multiple kinds of crops, Semen Tritici aestivi, Oryza sativa L., Brassica campestris L, Helianthi etc.), model plant arabidopsis and hundreds of weeds all have been reported that。
Wherein, the anti-ALS inhibitor mutational site that Oryza sativa L. is known includes Gly95, Ala96, Ala122, Trp548, Ser627, Ser653 and Gly654。ALS mutant antiweed level is relevant with the position of ALS amino acid mutation, also relevant with the number of the amino acid classes after sudden change and mutating acid。
At present, the mechanism of action of ALS inhibitor class herbicide is not yet determined, whether the sudden change being difficult to look-ahead ALS other amino acid sites of albumen can produce Herbicid resistant, rely only on the practical exploration that scientific research personnel is long-term, arduous, and be only possible to the Herbicid resistant site finding that ALS albumen is new by some fortune。
Summary of the invention
First technical problem to be solved by this invention is to provide a kind of ALS mutated genes。
The present invention also to solve the technical problem that the ALS albumen being to provide coded by above-mentioned ALS mutated genes。
The present invention also to solve the technical problem that and there is provided containing the above-mentioned expression cassette of ALS mutated genes, recombinant vector or cell。
The present invention also to solve the technical problem that the application that there is provided above-mentioned ALS mutated genes, albumen, expression cassette, recombinant vector or cell in preparation green plants herbicide。
The present invention also to solve the technical problem that and there is provided the method obtaining the green plants with Herbicid resistant。
The present invention finally to solve the technical problem that and there is provided the method identifying the green plants with Herbicid resistant。
The present inventor is by long-term, arduous research, the EMS mutant plant of Indica elite restorer 9311 is carried out for a long time, constantly screens, it is found that a series of ALS mutain, including some known ALS mutain previously described and new ALS mutain, it is insensitive to ALS inhibitor class herbicide, so that plant has ALS inhibitor class Herbicid resistant。Present invention application in plant breeding, can be used for cultivating the plant with Herbicid resistant, especially crops, also developed the application in the crops such as transgenic or non-transgenic such as Oryza sativa L. of these albumen and encoding gene thereof。
For solving above-mentioned technical problem, the technical solution used in the present invention is as follows: a kind of ALS mutated genes, it is become nucleotide C at the 75th nucleotide of the als gene sequence of Oryza sativa L. by G, the 339th nucleotide is become 710 nucleotide of nucleotide C and the by A and sported nucleotide T by C。
Above-mentioned its nucleotide sequence of ALS mutated genes is such as shown in SEQIDNo:3。
The above-mentioned ALS albumen coded by ALS mutated genes。This mutagenic origin is in Oryza sativa L., compared with the wild rice ALS (such as Genbank accession number BAB20812.1) to ALS inhibitor class herbicide sensitive, its aminoacid is undergone mutation on Gln25, Gln113, Ala237 (mutant plant 2) site；Or its become nucleotide C at the 75th nucleotide of the als gene sequence of Oryza sativa L. by G, the 339th nucleotide is become nucleotide C by A, this its nucleotide sequence of ALS mutated genes is such as shown in SEQIDNo:1, and namely its aminoacid undergos mutation (its aminoacid sequence is such as shown in SEQIDNO.2) on Gln25, Gln113 (mutant plant 1) site。
There is presently no report to show, at Gln25, Gln113 and the Ala237 site mutation of the ALS from Oryza sativa L., Oryza sativa L. can be made to have ALS inhibitor class Herbicid resistant。
Above-mentioned ALS albumen, its aminoacid sequence is such as shown in SEQIDNO.4。
Present invention also includes expression cassette, recombinant vector or cell, and it contains above-mentioned ALS mutated genes。
Present invention also includes above-mentioned ALS mutated genes, albumen, expression cassette, recombinant vector or cell application in green plants antiweed。
Above-mentioned green plants is Oryza sativa L., Nicotiana tabacum L., arabidopsis, Cotton Gossypii etc.。
