CN102936591A - Acetolactic acid synthetase mutants and application thereof - Google Patents
Acetolactic acid synthetase mutants and application thereof Download PDFInfo
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Abstract
The invention discloses acetolactic acid synthetase mutants and application thereof. Compared with the wild type acetolactic acid synthetase, at least one of the acetolactic acid synthetase mutants Ala173 and Trp542 is mutated. When the acetolactic acid synthetase mutants are expressed in a plant, the plant can have weedicide resistance.
Description
Technical field
The present invention relates to the plant genetics and breeding field.Particularly, the present invention relates to acetolactate synthestase mutant and application thereof.More specifically, the present invention relates to the method for nucleic acid, expression cassette, carrier or the cell of acetolactate synthestase mutant, separation, plant that acquisition has Herbicid resistant and the authentication method of the plant that obtained by claim 8 or 9 described methods.
Background technology
Weeds are one of greatest factor that affect crop yield in the farmland, and the manpower of the required consumption of artificial weeding and cost are very high, and be not easy to intensive agriculture production, seriously restricted the development process of proportion of crop planting to high yield, high-quality and low-cost direction.Therefore, weedicide is just transported Ying Ersheng.Yet weedicide also can kill farm crop itself usually, and therefore for generally not having the farm crop of Herbicid resistant (tolerance), the use of weedicide has been subject to very big restriction in time and space.For this reason, the plant (farm crop) that people's research and development have Herbicid resistant (tolerance) can be used weedicide like this between planting season, kill weeds, but do not affect the growth of plant itself, has widened thus the use range of weedicide.Yet plant and conclusive material (such as albumen, gene etc.) thereof with Herbicid resistant also do not have the theory rational design to go out at present.
Therefore, at present searching makes plant have the protein of Herbicid resistant and its method that is applied is still remained to be improved.
Summary of the invention
The present invention is intended to solve at least one of technical problem that exists in the prior art.For this reason, one object of the present invention is to propose a kind of new plant that makes and has the protein of Herbicid resistant, and is applied to have weedicide.
For this reason, according to an aspect of the present invention, the present invention proposes a kind of acetolactate synthestase mutant.According to embodiments of the invention, compare with the wild-type acetolactate synthestase, the Ala173 of acetolactate synthestase mutant of the present invention and Trp542 have one of at least a sudden change.According to embodiments of the invention, the contriver is separated to the new mutant body of acetolactate synthestase, and Analysis deterrmination this mutant can make plant possess Herbicid resistant, thereby by making expression of plants acetolactate synthestase mutant of the present invention, can possess Herbicid resistant by this plant.
According to a further aspect in the invention, the invention allows for a kind of nucleic acid of separation.According to embodiments of the invention, the foregoing acetolactate synthestase mutant of the nucleic acid encoding of this separation.According to embodiments of the invention, the nucleic acid of the encoding acetolactate synthase mutant that the present invention is separated to imports plant, makes it express aforementioned acetolactate synthestase mutant of the present invention, can make this plant possess Herbicid resistant.
According to another aspect of the invention, the invention allows for a kind of expression cassette, carrier or cell.According to embodiments of the invention, this expression cassette, carrier or cell comprise foregoing nucleic acid of the present invention.Thus, utilize expression cassette of the present invention and carrier effectively with the nucleic acid importing plant of encoding acetolactate synthase mutant of the present invention, to make the aforementioned acetolactate synthestase mutant of the present invention of this expression of plants, thereby to make it possess Herbicid resistant.In addition, the contriver finds that cell of the present invention can be expressed acetolactate synthestase mutant of the present invention, possesses Herbicid resistant, can effectively become the plant that possesses Herbicid resistant after cultivating.
In accordance with a further aspect of the present invention, the present invention also provides foregoing acetolactate synthestase mutant, nucleic acid and expression cassette, carrier or cell, the purposes in obtaining to have the plant of Herbicid resistant.According to embodiments of the invention, as previously mentioned, utilize expression cassette of the present invention, carrier and cell can effectively obtain to possess the plant of Herbicid resistant.
According to another aspect of the invention, the present invention also provides a kind of acquisition to have the method for the plant of Herbicid resistant.According to embodiments of the invention, the method realizes one of at least by following approach: (1) makes plant comprise the nucleic acid of encoding acetolactate synthase mutant of the present invention; (2) make expression of plants acetolactate synthestase mutant of the present invention.According to embodiments of the invention, utilize the method can effectively obtain to possess the plant of Herbicid resistant.
