CN103103176A - Herbicide-resistant corn protein and application thereof in plant breeding - Google Patents
Herbicide-resistant corn protein and application thereof in plant breeding Download PDFInfo
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- CN103103176A CN103103176A CN2013100423834A CN201310042383A CN103103176A CN 103103176 A CN103103176 A CN 103103176A CN 2013100423834 A CN2013100423834 A CN 2013100423834A CN 201310042383 A CN201310042383 A CN 201310042383A CN 103103176 A CN103103176 A CN 103103176A
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Abstract
The invention provides an ALS (Acetolactate Synthase) protein capable of endowing plants with herbicide resistance. The amino acid sequence of the herbicide-resistant corn protein is as shown in SEQ ID NO: 2 or 4. Furthermore, the invention further provides corresponding nucleic acid, expression cassettes, vectors, cells, the plants, application and a method for obtaining the plants with herbicide resistance, and a method for identifying the plants in the invention.
Description
Technical field
The invention belongs to the plant protein field, particularly, the present invention relates to give the albumen of plant (especially corn) Herbicid resistant (tolerance) and the application in plant breeding thereof.
Background technology
In the farmland, weeds are one of most important factor that affect crop yield, 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, and it uses as playing a significantly greater role in the innovation that solves Farmland weed, promotion planting type and volume increase.
Common weedicide includes the weedicides such as sulfonylurea, imidazolone type, Kui Linpyrimido quinoline triazole species, Whitfield's ointment miazines, and big area business promotion is at present used.Yet, because these weedicides also can kill farm crop itself usually, therefore for generally not having the farm crop of Herbicid resistant (tolerance), the use of weedicide has been subject to very big restriction on time and space, use for the previous period weedicide as needs in crop seeding.
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.although current have many bibliographical informations to suddenly change on some gene to import again wild-type plant become transgenic plant, can bring Herbicid resistant to plant, but the experiment spininess model plant more clearly to genetic backgrounds such as Arabidopis thalianas, but the farm crop for the reality plantation, especially the farm crop with good proterties (genetic background complexity simultaneously) that process transgenosiss in a large amount of early stage and/or non-transgenic breeding obtain, often situation is very pessimistic, find as us, import at external and/or Arabidopis thaliana the mutator gene that test has Herbicid resistant, after importing corn etc., the situation of losing Herbicid resistant but occurs to have occured in a large number to reply, although some has kept Herbicid resistant, use in putting into practice but corn growth is undesired at all, this is likely due to the synergistic result of lots of genes in corn.So, for the plant of complex inheritance background (as corn, especially the corn of improveing), have the plant of Herbicid resistant and truly can also not have at present the theory rational design to go out by conclusive material (as albumen, gene etc.), can only depend on the arduous practice of scientific research personnel and obtain, especially rely on long-term screening, and rely on some fortune to obtain.
The inventor is long-term and arduous research practice just, rely on fortune to find a series of (sudden change) albumen by accident in 21 mutant plants of corn inbred line capital, it can keep the normal plants physiological function of original albumen substantially under the condition that weedicide exists, thereby makes corn (especially the capital 21) have Herbicid resistant (tolerance).The inventor has also developed these albumen and the application of encoding gene in transgenosis or non-transgenic plant breeding thereof, can be used for cultivating the plant with Herbicid resistant, and farm crop especially are as corn etc.
Summary of the invention
The technical problem to be solved in the present invention is that the plant that makes that provides new has the protein of Herbicid resistant.In addition, the present invention also provide the nucleic acid of these protein of encoding and genetically engineered intermediate (as, expression cassette, carrier and cell etc.), acquisition has the methods and applications of the plant of Herbicid resistant, and the authentication method that judges whether plant adopts the inventive method to obtain is provided.
Particularly, in first aspect, the invention provides the ALS mutein, its aminoacid sequence is as shown in SEQ ID NO:2 or 4.This albumen mass-energy makes plant (as corn, especially the capital 21) have Herbicid resistant.In this article, ALS is exactly the abbreviation of acetolactate synthestase (acetolactate synthase), and 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.