Present invention also includes the method obtaining the green plants with Herbicid resistant, comprises the steps:
1) green plants is made to comprise described ALS mutated genes；Or
2) green plants is made to express described ALS albumen。
Above-mentioned method, including transgenic, hybridizes, backcrosses or asexual propagation step。
The method identifying the green plants of the method acquisition of preceding claim, comprises the following steps:
1) measure whether described green plants comprises above-mentioned ALS mutated genes；Or,
2) measure whether described green plants expresses above-mentioned ALS albumen。
Beneficial effect: field sprays test result indicate that of ALS inhibitor class herbicide " hundred ridges leading to ", the Oryza sativa L. 3-4 leaf seedling of the ALS mutain containing the present invention use 3mL hundred ridge logical/L water (9 times recommend concentration) after, plant still normal growth is grown and solid, and wild rice 3-4 leaf seedling use 1mL hundred ridge logical/3L water (1 times recommends concentration) 30 days after to show as whole strain dead。
Accompanying drawing explanation
The resistant rice mutant that the logical herbicide screening in Fig. 1 hundred ridge obtains；
Fig. 2 pcr amplification als gene 5 ' end result figure；1st swimming lane is Marker；Marker molecular weight is followed successively by 5kb, 3kb, 2kb, 1kb, 750bp, 500bp, 250bp, 100bp from top to bottom, 2nd swimming lane is 9311 wild rice DNA, 3rd～13 swimming lane is the DNA of antiweed mutant plant 1, and other swimming lane is the DNA of antiweed mutant plant 2；Purpose fragment length 1162bp；
Fig. 3 pcr amplification als gene 3 ' end result figure；1st swimming lane is Marker；Marker molecular weight is followed successively by 5kb, 3kb, 2kb, 1kb, 750bp, 500bp, 250bp, 100bp from top to bottom, 2nd swimming lane is 9311 wild rice DNA, 3rd～13 swimming lane is the DNA of antiweed mutant plant 1, and other swimming lane is the DNA of antiweed mutant plant 2；Purpose fragment length 1207bp；
Fig. 4 A and Fig. 4 B respectively hundred ridges lead to the herbicide ALS inhibition of enzyme activity to wild type and antiweed mutant plant 1 and mutant plant 2；
The light absorption value of wild type and the ALS inhibition of enzyme activity of Herbicide Resistant Rice mutant mutant plant 1 and mutant plant 2 is measured by the logical herbicide in Fig. 5 hundred ridge；
Fig. 6 A and Fig. 6 B respectively sulfometuronmethyl herbicide ALS inhibition of enzyme activity to wild type and Herbicide Resistant Rice mutant mutant plant 1 and mutant plant 2；
The light absorption value of wild type and the ALS inhibition of enzyme activity of Herbicide Resistant Rice mutant mutant plant 1 and mutant plant 2 is measured by Fig. 7 sulfometuronmethyl herbicide。
The Overexpression vector BamHI/SacI double digestion proof diagram of the als gene of Fig. 8 A sudden change mutant plant 1；1st swimming lane is Marker；Marker molecular weight is followed successively by 19329 from top to bottom, 7743,6223,4254,3472,2690,1882,1489,925bp, 2nd swimming lane is the recombinant vector without enzyme action, and the 3rd swimming lane is the genetic fragment that produces after BamHI/SacI double digestion of recombinant vector and plasmid fragments DNA；The Overexpression vector BamHI/SacI double digestion proof diagram of the als gene of Fig. 8 B sudden change mutant plant 2；1st swimming lane is the recombinant vector without enzyme action, 2nd swimming lane is Marker, molecular weight is followed successively by 5kb, 3kb, 2kb, 1kb, 750bp, 500bp, 250bp, 100bp from top to bottom, 3rd, 4 swimming lanes are the genetic fragment that produces after BamHI/SacI double digestion of recombinant vector and plasmid fragments DNA, genetic fragment size meets expection, it was demonstrated that vector construction success；
Fig. 9 transposon mutant type als gene Oryza sativa L. PCR detects figure；Always having 21 swimming lanes, the 1st swimming lane, the 9th swimming lane, the 19th swimming lane are Marker；Marker molecular weight is followed successively by 5kb, 3kb, 2kb, 1kb, 750bp, 500bp, 250bp, 100bp from top to bottom, 8th swimming lane, the 18th swimming lane are wild type DNA, as negative control, 20th, 21 swimming lanes are plasmid DNA, as positive control, 3rd～7 swimming lane is the transgenic DNA of mutant plant 1, and other swimming lanes are the transgenic DNA of mutant plant 2。