According to a further aspect in the invention, the authentication method of the plant that the invention allows for the method for a kind of plant that is had Herbicid resistant by foregoing acquisition and obtain.According to embodiments of the invention, the method comprise the steps one of at least: (1) measures the nucleic acid whether described plant comprises encoding acetolactate synthase mutant of the present invention; (2) measure described plant and whether express acetolactate synthestase mutant of the present invention.Thus, utilize the method, can identify effectively whether have the plant that the method for the plant of Herbicid resistant obtains by acquisition of the present invention possesses Herbicid resistant.
Need to prove, acetolactate synthestase mutant of the present invention and application thereof, the present inventor finishes through arduous creative work and the work of optimization.
Additional aspect of the present invention and advantage in the following description part provide, and part will become obviously from the following description, or recognize by practice of the present invention.
Description of drawings
Above-mentioned and/or additional aspect of the present invention and advantage are from obviously and easily understanding becoming the description of embodiment in conjunction with following accompanying drawing, wherein:
Fig. 1 has shown according to an embodiment of the invention No. 21 acetolactate synthestase mutant plants in 2 strain corn capital.
Embodiment
The below describes embodiments of the invention in detail, and the example of described embodiment is shown in the drawings, and wherein identical or similar label represents identical or similar element or the element with identical or similar functions from start to finish.Be exemplary below by the embodiment that is described with reference to the drawings, only be used for explaining the present invention, and can not be interpreted as limitation of the present invention.
At first, need to prove, acetolactate synthestase (acetolactate synthase, ALS), its enzyme classification is numbered EC4,1.3.18.Acetolactate synthestase is a key enzyme during branched-chain amino acid synthesizes, as α-amino-isovaleric acid and leucic synthetic in the catalysis pyruvic acid generate acetylactis and carbonic acid gas, catalysis pyruvic acid and batanone acid generation 2-aldehyde-base-2-hydroxybutyric acid in Isoleucine synthetic.
At present, have been found that the ALS mutain (comprises corn different plants, wheat, paddy rice, rape, with Sunflower Receptacle etc.) homologous protein the 122nd, 155,197,205,574,653, and/or 654 etc. the site can produce sudden change, thereby so that corresponding plant is to imidazolone type, sulfonylurea, the weedicides such as triazolo pyrimidine class and pyrimidine salicylic acid produce resistance (can be referring to Tan S, Evzns R R, Dahmer M L, et al.Imidazolinone-tolerant crops:History, current status and Future.Pest Management Science.2005,61:246-257; Tan S, Evzns R R, Dahmer M L, et al.Imidazolinone-tolerant crops:History, current status and Future[J] .Pest Management Science.2005,61:246-257 incorporates it into this paper in full by reference).Yet report does not show, in the 173rd or 542 sudden change from the ALS of corn, meeting is so that corresponding plant (especially corn) has Herbicid resistant.Yet the present inventor finds and separates to have obtained the new acetolactate synthestase mutant that has sudden change at the 173rd or 542 from the milpa of sudden change, and finds that these sudden changes can make plant possess Herbicid resistant.
Acetolactate synthestase mutant and coding nucleic acid thereof
For this reason, according to an aspect of the present invention, the present invention proposes a kind of acetolactate synthestase mutant.According to embodiments of the invention, compare with the wild-type acetolactate synthestase, the Ala173 of acetolactate synthestase mutant of the present invention and Trp542 have one of at least a sudden change.According to embodiments of the invention, the contriver is separated to the new mutant body of acetolactate synthestase, and Analysis deterrmination this mutant can make plant possess Herbicid resistant, thereby by making expression of plants acetolactate synthestase mutant of the present invention, can make this plant possess Herbicid resistant.
At present, the weedicide take inhibition ALS as target position comprises the weedicides such as sulfonylurea, imidazolone type, Kui Linpyrimido quinoline triazole species, Whitfield's ointment miazines.The contriver finds that acetolactate synthestase mutant of the present invention can make plant that above-mentioned weedicide is all possessed resistance.According to embodiments of the invention, acetolactate synthestase mutant of the present invention can make plant possess good imidazolinone herbicide resistance, especially Imazethapyr resistance.Wherein the chemical name of Imazethapyr is (RS) 5-ethyl-2-(4-isopropyl-4-methyl-5-oxo-3-tetrahydroglyoxaline-2-yl) nicotinic acid, and CAS number is 81335-77-5.