The protein of first aspect present invention is to find from the milpa of inventor sudden change (as, capital 21) through a large amount of screenings, can make corresponding plant have Herbicid resistant.At present, also report does not show, at the mutain of aminoacid sequence as shown in SEQ ID NO:2 or 4 from corn, can make corresponding plant (especially corn) have Herbicid resistant, more unpredictable when keeping Herbicid resistant, can also keep normal growing state.The characteristic traits such as in this article, capital 21 claims again the capital No. 21, is the corn selfing strain by the exploitation of Institute of Crop Science, Chinese Academy of Agricultural Science's corn research centre, and it has the plant type compactness, disease resistance is good, resistant to lodging and combining ability is strong.The inventor finds that this strain is can be in the seed immersion test of high density Imazethapyr screened and goes out anti-Imazethapyr mutant plant.The public can obtain capital 21 by the commercial channel, for example can buy from Institute of Crop Science, Chinese Academy of Agricultural Science's corn research centre.
Except the described mutant plant screening of the specific embodiment of the invention and amplification method, in the situation that protein sequence is known, 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, add or lacked the protein of amino-acid residue, these methods are recorded in the documents such as " molecular cloning experiment guide " (Beijing: Science Press, 2002).By transgenic technology, the gene of these protein of encoding can import in plant, preferably imports in corn, for example imports in (wild-type) capital 21, thereby gives Genes For Plant Tolerance weedicide performance.
At present, weedicide take inhibition ALS as target position comprises weedicide (the Peter Babczinski such as sulfonylurea, imidazolone type, Kui Linpyrimido quinoline triazole species, Whitfield's ointment miazines, Thomas Zelinski.Mode of action herbicidal ALS-inhibitors on acetolactate synthase from green plant cell cultures, yeast, and Escherichia coli.Pesticide science, 1991,31:305-323; Zheng Peizhong, Shen Jianying. the kind of inhibitor of acetolactate synthetase and Advance in Drug Resistance thereof. Weed Science, 2009,2:4-8).Therefore, in this article, the weedicide that Herbicid resistant refers to comprising sulfonylurea, imidazolone type, Kui Linpyrimido quinoline triazole species, Whitfield's ointment miazines has tolerance.Preferably in the present invention, weedicide is imidazolinone herbicide, as in the specific embodiment of the present invention, weedicide is Imazethapyr (its chemical name is (RS) 5-ethyl-2-(4-isopropyl-4-methyl-5-oxo-3-tetrahydroglyoxaline-2-yl) nicotinic acid, and No. CAS is 81335-77-5).Preferably in the present invention namely, Herbicid resistant is the imidazolinone herbicide resistance, is more preferably the Imazethapyr resistance.
In second aspect, the invention provides nucleic acid, the protein of its coding first aspect present invention.In this article, nucleic acid can be DNA, can be also RNA, preferably DNA.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 the protein of Optimized Coding Based first aspect present invention.In case obtain this nucleic acid, just it can be cloned into carrier, then transform or be transfected into corresponding cell, then breed by the host cell of routine, therefrom separate obtaining a large amount of nucleic acid.The nucleotide sequence of the nucleic acid of preferred second aspect present invention is as shown in SEQ ID NO:1 or 3.
In the third aspect, the invention provides expression cassette (cassette), it comprises the nucleic acid of second aspect present invention.The terminator that the expression cassette of third aspect present invention preferably comprises the nucleic acid of promoter region, second aspect present invention and transcribes and translate on 5 '-3 ' transcriptional orientation.The expression cassette of third aspect present invention preferably is adapted at expressing in plant the nucleic acid of second aspect present invention.Exemplary promotor comprises from the stunt promotor of I gene or tobacco mosaic virus (TMV) (TMV) Ω element of CaMV35S promotor or rice Actin muscle I gene, corn alcohol dehydrogenase gene, corn; The exemplary terminator of transcribing and translating comprises Transcription Termination element or polyadenylation signal etc.The expression cassette of third aspect present invention can also comprise, (for example copy required replication orgin in bacterium, ORI district from pBR322 or P15Aori), 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.In addition; the third aspect present invention expression cassette can also comprise enhanser, intron, multiple clone site, operator gene, repressor binding site, transcription factor binding site point etc.; such as from leader sequence of TMV, corn chlorisis mottle virus (MCMV) or alfalfa mosaic virus (AMV) etc. as enhanser, and for example can comprise the intron from Adh1, bronze1, Actin muscle 1 or Actin muscle 2.