Detailed description of the invention
Below in conjunction with embodiment, embodiment of the present invention are described in detail, it will be appreciated by those skilled in the art that the following example is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention。Unreceipted actual conditions person in embodiment, conventionally the condition of condition or manufacturer's suggestion carries out。Agents useful for same or the unreceipted production firm person of instrument, be can pass through city available from conventional products。
Embodiment 1: Rice Resistance imidazolinone herbicide mutant acquisition process (hundred ridges lead to)
By indica type conventional rice 9311 seed (purchased from Jiangsu Province's agricultural plasm resource protection with utilize platform) (this be M0; with clear water soak 2 hours) 150kg divide 6 times with under 0.5-1.0% (w/w) ethyl methane sulfonate (EMS) room temperature soak 6-9 hour, every 1 hour of period shake seed；Discarding EMS solution, tap water stirs immersion seed 5 times, each 5 minutes, then with tap water seed overnight, carries out field sowing next day, and carries out conventional fertilizer water management (this is M1)。After plant maturation, seed mixes receipts, dries, preservation of passing the winter。Next year sowing field。When Oryza sativa L. (this is M2) seedling length is to the 3-4 leaf phase, spray for 3mL hundred ridge logical/(" hundred ridges lead to " is that BASF Aktiengesellschaft produces a kind of water aqua type imidazolinone herbicide to L water, recommending minimum working concentration is logical/1.5～3L water in 1mL hundred ridge), the Oryza sativa L. mutant plant (Fig. 1) of anti-imidazolinone herbicide also it is after 30 days in normal green plant。Amounting to M2 individual plant 370 strain obtaining antiweed, these resistance individual plants carry out conventional fertilizer water management, have 320 M2 individual plants can be normally solid, after seed maturity, individual plant sowing, dry, preservation of passing the winter。
Embodiment 2: anti-imidazolinone herbicide rice mutant mutational site is analyzed
Take in the Herbicide Resistant Rice mutant plant that above-described embodiment 1 obtains and choose mutant plant 1 and the blade of mutant plant 2, extract genomic DNA respectively, serve Hai Han space bio tech ltd and carry out gene order-checking。Sequencing result and 9311 is compared with reference to genome sequence (http://rise2.genomics.org.cn/page/rice/download.jsp), find that above-mentioned Herbicide Resistant Rice mutant there occurs the sudden change in multiple site on als gene, wherein antiweed mutant plant 1 is the 75th of als gene the, 339 site bases are undergone mutation, C is become respectively by G, A becomes C, cause its corresponding encoded aminoacid sequence the 25th, 113 sites are become histidine from glutamine, glutamine becomes histidine, namely the nucleotide sequence of the als gene of antiweed mutant plant is such as shown in SEQIDNO.1, the aminoacid sequence of the ALS albumen of its coding is such as shown in SEQIDNO.2；Antiweed mutant plant 2 the 75th of als gene the, 339,710 site bases undergo mutation, become C, A by G respectively to become C, C and become T, cause the 25th of aminoacid sequence the of its corresponding encoded the, 113,237 sites are become histidine from glutamine respectively, glutamine becomes histidine, alanine becomes valine, namely the nucleotide sequence of the als gene of antiweed mutant plant is such as shown in SEQIDNO.3, and the aminoacid sequence of the ALS albumen of its coding is such as shown in SEQIDNO.4。Mutant plant 1 (9311M1) its Classification And Nomenclature of the present invention is the antiweed mutant of indica type conventional rice 9311 (OryzasativaindicaGroupcultivar9311), this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) on March 30th, 2016, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica, postcode: 100101, deposit number is CGMCCNo.12265。Mutant plant 2 (9311M2) its Classification And Nomenclature of the present invention is the antiweed mutant of indica type conventional rice 9311 (OryzasativaindicaGroupcultivar9311), this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) on March 30th, 2016, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica, postcode: 100101, deposit number is CGMCCNo.12266。