According to embodiments of the invention, the mutation type of the Ala173 of acetolactate synthestase mutant of the present invention and Trp542 sudden change is not particularly limited.According to concrete examples more of the present invention, compare with the wild-type acetolactate synthestase, acetolactate synthestase mutant of the present invention has and is selected from following at least a sudden change: Ala173Val and Trp542Cys namely sport Cys from the Trp that the 173rd Ala of wild-type acetolactate synthestase aminoacid sequence sports Val or the 542nd.Preferably, according to embodiments of the invention, acetolactate synthestase mutant of the present invention has the aminoacid sequence shown in SEQ ID NO:2 or 4.In addition, those skilled in the art understand, in the known situation of protein sequence, those skilled in the art have the ability to import expression vector by the coding gene sequence that changes known protein and with it, just can prepare replacement, adding or lacked the protein of amino-acid residue (can be referring to " molecular cloning experiment guide ", Beijing: Science Press, 2002, by with reference to incorporating in full it into this paper) etc. in the document, therefore, except the aminoacid sequence shown in the above-mentioned SEQ ID NO:2 or 4 that enumerates, acetolactate synthestase mutant of the present invention can also comprise aforementioned aminoacid sequence is carried out aminoacid replacement, add or disappearance and do not affect the protein of its character.
According to a further aspect in the invention, the invention allows for a kind of nucleic acid of separation.According to embodiments of the invention, the foregoing acetolactate synthestase mutant of the nucleic acid encoding of this separation.Wherein, in this article, sometimes also nucleic acid of the present invention is called the nucleic acid of encoding acetolactate synthase mutant of the present invention.According to embodiments of the invention, the nucleic acid of the encoding acetolactate synthase mutant that the present invention is separated to imports plant, can make it express aforementioned acetolactate synthestase mutant of the present invention, thereby can make this plant possess Herbicid resistant.
In this article, the type of the nucleic acid of separation of the present invention is not particularly limited, can be any deoxyribonucleotide corresponding with the encoding gene of acetolactate synthestase mutant and/or polymkeric substance of ribonucleotide of comprising, include but not limited to DNA, RNA or cDNA, preferred DNA.According to embodiments of the invention, to compare with the wild-type acetolactate synthase gene, the nucleic acid of separation of the present invention has and is selected from following at least a sudden change: c.C518T and c.G1626T.Wherein, according to some embodiments of the present invention, the nucleic acid of separation of the present invention has the nucleotide sequence shown in SEQ ID NO:1 or 3.In addition, under the prerequisite of knowing coded protein sequence or nucleotide sequence, codon corresponding relation and host expresses frequency by routine, use the method for PCR method, DNA recombination method or synthetic, those skilled in the art have the ability to obtain the also nucleic acid of Optimized Coding Based acetolactate synthestase mutant of the present invention.And then, obtain nucleic acid of the present invention after, it can be cloned into carrier, transform again or be transfected into corresponding cell, then breed by the host cell of routine, therefrom separate obtaining a large amount of nucleic acid.
Expression cassette, carrier or cell
According to another aspect of the invention, the invention allows for a kind of expression cassette, carrier or cell.According to embodiments of the invention, this expression cassette, carrier or cell comprise foregoing nucleic acid of the present invention.Thus, utilize expression cassette of the present invention and carrier effectively with the nucleic acid importing plant of encoding acetolactate synthase mutant of the present invention, to make the aforementioned acetolactate synthestase mutant of the present invention of this expression of plants, thereby to make it possess Herbicid resistant.In addition, the contriver finds that cell of the present invention can be expressed acetolactate synthestase mutant of the present invention, possesses Herbicid resistant, can effectively become the plant that possesses Herbicid resistant after cultivating.