In fourth aspect, the invention provides carrier, it comprises the nucleic acid of second aspect present invention.In this article, carrier refers to bacterial plasmid commonly used in this area, clay, phagemid, yeast plasmid, vegetable cell virus, animal virus and other various virus vector.Different according to applied purpose, carrier can be divided into cloning vector, expression vector and conversion carrier, refers to the purpose used respectively for clone and verify gene, expression corresponding gene and corresponding gene being transformed.Carrier available in the present invention includes but not limited to: express the carrier (prokaryotic expression carrier) of use in bacterium, express the carrier (as pichia vector) of use 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 use in mammalian cell, expressing the plant viral vector of use and express use in mammal galactophore in plant.The carrier of fourth aspect present invention is conversion carrier, especially plant conversion carrier, the particularly carrier of suitable Agrobacterium-mediated Transformation plant.The much commercializations of these carriers.
Aspect the 5th, the invention provides cell, it comprises the nucleic acid of second aspect present invention.Cell can be prokaryotic cell prokaryocyte, can be also eukaryotic cell, as, bacterial cell, yeast cell, vegetable cell, insect cell, mammalian cell etc.In this article, the cell that vegetable cell refers to 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.Vegetable cell can be to use organism (as the certain plants hormone) just developable vegetable cell or cell mass, can be also fully without the vegetable cell or the cell mass that develop into the plant ability, generally, i.e. dead vegetable cell or cell mass, the commercially available wheat flour of crossing as drying and crushing etc.
The nucleic acid of a second aspect of the present invention can be inserted in any carrier that transforms in cell.Preferred cell is bacterial cell, and in particular for the clone or store nucleic acid or be used for the bacterial cell of transformed plant cells, for example Agrobacterium, intestinal bacteria, comprise Agrobacterium tumefaciems and Agrobacterium rhizogenes etc.Preferred cell is vegetable cell in addition, maize cell preferably, be more preferably capital 21 cells, the nucleic acid of the second aspect present invention that comprises in described vegetable cell can import vegetable cell by transgenic technology, comprising in core, chloroplast(id), plastosome and/or the plastid that imports to vegetable cell, can be also to make the nucleic acid of second aspect present invention be present in vegetable cell by mutating technology (technology as described in the specific embodiment of the invention).
Aspect the 6th, the invention provides plant, it comprises the nucleic acid of second aspect present invention, and/or it expresses the protein of first aspect present invention.Due to the protein of the nucleic acid of having introduced second aspect present invention and first aspect present invention, the plant of sixth aspect present invention has Herbicid resistant.In this article, plant refers to by photosynthesis, maintain single plant, plant group or its reproductive material of existence with inorganicss such as water, carbonic acid gas and inorganic salt with regard to energy synthetic carbohydrate, protein, comprise the bred part of plant, plant variety, plant, plant event, plant offspring, plant seed or other plant.Wherein, the plant offspring itself is exactly plant, the plant offspring who comprise the plant offspring that produces by transgenic technology, produces with the other plant mixing breed and backcrossing or plant offspring that selfing produces.
The plant of sixth aspect present invention can be dicotyledons, can be also monocotyledons.Exemplary plant comprises wheat, paddy rice, corn, rye, oat, barley, soybean, potato, rape, beet, sugarcane, Chinese sorghum, tomato, pumpkin, capsicum, romaine lettuce, Sunflower Receptacle, coffee, tea, cotton, clover, tobacco or Arabidopis thaliana etc.In the specific embodiment of the present invention, the plant of preferred sixth aspect present invention is corn, is more preferably capital 21.Certainly, as in the specific embodiment of the present invention, Arabidopis thaliana is also optional plant variety.