The rice paddy seed of this preservation is consistent with the condition of culture of common rice seed, happiness high temperature, many wet, short-day, soil is required tight, 20-32 DEG C, damp soil (pH5.5-7.5), all can grow preferably under normal sunshine condition。After rice paddy seed is soaked with clear water, it is placed in constant incubator 25-30 DEG C and cultivates 48 hours, sooner or later respectively change a water；Then rice paddy seed is placed in the plate being lined with wet gauze, cover lid, is placed on constant incubator 25-30 DEG C cultivation, within about 3-4 days, just can detect germination。
The anti-imidazolinone herbicide rice mutant als gene clone of embodiment 3
Take the blade of above-mentioned Herbicide Resistant Rice mutant 9311M1 and 9311M2, extract genomic DNA respectively。The special primer of the chromosome sequence design amplification als gene 3 ' terminal sequence according to 9311 Oryza sativa L. wild type als genes (BAC clones Genbank accession number AAAA02006431.1) place is: forward primer 3F5 '-GGTCTTGCGTCTGGTTGGCGAGT-3 ', reverse primer 3R5 '-CTCTTTATGGGTCATTCAGGTCAA-3 ', the special primer of design amplification als gene 5 ' terminal sequence is: forward primer 5F5 '-ATCCGAGCCACACATCGCCTCAC-3 ', reverse primer 5R5 '-AGCAACAGGTCAGCCTTATCCAC-3 '。The sequence of these two pairs of special primer amplifications has the lap of 230bp, it is possible to be spliced into complete als gene sequence。
Adopting TakaraPrimerSTARMaxDNAPolymerase polymerase (purchased from Takara company) to expand als gene 5 ' terminal sequence, 3 ' terminal sequences, its reaction system is as follows:
Pcr amplification reaction program adopts two-step method, and annealing adopts 68 degree together with being combined into extension。
Program is as follows: denaturation: 98 DEG C of 3min；35 circulations: 98 DEG C of 10sec of degeneration；Extend 68 DEG C of 1min；Insulation: 72 DEG C of 10min。
Take 2 μ lPCR products to detect through 1% agarose gel electrophoresis, find have expection size fragment after (Fig. 2, Fig. 3), remaining PCR primer is after PCR cleaning agents box (purchased from Axygen company) cleaning reclaims, it is cloned into pMD19-T carrier (purchased from Takara company), then converts escherichia coli。Each conversion 12 Escherichia coli clones of random picking carry out PCR detection, take 6 monoclonals that PCR result is positive, send Jin Sirui bio tech ltd to check order, it is thus achieved that sudden change als gene sequence。
The embodiment 4 Oryza sativa L. anti-imidazolinone herbicide of M3 mutant (hundred ridges lead to)
The planting seed that 9311M1 and 9311M2 gathers in the crops is emerged (this is M3), when M3 rice seedling length to 3-4 leaf phase, spray 4mL hundred ridge and lead to/L water (recommending minimum working concentration is that 1mL hundred ridge leads to/3L water, is equivalent to 12 times of concentration)。After herbicide spraying 15 days, resistance Seedling M3 in normal green, can continue to grow tall to 20-30cm, and non-resistance seedling leaf to lose green even part withered and yellow, plant does not grow tall, only 5-9cm。After 30 days, M3 resistant strain is normal green plant, and the wild rice spraying same concentration herbicidal agent is all withered, it was shown that the logical herbicide in hundred ridges of at least anti-12 times of concentration of sudden change Oryza sativa L.。
The embodiment 5 anti-sulfonylurea herbicide of Oryza sativa L. M3 mutant (sulfometuronmethyl)
The planting seed that 9311M1 and 9311M2 gathers in the crops is emerged (this is M3), when M3 rice seedling length to 3-4 leaf phase, spray 4.5g/L sulfometuronmethyl (water dispersible granules that Fan Bang bio tech ltd produces, effective ingredient 75%；Recommending minimum working concentration is 0.5625g/L, is equivalent to 8 times and recommends concentration)。After herbicide spraying 15 days, resistance Seedling M3 in normal green, can continue to grow tall to 20-30cm, and non-resistance seedling leaf to lose green even part withered and yellow, plant does not grow tall, only 5-9cm。After 30 days, M3 resistant strain is normal green plant, and the wild rice spraying same concentration herbicidal agent is all withered, it was shown that the sulfometuronmethyl herbicide of at least anti-8 times of concentration of sudden change Oryza sativa L.。