According to embodiments of the invention, preferably, expression cassette of the present invention is suitable for expressing nucleic acid of the present invention in plant.According to some embodiments of the present invention, expression cassette of the present invention comprises successively promoter region, nucleic acid of the present invention and transcribes and translate on 5 '-3 ' transcriptional orientation terminator.According to concrete examples more of the present invention, in expression cassette of the present invention, promotor can be selected from CaMV 35S promoter, rice Actin muscle I gene, corn alcohol dehydrogenase gene, corn stunt I gene and tobacco mosaic virus (TMV) (TMV) Ω element promotor one of at least.According to some embodiments of the present invention, in expression cassette of the present invention, the terminator of transcribing and translating can comprise Transcription Termination element or polyadenylation signal.According to concrete examples more of the present invention, expression cassette of the present invention may further include and (for example copies required replication orgin in bacterium, ORI district from pBR322 or P15A ori), and edaphic bacillus T-DNA (for example shifts needed element, the left margin of T-DNA and/or right margin), thus the conversion process conveniently from bacterium toward plant.According to some embodiments of the present invention; expression cassette of the present invention can further include enhanser, intron, multiple clone site, operator gene, repressor binding site and transcription factor binding site point; for example can adopt the leader sequence that is selected from TMV, corn chlorisis mottle virus (MCMV) and alfalfa mosaic virus (AMV) one of at least as enhanser, can adopt be selected from Adh 1, bronze 1, Actin muscle 1 and Actin muscle 2 one of at least as intron.
Need to prove that employed term " carrier " refers to bacterial plasmid commonly used in this area, clay, phagemid, yeast plasmid, vegetable cell virus, animal virus and other various virus vector in this article.Different according to applied purpose, carrier can be divided into cloning vector, expression vector and conversion carrier, and its purpose is respectively for clone and checking gene, expresses corresponding gene and corresponding gene is transformed.According to embodiments of the invention, carrier of the present invention can include but not limited to: express the carrier (prokaryotic expression carrier) of usefulness in bacterium, express the carrier (such as pichia vector) of usefulness in yeast, at the baculovirus vector of expressed in insect cells, the various carriers of expressing the carrier (vaccinia virus vector, retroviral vector, adenovirus carrier, adeno-associated virus carrier etc.) of usefulness in mammalian cell, expressing the plant viral vector of usefulness and express usefulness in mammal galactophore in plant.According to embodiments of the invention, carrier of the present invention can be conversion carrier, is preferably plant conversion carrier, more preferably is fit to the carrier of Agrobacterium-mediated Transformation plant.
According to embodiments of the invention, the type of cell of the present invention is not particularly limited.According to concrete examples more of the present invention, cell of the present invention can be prokaryotic cell prokaryocyte, also can be eukaryotic cell, as, bacterial cell, yeast cell, vegetable cell, insect cell, mammalian cell etc.Need to prove, aforesaid " vegetable cell " refers to that the cell from plant (can comprise cell mass, but individual cells preferably), but these cells can not only develop into the single plant of survival with inorganics synthetic carbohydrate, protein such as water, carbonic acid gas and inorganic salt by photosynthesis.
According to embodiments of the invention, the nucleic acid of encoding acetolactate synthase mutant of the present invention can be inserted into any carrier that is suitable for transforming into cell.According to embodiments of the invention, preferably, cell of the present invention is bacterial cell, and in particular for the bacterial cell of clone or storage nucleic acid or transformed plant cells, for example Agrobacterium, intestinal bacteria comprise Agrobacterium tumefaciems and Agrobacterium rhizogenes etc.According to other embodiment of the present invention, cell of the present invention is vegetable cell, preferred maize cell, more preferably capital 21 maize cells.According to embodiments of the invention, the nucleic acid of the encoding acetolactate synthase mutant of the present invention that comprises in the aforementioned vegetable cell can import vegetable cell by transgenic technology, for example can import in nuclear, chloroplast(id), plastosome and/or the plastid of vegetable cell, also can be by mutating technology so that nucleic acid of the present invention be present in the vegetable cell.
Purposes
In accordance with a further aspect of the present invention, the present invention also provides foregoing acetolactate synthestase mutant, nucleic acid and expression cassette, carrier or cell, the purposes in obtaining to have the plant of Herbicid resistant.According to embodiments of the invention, as previously mentioned, utilize expression cassette of the present invention, carrier and cell can effectively obtain to possess the plant of Herbicid resistant.
According to embodiments of the invention, described weedicide is the weedicide take inhibition ALS as target position herein, weedicides such as sulfonylurea, imidazolone type, Kui Linpyrimido quinoline triazole species, Whitfield's ointment miazines, preferred imidazolinone herbicide resistance, more preferably Imazethapyr.