Aspect the 7th, the invention provides the application of aforementioned aspects of the present invention in obtaining to have the plant process of Herbicid resistant.In obtaining to have the plant process of Herbicid resistant, can be applied to aforementioned aspects of the present invention.what seventh aspect present invention provided comprises, the application of the protein of first aspect present invention in obtaining to have the plant process of Herbicid resistant, the application of the nucleic acid of second aspect present invention in obtaining to have the plant process of Herbicid resistant, the application of the expression cassette of third aspect present invention in obtaining to have the plant process of Herbicid resistant, the application of the carrier of fourth aspect present invention in obtaining to have the plant process of Herbicid resistant, the application of the cell of fifth aspect present invention in obtaining to have the plant process of Herbicid resistant, the application of the plant of sixth aspect present invention in obtaining to have the plant process of Herbicid resistant.Preferably in a seventh aspect of the present invention, weedicide is imidazolinone herbicide, preferably Imazethapyr.In the specific embodiment of the present invention, the plant with Herbicid resistant that obtains in preferred seventh aspect present invention is corn, is more preferably capital 21.Certainly, as in the specific embodiment of the present invention, Arabidopis thaliana is also optional plant variety.
In this article, obtain to comprise preparation, cultivation, production or otherwise produce to obtain, comprise by the transgenic breeding mode obtaining, be transformed into the protein that makes it to express first aspect present invention after plant as the nucleic acid with second aspect present invention; Also comprise by non-transgenic breeding mode obtaining, as by hybridize, backcross, the plant of selfing or vegetative propagation sixth aspect present invention and sub-elect the nucleic acid that still comprises second aspect present invention and express the plant of the protein of first aspect present invention.Therefore, the application that a seventh aspect of the present invention provides comprises, the application in obtaining to have the transgenic plant process of Herbicid resistant and obtaining has the application in the non-transgenic plant process of Herbicid resistant.
First independently aspect, the invention provides (wild-type) capital 21 the plant that obtains to have Herbicid resistant (as, corn) application in, the preferably application in the corn with Herbicid resistant of the protein that obtains to comprise the nucleic acid of second aspect present invention and/or express first aspect present invention.Capital 21 itself has good proterties, but the more important thing is, the inventor finds, to it seed with weedicide (as, imidazolinone herbicide, preferably Imazethapyr) carry out mutagenesis, can obtain to have the corn of Herbicid resistant.
In eight aspect, the invention provides the method that acquisition has the plant of Herbicid resistant, it comprises the steps:
(1) make plant comprise the nucleic acid of second aspect present invention; And/or,
(2) make the protein of expression of plants first aspect present invention.
Preferably in the method for eighth aspect present invention, plant is corn, is more preferably capital 21.Certainly, as in the specific embodiment of the present invention, Arabidopis thaliana is also optional plant variety.
Can adopt transgenosis and non-transgenic method aforementioned or that those skilled in the art can be known, make plant comprise the nucleic acid of second aspect present invention, perhaps make the protein of expression of plants first aspect present invention.Implement the method for eighth aspect present invention, can obtain the plant of sixth aspect present invention.Preferably in a eighth aspect of the present invention, the step that can adopt comprises transgenosis, hybridizes, backcrosses, selfing or vegetative propagation step.These steps itself all can be known and be implemented for the technician in transgenosis or non-transgenic breeding field.
Aspect the 9th, the method for the plant that the method for the plant of identifying sixth aspect present invention or eighth aspect present invention of the invention provides obtains, it comprises the steps:
(1) measure the nucleic acid whether described plant comprises second aspect present invention; And/or,
(2) measure the protein whether described plant expresses first aspect present invention.
By the method, can judge whether plant belongs to plant of the present invention, namely whether belongs to the plant of sixth aspect present invention, perhaps whether belong to the plant of the method acquisition of eighth aspect present invention.The step of measuring can be undertaken by detection of nucleic acids and/or the protein detection method of routine, only need to be able to detect protein or its coding nucleic acid band and all include the present invention in just like the method for the mutain shown in SEQ ID NO:2 or 4 or the sudden change of its corresponding nucleic.Exemplary method comprises protein sequencing, nucleic acid sequencing, polymerase chain reaction (PCR) detection, probe hybridization detection etc.