The ALS enzyme activity determination of embodiment 6 Oryza sativa L. M3 mutant
In order to verify that the Herbicid resistant of rice mutant is whether caused by ALS suddenlys change, and present inventor has performed ALS enzyme assay。Assay method is with reference to the method (SinghB.K., StidhamM.A., ShanerD.L.Assayofacetohydroxyacidsynthase.AnalyticalBioc hemistry, 1988,171:173-179.) of Singh etc.。Concrete, take the M3 plant leaf 0.2g of wild type, 9311M1 and 9311M2 respectively, mortar is pulverized by liquid nitrogen grinding, add 2mL extracting solution (100mMK2HPO4, pH7.5,10mM Sodium Pyruvate, 5mMEDTA, 1mM valine, 1mM leucine, 10mM cysteine, 0.1mM flavin adenine dinucleotide (FAD), 5mM magnesium chloride, 10% (V/V) glycerol, 1% (w/v) polyvinylpyrrolidone), lyolysis to be extracted continues to grind about 1min after freezing。12000rpm, 4 DEG C of centrifugal 30min, Aspirate supernatant, add ammonium sulfate and make up to 50% saturation, place half an hour on ice, 12000rpm, 4 DEG C of centrifugal 30min, abandon supernatant, will be precipitated and dissolved in 0.2mL reaction buffer (100mMK2HPO4, pH7.0,1mMEDTA, 10mM magnesium chloride, 100mM Sodium Pyruvate, 1mM diphosphothiamine, 0.1mM flavin adenine dinucleotide (FAD)), respectively the ALS extracting solution of each plant。
10 μ L herbicides " hundred ridges lead to " (water preparation it is separately added in obtaining ALS extracting solution, effective ingredient 240g/L), or the 1mg sulfometuronmethyl (water dispersible granules that Fan Bang bio tech ltd produces, effective ingredient 75%), mixing, hatching 1h for 37 DEG C, add 0.1ml3M sulphuric acid and terminate reaction, reactant mixture is hatched 30 minutes 60 DEG C of reactions and is easy to decarboxylation。Then 0.4mL nitrite ion (0.09g/L1-naphthols and 0.009g/L creatine, dissolve with 2.5MNaOH) is added。Mixed liquor is hatched 30 minutes at 37 DEG C and is carried out (2 the acetone acid formation acetolactic acids of ALS catalysis that develop the color, acetolactic acid decarboxylation forms 3-Hydroxybutanone, pink is formed again with creatine and 1-naphthols, this complex has obtained the maximum absorption at 530nm place), measure the absorbance of its 530nm subsequently, ALS activity A530 light absorption value represents, the height of the height reflection ALS activity of A530 light absorption value。Testing with water for comparison, wild type, 9311M1 strain and 9311M2 strain all respectively survey 5 individual plants。
A530 light absorption value measurement result finds, when wild type, 9311M1 and 9311M2 ALS extracting solution in do not have ALS inhibitor hundred ridge to lead to time, their A530 light absorption value is all between 1.2-1.4, it was shown that the ALS enzymatic activity of wild type and mutant there was no significant difference (Fig. 4, Fig. 5)；And add ALS inhibitor hundred ridge logical after, the A530 light absorption value of wild type is only 0.3, the A530 light absorption value of 9311M1 and 9311M2 is about 1.1, namely the ALS enzymatic activity of wild type is only about the 25% of comparison, and the ALS enzymatic activity of 9311M1 and 9311M2 still has about 80% (Fig. 4, Fig. 5), the ALS enzymatic activity of mutant is more than 3 times of wild type, it was shown that hundred ridges are led to insensitive by the sudden change ALS of 9311M1 and 9311M2, thus imparting resistance。
Same, A530 light absorption value measurement result finds, when wild type, 9311M1 and 9311M2 ALS extracting solution in there is no ALS inhibitor sulfometuronmethyl time, their A530 light absorption value is all between 1.3-1.5, it was shown that the ALS enzymatic activity of wild type and mutant there was no significant difference (Fig. 6, Fig. 7)；And after adding ALS inhibitor sulfometuronmethyl, the A530 light absorption value of wild type is only 0.2, the A530 light absorption value of 9311M1 and 9311M2 is about 0.7-0.8, namely the ALS enzymatic activity of wild type is only about the 16% of comparison, and the ALS enzymatic activity of 9311M1 and 9311M2 still has about 54% (Fig. 6, Fig. 7), the ALS enzymatic activity of mutant is more than 3 times of wild type, it was shown that the sudden change ALS of 9311M1 and 9311M2 is insensitive to sulfometuronmethyl, thus imparting resistance。