Based on aforesaid purposes, according to another aspect of the invention, the present invention also provides a kind of acquisition to have the method for the plant of Herbicid resistant.According to embodiments of the invention, acquisition of the present invention has the method for the plant of Herbicid resistant can realizing one of at least by following approach: (1) makes plant comprise the nucleic acid of encoding acetolactate synthase mutant of the present invention; (2) make expression of plants acetolactate synthestase mutant of the present invention.The contriver is surprised to find, and utilizes the method can effectively obtain to possess the plant of Herbicid resistant.
According to embodiments of the invention, the kind of the plant that method was suitable for that acquisition of the present invention has the plant of Herbicid resistant is not particularly limited.According to concrete examples more of the present invention, acquisition of the present invention has the method for Herbicid resistant to being that single, double cotyledon plant is all applicable, is particularly useful for paddy rice, wheat, rape, corn, cotton, millet and clover, more preferably corn.
According to embodiments of the invention, has the plant that the method for the plant of Herbicid resistant obtains by acquisition of the present invention, the weedicides such as weedicide such as the sulfonylurea take inhibition ALS as target position, imidazolone type, Kui Linpyrimido quinoline triazole species, Whitfield's ointment miazines had good tolerance, wherein, preferred imidazolone type, more preferably Imazethapyr.
According to embodiments of the invention, approach and the concrete steps of method that obtain to have the plant of Herbicid resistant are not particularly limited, as long as can realize by one of above-mentioned approach.According to other embodiment of the present invention, can or otherwise produce the plant that obtains to have Herbicid resistant by preparation, cultivation, production, particularly, comprise by the transgenic breeding mode obtaining, make it to express acetolactate synthestase mutant of the present invention after for example the nucleic acid of encoding acetolactate synthase mutant of the present invention being transformed into plant; Also comprise by non-transgenic breeding mode obtaining, as by hybridize, backcross, selfing or vegetative propagation obtain the plant that sub-elects the nucleic acid that still comprises encoding acetolactate synthase mutant of the present invention behind the plant and express acetolactate synthestase mutant of the present invention.According to concrete examples more of the present invention, can by be selected from transgenosis, hybridize, backcross, selfing and the vegetative plant that one of at least obtains to have Herbicid resistant.Wherein, above steps all can be known and be implemented to those skilled in the art.Thus, utilize aforesaid method of the present invention can effectively obtain to have the plant of Herbicid resistant.
Authentication method
According to a further aspect in the invention, the authentication method of the plant that the invention allows for the method for a kind of plant that is had Herbicid resistant by acquisition of the present invention and obtain.According to embodiments of the invention, the method comprise the steps one of at least: (1) measures the nucleic acid whether described plant comprises encoding acetolactate synthase mutant of the present invention; (2) measure described plant and whether express acetolactate synthestase mutant of the present invention.Thus, pass through the method, can judge effectively whether plant belongs to by acquisition of the present invention and have the plant that the method for the plant of Herbicid resistant obtains, can identify effectively namely whether have the plant that the method for the plant of Herbicid resistant obtains by acquisition of the present invention possesses Herbicid resistant.
According to embodiments of the invention, the step of evaluation is not particularly limited.According to concrete examples more of the present invention, can be undertaken by detection of nucleic acids and/or the protein detection method of routine, as long as can acetolactate synthestase or its coding nucleic acid of plant be detected, particularly, whether whether the acetolactate synthestase of wanting to detect plant carries Ala173 or Trp542 sudden change or its coding nucleic acid has the corresponding nucleic sudden change and get final product, preferably identifies by detecting the method that Ala173Val or Trp542Cys sudden change or its corresponding nucleic suddenly change.Some concrete examples according to the present invention can be by being selected from that protein sequencing, nucleic acid sequencing, polymerase chain reaction (PCR) detect and the plant that one of at least the method that has the plant of Herbicid resistant by acquisition of the present invention is obtained of probe hybridization detection is identified.
According to embodiments of the invention, had the method for plant of Herbicid resistant by acquisition of the present invention and the kind of the plant that authentication method was suitable for of the plant that obtains is not particularly limited.According to concrete examples more of the present invention, above-mentioned authentication method is all applicable to single, double cotyledon plant, is particularly useful for paddy rice, wheat, rape, corn, cotton, millet and clover, more preferably corn.
Below with reference to specific embodiment, the present invention will be described, need to prove, these embodiment only are illustrative, and can not be interpreted as limitation of the present invention.If do not specialize, the conventional means that the technique means that adopts among the embodiment is well known to those skilled in the art, for example can carry out with reference to " molecular cloning experiment guide " third edition or related products, the reagent that adopts and product also are and can commercial obtain.Various processes and the method do not described in detail are ordinary methods as known in the art, the source of agents useful for same, trade(brand)name and be necessary to list its moiety person, all when occurring first, indicate, thereafter used identical reagent if no special instructions, all identical with the content of indicating first.