The protein, nucleic acid, expression cassette, carrier, cell, plant, the acquisition that make plant have Herbicid resistant that the beneficial effect that the present invention obtains is to obtain alternative prior art has application and the method for plant of Herbicid resistant and the method for identifying plant of the present invention, and can obtain to have the plant variety of Herbicid resistant by transgenosis or non-transgenic method, especially can inherit the corn variety of No. 21 good characteristics in capital.
The present invention has quoted open source literature, and these documents are in order more clearly to describe the present invention, and their full text content is all included this paper in and carried out reference, just looks like that repeated description is excessively the same in this article for their full text.For the ease of understanding, below will the present invention be described in detail by concrete drawings and Examples.It needs to be noted, these descriptions are only exemplary descriptions, do not consist of limitation of the scope of the invention.According to the discussion of this specification sheets, many variations of the present invention, change are all apparent concerning one of ordinary skill in the art.
Description of drawings
Fig. 1 has shown the maize mutant plant of anti-imidazolinone herbicide.
Embodiment
In following embodiment, method therefor if no special instructions, is the method that molecular biology commonly used, tissue culture technique and agronomy handbook are put down in writing.For example, concrete steps can be referring to " Molecular Cloning:A Laboratory Manual (3
rdEdition) " (Sambrook, J., Russell, David W., 2001, Cold Spring Harbor), " Plant Propagation by Tissue Culture " (EdwinF.George, Michael A.Hall, Geert-Jan De Klerk, 2008, Springer).
Embodiment 1 extracts the protein mutant of anti-imidazolinone herbicide from the mutant plant of No. 21, capital
Sow each kind/strain corn, comprising can be available from Institute of Crop Science, Chinese Academy of Agricultural Science's corn research centre with corn inbred line capital 21() sowing, before sowing, with various corn seeds 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.Through the great many of experiments screening, finally chance on, wherein there is green rice shoot to continue growth (Fig. 1), keep and breed these green rice shoots, confirm as No. 21 mutant plant 2 strains in capital of anti-imidazolinone herbicide, these plant can grow by normal growth, and all the other corns are all dead.
get respectively the blade of each No. 21 mutant plants in capital, No. 21, capital plant leaf together with wild-type, extract respectively their genomic dna, entrust Beijing three rich polygala root Bioisystech Co., Ltd check order and carry out sequence relatively, find: 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 as shown in SEQ ID NO:1, the aminoacid sequence of the ALS albumen of its coding is as shown in SEQ ID 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 as shown in SEQ ID NO:3, and the aminoacid sequence of the ALS albumen of its coding is as shown in SEQ ID NO:4.
The isolated activity of the anti-imidazolinone herbicide of embodiment 2 protein mutants is measured
Method (Specific activity determination of acetolactate synthase from maize(Zea mays L.) with reference to Fan ZJ etc. compile .The Proceedings of the18th Asia-Pacific Weed Science Society Conference.515-522.May28-June2 referring to Han WN etc., 2001.Beijing:Standards Press of China.), extract the wild-type in No. 21, above-mentioned capital and the ALS enzyme of each mutant plant, and measure the ratio that corresponding enzymic activity is suppressed by imidazolinone herbicide.Process after getting respectively each plant seedling 5g chopping, adds the 50mmol/LK of 10mL pH7 in brief
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), after pulverizing with quartzite sand grind, with 8 layers of filtered through gauze, filtrate was in centrifugal 30 minutes of 4 ℃, 20000rpm.Get supernatant liquor, add ammonium sulfate to 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 pH7
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.0.1mL0.5% Imazethapyr aqua (contrast water consumption substitution), 0.5mL enzyme reaction solution (are wherein contained the 24mmol/L Sodium.alpha.-ketopropionate, 0.6mmol/LMgCl
2, 1mmol/L TPP, 20 μ mol/L FAD, 50mmol/L K
2HPO
4-KH
2PO
4, pH7) and 0.4mL ALS enzyme liquid mix, with 35 ℃ of lucifuge standing and reacting 1 hour, then add 0.1mL3mol/L sulfuric acid termination reaction, place decarboxylation in 15 minutes in 60 ℃.Then add developer (0.5ml0.5%(w/w) creatine and 0.5ml5%(w/w) mixing solutions (being dissolved in the NaOH solution of 2.5mol/L) of naphthyl alcohol) place colour developing in 15 minutes in 60 ℃, measure the absorbancy at 525nm place.To add the ALS enzymic activity in No. 21, wild-type capital of contrast to be designated as respectively 100%, calculate imidazolinone herbicide to the impact of the activity of the wild-type in No. 21, capital and each sudden change ALS enzyme.