Embodiment 7 transgenic ALS Rice Resistance hundred ridge is logical, sulfometuronmethyl
Design special primer 5 '-CGCGGATCCATCCGAGCCACACATCGCCTC-3 ' and 5 '-TCCCCGCGGCCTACGGAAAACAACACAC-3 ', it 5 ' is separately added into BamHI and SacI enzyme action decorating site。The method of reference example 3, from the genomic DNA of above-mentioned rice mutant 9311M1 and 9311M2, sudden change als gene is amplified by PCR, after order-checking is correct, with BamHI and SacI double digestion sudden change als gene fragment and plant expression vector pCAMBIA1301 plasmid (purchased from pcambia company) respectively, digestion products T4-DNA enzyme (purchased from TaKaRa company) connects, and connects product and converts escherichia coli。Recombiant plasmid extracts DNA, verify with BamHI and SacI double digestion, big plasmid fragments and little genetic fragment (Fig. 8) can be produced, it was demonstrated that be cloned in plant expression vector pCAMBIA1301 plasmid (purchased from pcambia company) by the als gene shown in nucleotide sequence such as SEQIDNO.1 or 3。The plasmid vector built is converted Agrobacterium EHA105, cultivates thalline。Adopt conventional Agrobacterium-mediated transformation Japonica rice Japan fine (purchased from Jiangsu Province's agricultural plasm resource protection with utilize platform); after obtaining transfer-gen plant sowing; when Progeny plants length is to the 3-4 leaf phase, PCR detects transfer-gen plant (Fig. 9)。PCR detection primer is forward primer 35SF5 '-ATGGTTAGAGAGGCTTACGC-3 ', reverse primer 5R5 '-AGCAACAGGTCAGCCTTATCCAC-3 ', and amplified fragments includes 5 ' terminal sequences of CaMV35S promoter and als gene, and size is about 2kb。Pcr amplification reaction system reference example 3, pcr amplification reaction program adopts two-step method, and annealing adopts 68 degree together with being combined into extension。Amplification program is as follows: denaturation: 98 DEG C of 3min；30 circulations: 98 DEG C of 10sec of degeneration；Extend 68 DEG C of 2min；Insulation: 72 DEG C of 10min。After PCR identifies and is positive, spray 4mL hundred ridge logical/L water (12 times recommend concentration) or spray 4.5g/L sulfometuronmethyl (8 times recommend concentration), after 7 days, ALS enzymatic activity is measured, it has been found that the ALS enzymatic activity of transgenic paddy rice is significantly higher than non-transgenic Oryza sativa L. with reference to method described in embodiment 6；Finding after 30 days that transgenic paddy rice growth conditions is good, the fine Oryza sativa L. of non-transgenic Japan is then all withered。
Anti-hundred ridges of embodiment 8 transgenic ALS Nicotiana tabacum L. are logical, sulfometuronmethyl
Design special primer 5 '-CGCGGATCCATCCGAGCCACACATCGCCTC-3 ' and 5 '-TCCCCGCGGCCTACGGAAAACAACACAC-3 ', it 5 ' is separately added into BamHI and SacI enzyme action decorating site。From the genomic DNA of above-mentioned rice mutant 9311M1 and 9311M2, sudden change als gene is amplified by PCR, after order-checking is correct, with reference to embodiment 7 method, the als gene shown in nucleotide sequence such as SEQIDNO.1 or 3 is cloned in plant expression vector pCAMBIA2301 plasmid (purchased from pcambia company)。Select positive colony and convert Agrobacterium EHA105, adopt conventional Agrobacterium-mediated transformation Ben Shi Nicotiana tabacum L. dish, after obtaining transfer-gen plant sowing, when Progeny plants length is to the 3-4 leaf phase, after PCR identifies and is positive, spray 4mL hundred ridge logical/L water (12 times recommend concentration) or spray 4.5g/L sulfometuronmethyl (8 times recommend concentration), after 7 days, ALS enzymatic activity is measured, it has been found that the ALS enzymatic activity of transgene tobacco is significantly higher than non-transgenic tobacco with reference to method described in embodiment 6；Finding after 30 days that transgene tobacco growth conditions is good, non-transgenic tobacco is then all withered。
Embodiment 9 transgenic arabidopsis obtains