Embodiment 1: anti-imidazolinone herbicide mutant genetics compartment analysis
With 5 mu of ground of 21 sowings, corn inbred line capital, application rate 5000kg/ mu.Pollinating season, with male flower and female flower difference bagging, collect fresh male flower pollen, with containing 0.067%(w/w) paraffin oil of ethyl methane sulfonate (EMS) concentration processed pollen 45 minutes, wherein pollen and paraffin oil volume ratio are about 1:10, then are coated onto on the female flower filigree and bagging at the mixture of handling in 30 minutes pollen and paraffin oil.Gather in the crops after the plant to be planted maturation.Then, with the seed of results imidazolinone herbicide Imazethapyr ((RS) 5-ethyl-2-(4-isopropyl-4-methyl-5-oxo-3-tetrahydroglyoxaline-2-yl) nicotinic acid, CAS 81335-77-5) (concentration is 0.5% to aqua, available from Shandong Cynda Chemical Co., Ltd) soaked 18 hours, be seeded in the experimental plot after draining away the water.Observe the Course of Corn Seed Germination situation after 10 days, find wherein to have the green rice shoot of 2 strains to continue the growth (see figure 1), keep and breed this green rice shoot, it is No. 21 mutant plants in capital of anti-imidazolinone herbicide.
Get respectively the blade of No. 21 mutant plants in above-mentioned each capital (mutant plant 1 and mutant plant 2), and take the wild-type capital No. 21 plant as contrast, extract respectively genomic dna, and entrust Beijing three rich polygala root Bioisystech Co., Ltd to check order, found that: mutant plant 1 is with respect to No. 21, wild-type capital, undergo mutation in the 518th site at acetolactate synthestase (ALS) gene, become T by C, cause the 173rd of corresponding encoded albumen to become α-amino-isovaleric acid by L-Ala, namely the nucleotide sequence of the als gene of this mutant plant is shown in SEQ ID NO:1, and the aminoacid sequence of the ALS albumen of its coding is shown in SEQID NO:2; Mutant plant 2 is with respect to No. 21, wild-type capital, undergo mutation in the 1626th site at als gene, become T by G, cause the 542nd of corresponding encoded albumen to become halfcystine by tryptophane, namely the nucleotide sequence of the als gene of this mutant plant is shown in SEQ ID NO:3, and the aminoacid sequence of the ALS albumen of its coding is shown in SEQ ID NO:4.Above-mentioned sudden change als protein from No. 21, capital is acetolactate synthestase mutant of the present invention.
The isolated activity of the anti-imidazolinone herbicide of embodiment 2 acetolactate synthestase mutant is measured
Method (Specific activity determination of acetolactate synthase from maize with reference to Fan ZJ etc., Zea mays L., incorporate in full it into this paper by reference), and book (the The Proceedings of the 18 th Asia-Pacific Weed Science Society Conference.515-522.May 28-June 2 that write referring to Han WN etc., 2001.Beijing:Standards Press of China., incorporate in full it into this paper by reference), the wild-type that No. 21, the capital among the extraction embodiment 1 and the ALS enzyme (being acetolactate synthestase) of each mutant plant, and measure the ratio that corresponding enzymic activity is suppressed by imidazolinone herbicide.