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 in the situation that there is imidazolinone herbicide, the activity of wild-type ALS enzyme has decline very significantly, 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.
The affect table of imidazolinone herbicide on the wild-type in No. 21, capital and sudden change ALS enzymic activity
The acquisition of the transgenic plant of the embodiment 3 white mutant genes of turning egg(s)
Entrust the Weimingkaituo Agro-Biological Technology Co., Ltd., Beijing to change respectively the encoding gene of the sudden change ALS enzyme in No. 21, above-mentioned capital over to wild-type Arabidopis thaliana plant with the agrobacterium-mediated transformation of routine.process in brief, utilize forward primer (5 ’ – CCGTCCGGTCTGTAGCGTGTAC-3 ') and reverse primer (5 '-CATAACAGATAGCTGACGGCCTAC-3 '), 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 as shown in SEQ ID NO:1 and 3 is cloned in 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.
Claims (10)
1.ALS mutein, its aminoacid sequence is as shown in SEQ IDNO:2 or 4.
2. protein claimed in claim 1, wherein weedicide is imidazolinone herbicide, preferably Imazethapyr.
3. the described protein of claim 1 or 2, it is from corn, and especially the capital 21.
4. nucleic acid, the arbitrary described protein of its coding claim 1 ~ 3, preferably its nucleotide sequence is as shown in SEQ ID NO:1 or 3.
5. expression cassette, carrier or cell, it comprises nucleic acid claimed in claim 4.
6. the arbitrary described protein of claim 1 ~ 3, nucleic acid claimed in claim 4, expression cassette claimed in claim 5, carrier or cell, the plant that comprises nucleic acid claimed in claim 4, the plant of arbitrary described protein of expressing claim 1 ~ 3 or capital 21 application in obtaining to have the plant of Herbicid resistant, preferably wherein weedicide is imidazolinone herbicide, preferably Imazethapyr.
7. acquisition has the method for the plant of Herbicid resistant, and it comprises the steps:
(1) make plant comprise nucleic acid claimed in claim 4; Or,
(2) make the arbitrary described protein of expression of plants claim 1 ~ 3.
8. method claimed in claim 7, it comprises transgenosis, hybridizes, backcrosses, selfing or vegetative propagation step.
9. the described application of claim 6 or 7 or method, wherein said plant is corn.
10. the method for plant identification, wherein plant be the plant that obtains of the corn of the corn that comprises nucleic acid claimed in claim 4, arbitrary described protein of expressing claim 1 ~ 3 or claim 7,8 or 9 described methods (as, corn), it comprises the steps:
(1) measure described plant and whether comprise nucleic acid claimed in claim 4; Or,
(2) measure the arbitrary described protein whether described plant expresses claim 1 ~ 3.
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| CN106467908A (en) * | 2015-08-18 | 2017-03-01 | 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 |
| 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 |
| 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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| CN105695493A (en) * | 2016-04-12 | 2016-06-22 | 江苏省农业科学院 | Application of ALS mutant type genes in aspect of herbicide resistance |
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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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| 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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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106467908A (en) * | 2015-08-18 | 2017-03-01 | 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 |
| 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 |
| 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 |
| 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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| CN103103176B (en) | 2014-03-26 |
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