Design special primer 5 '-CGCGGATCCATCCGAGCCACACATCGCCTC-3 ' and 5 '-TCCCCGCGGCCTACGGAAAACAACACAC-3 ', it 5 ' is separately added into BamHI and SacI enzyme action decorating site。From the genomic DNA of above-mentioned rice mutant 9311M1 and 9311M2, sudden change als gene is amplified by PCR, after order-checking is correct, als gene shown in nucleotide sequence such as SEQIDNO.1 or 3 is cloned in plant expression vector pCAMBIA2301 plasmid (purchased from pcambia company) by the method with reference to embodiment 7, select positive colony and convert Agrobacterium EHA105, cultivate thalline, by agriculture bacillus mediated method arabidopsis thaliana transformation (CloughS, BentA.Floraldip:asimplifiedmethodforAgrobacterium-mediat edtransformationofArabidopsisthaliana.PlantJournal, 1998, 16 (6): 735-743.), after the arabidopsis maturation sowing converted, sow immediately, the Arabidopsis thaliana Seedlings of non-bolting is sprayed 3mL hundred ridge logical/L water (9 times recommend concentration), find after 30 days that non-transgenic arabidopsis is withered, and transgenic arabidopsis growth conditions is good。
Embodiment 10 transgene cotton obtains
Design special primer 5 '-CGCGGATCCATCCGAGCCACACATCGCCTC-3 ' and 5 '-TCCCCGCGGCCTACGGAAAACAACACAC-3 ', it 5 ' is separately added into BamHI and SacI enzyme action decorating site。From the genomic DNA of above-mentioned rice mutant 9311M1 and 9311M2, sudden change als gene is amplified by PCR, after order-checking is correct, als gene shown in nucleotide sequence such as SEQIDNO.1 or 3 is cloned in plant expression vector pCAMBIA2301 plasmid (purchased from pcambia company) by the method with reference to embodiment 7, select positive colony and convert Agrobacterium LAB4404, cultivate thalline, by agriculture bacillus mediated method converting cotton hypocotyl and cotyledon, after obtaining transfer-gen plant sowing, when Progeny plants length is to the 2-3 leaf phase, after PCR identifies and is positive, seedling is sprayed 3mL hundred ridge and leads to/L water (9 times recommend concentration), find after 30 days that non-transgenic Cotton Gossypii is withered, and transgene cotton growth conditions is good。
Although the specific embodiment of the present invention is described in detail, it will be understood to those of skill in the art that。According to disclosed all instructions, it is possible to those details carry out various amendment and replacement, and these are all in protection scope of the present invention。The four corner of the present invention is provided by the extremely any equivalent of appended patent requirements。
Claims (11)
1. an ALS mutated genes, it is become nucleotide C at the 75th nucleotide of the als gene sequence of Oryza sativa L. by G, the 339th nucleotide is become 710 nucleotide of nucleotide C and the by A and sported nucleotide T by C。
2. ALS mutated genes according to claim 1, it is characterised in that described its nucleotide sequence of ALS mutated genes is such as shown in SEQIDNo:3。
3. the ALS albumen coded by ALS mutated genes described in claim 1 or 2。
4. ALS albumen according to claim 3, it is characterised in that its aminoacid sequence is such as shown in SEQIDNO.4。
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 ~ 2, the albumen described in claim 3 or 4, expression cassette described in claim 5, recombinant vector or cell application in green plants antiweed。
7. application according to claim 6, it is characterised in that described green plants is Oryza sativa L., Nicotiana tabacum L., arabidopsis or Cotton Gossypii。
8. the method obtaining the green plants with Herbicid resistant, it is characterised in that comprise the steps:
1) green plants is made to comprise the ALS mutated genes described in claim 1 or 2；Or
2) green plants is made to express arbitrary described ALS albumen of claim 3 or 4。
9. method according to claim 8, it is characterised in that it includes transgenic, hybridizes, backcrosses or asexual propagation step。
10. the method for the green plants that the method described in claim 8 or 9 of identifying obtains, it is characterised in that comprise the following steps:
1) measure whether described green plants comprises the ALS mutated genes described in claim 1 or 2；Or,
2) measure whether described green plants expresses arbitrary described ALS albumen of claim 3 or 4。
11. the protective agent of a herbicide, it is characterised in that this protective agent is made up of arbitrary described ALS albumen of claim 3 or 4。
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