Concrete steps are as follows:
After getting respectively each plant seedling 5g chopping, add the 50mmol/L K of 10mL pH 7
2HPO
4-KH
2PO
4Damping fluid (wherein contains the 1mmol/L Sodium.alpha.-ketopropionate, 0.5mmol/L MgCl
2, 0.5mmol/L TPP, 10 μ mol/L FAD), with 8 layers of filtered through gauze, filtrate was in centrifugal 30 minutes of 4 ℃, 20000rpm after then pulverizing with quartzite sand grind.Get supernatant liquor, add ammonium sulfate and make it to reach 50% saturation ratio, then place after 2 hours for 0 ℃, in centrifugal 30 minutes of 4 ℃, 20000rpm.Abandon supernatant liquor, will be precipitated and dissolved in the 50mmol/L K of 5mL pH 7
2HPO
4-KH
2PO
4Damping fluid (wherein contains the 20mmol/L Sodium.alpha.-ketopropionate, 0.5mmol/L MgCl
2), respectively the ALS enzyme liquid of each plant.Then, 0.1mL 0.5% Imazethapyr aqua (contrast water consumption substitution), 0.5mL enzyme reaction solution (are wherein contained the 24mmol/L Sodium.alpha.-ketopropionate, 0.6mmol/L MgCl
2, 1mmol/L TPP, 20 μ mol/LFAD, 50mmol/L K
2HPO
4-KH
2PO
4, pH 7) and 0.4mLALS enzyme liquid mix, with 35 ℃ of lucifuge standing and reacting 1 hour, then add 0.1mL 3mol/L sulfuric acid termination reaction, place decarboxylation in 15 minutes in 60 ℃.Then adding developer (0.5ml 0.5%(w/w) creatine and 0.5ml 5%(w/w) mixing solutions (being dissolved in the NaOH solution of 2.5mol/L) of naphthyl alcohol places colour developing in 15 minutes in 60 ℃, measures the absorbancy at 525nm place.The ALS enzymic activity that adds No. 21, wild-type capital of contrast is designated as respectively 100%, calculates imidazolinone herbicide to the impact of the activity of the wild-type acetolactate synthestase that derives from No. 21, capital and each mutant acetolactate synthetic enzyme.
The result is as shown in the table, the ALS enzyme that respectively suddenlys change that No. 21, the capital has all kept the activity of wild-type ALS enzyme substantially, and exist in the situation of imidazolinone herbicide, the activity of wild-type ALS enzyme has very significant decline, and the ALS enzyme that respectively suddenlys change has all kept constitutive enzyme active, shows that sudden change ALS endonuclease capable brings the performance of anti-imidazolinone herbicide.
Imidazolinone herbicide is on the table that affects of the wild-type in No. 21, capital and sudden change ALS enzymic activity
The acquisition of the transgenic plant of the white mutant gene of embodiment 3 turning egg(s)s
The contriver entrusts the Weimingkaituo Agro-Biological Technology Co., Ltd., Beijing to change respectively wild-type Arabidopis thaliana plant over to the encoding gene (SEQ ID NO:1 and 3) that conventional agrobacterium-mediated transformation will derive from the sudden change ALS enzyme of No. 21 acetolactate synthestase mutant plants in capital among the embodiment 1, and concrete steps are as follows:
Utilize forward primer (5 '-CCGTCCGGTCTGTAGCGTGTAC-3 ': SEQ ID NO:5) and reverse primer (5 '-CATAACAGATAGCTGACGGCCTAC-3 ': SEQ ID NO:6), the pcr amplification als gene that goes out to suddenly change from the genomic dna of each mutant plant in No. 21, above-mentioned capital respectively, after order-checking is correct, the als gene of nucleotide sequence shown in SEQ IDNO:1 and 3 is cloned in the pCAMBIA1303 plasmid (available from Cambia company), select positive colony and transform Agrobacterium AGL0, cultivate thalline and arabidopsis thaliana transformation, PCR obtains the T1 transgenic seed in generation after identifying and transforming positive Arabidopis thaliana sowing, process with the spraying of imidazolinone herbicide Imazethapyr during plantation, the discovery growth conditions is good, and not genetically modified Arabidopis thaliana seedling is all withered.Thus, obtain transgenic arabidopsis with imidazolinone herbicide resistance.
In the description of this specification sheets, the description of reference term " embodiment ", " some embodiment ", " example ", " concrete example " or " some examples " etc. means to be contained at least one embodiment of the present invention or the example in conjunction with specific features, structure, material or the characteristics of this embodiment or example description.In this manual, the schematic statement of above-mentioned term not necessarily referred to identical embodiment or example.And the specific features of description, structure, material or characteristics can be with suitable mode combinations in any one or more embodiment or example.
Although illustrated and described embodiments of the invention, those having ordinary skill in the art will appreciate that: can carry out multiple variation, modification, replacement and modification to these embodiment in the situation that does not break away from principle of the present invention and aim, scope of the present invention is limited by claim and equivalent thereof.
Claims (10)
1. an acetolactate synthestase mutant is characterized in that, compares with the wild-type acetolactate synthestase, and the Ala173 of described acetolactate synthestase mutant and Trp542 have one of at least a sudden change.
2. acetolactate synthestase mutant according to claim 1 is characterized in that, compares with the wild-type acetolactate synthestase, and described acetolactate synthestase mutant has and is selected from following at least a sudden change: Ala173Val and Trp542Cys.
3. acetolactate synthestase mutant according to claim 1 and 2 is characterized in that, described acetolactate synthestase mutant has the aminoacid sequence shown in SEQ ID NO:2 or 4.
4. the nucleic acid of a separation is characterized in that, each described acetolactate synthestase mutant of the nucleic acid encoding claim 1 ~ 3 of described separation,
Randomly, compare with the wild-type acetolactate synthase gene, the nucleic acid of described separation has and is selected from following at least a sudden change: c.C518T and c.G1626T.
5. the nucleic acid of separation according to claim 4 is characterized in that, the nucleic acid of described separation has the nucleotide sequence shown in SEQ ID NO:1 or 3.
6. an expression cassette, carrier or cell, it comprises claim 4 or 5 described nucleic acid.
7. each described acetolactate synthestase mutant of claim 1 ~ 3, claim 4 or 5 described nucleic acid and expression cassette claimed in claim 6, carrier or cell, the purposes in obtaining to have the plant of Herbicid resistant,
Randomly, described weedicide is imidazolinone herbicide, preferred Imazethapyr.
8. an acquisition has the method for the plant of Herbicid resistant, it is characterized in that, by realizing one of at least of following approach:
(1) make described plant comprise claim 4 or 5 described nucleic acid;
(2) make each described acetolactate synthestase mutant of described expression of plants claim 1 ~ 3,
Preferably, described plant is at least a of paddy rice, wheat, rape, corn, cotton, millet and clover, and more preferably, described plant is corn,
Randomly, described weedicide is imidazolinone herbicide, preferred Imazethapyr.
9. method according to claim 8 is characterized in that, by be selected from transgenosis, hybridize, backcross, selfing and the vegetative plant that one of at least obtains to have Herbicid resistant.
10. the authentication method of a plant that is obtained by claim 8 or 9 described methods is characterized in that, comprise the steps one of at least:
(1) measures described plant and whether comprise claim 4 or 5 described nucleic acid;
(2) measure described plant and whether express each described acetolactate synthestase mutant of claim 1 ~ 3,
Preferably, described plant is at least a of paddy rice, wheat, rape, corn, cotton, millet and clover, and more preferably, described plant is corn.
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| CN 201210479351 CN102936591A (en) | 2012-11-22 | 2012-11-22 | Acetolactic acid synthetase mutants and application thereof |
| CN201310042383.4A CN103103176B (en) | 2012-11-22 | 2013-02-01 | Herbicide-resistant corn protein and application thereof in plant breeding |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106636026A (en) * | 2017-02-08 | 2017-05-10 | 上海市农业科学院 | Corn protein having herbicide resistance activity and encoding gene and application thereof |
| WO2017177765A1 (en) * | 2016-04-12 | 2017-10-19 | 江苏省农业科学院 | Applications of als mutant type gene in resisting against herbicides |
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| CN106467908B (en) * | 2015-08-18 | 2019-06-21 | 未名兴旺系统作物设计前沿实验室（北京）有限公司 | The plant of herbicide-tolerant and its application |
| CN106701726A (en) * | 2017-02-08 | 2017-05-24 | 上海市农业科学院 | Rapeseed protein having anti-imidazolone weedicide activity, and coding gene and application thereof |
| CN106754810A (en) * | 2017-02-08 | 2017-05-31 | 上海市农业科学院 | Rape protein, its encoding gene and its application with antiweed activity |
| CN107267480A (en) * | 2017-07-13 | 2017-10-20 | 未名兴旺系统作物设计前沿实验室（北京）有限公司 | The application of herbicide resistant protein and its gene in plant breeding |
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| CN102533844A (en) * | 2011-12-21 | 2012-07-04 | 中国农业科学院生物技术研究所 | Corn chloroplast transgene expression vector containing herbicide resistant gene and application thereof |
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| WO2017177765A1 (en) * | 2016-04-12 | 2017-10-19 | 江苏省农业科学院 | Applications of als mutant type gene in resisting against herbicides |
| CN106636026A (en) * | 2017-02-08 | 2017-05-10 | 上海市农业科学院 | Corn protein having herbicide resistance activity and encoding gene and application thereof |
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| CN103103176A (en) | 2013-05-15 |
| CN103103176B (en) | 2014-03-26 |
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