CN103103176B - 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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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 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 farmland, weeds are one of greatest 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 solution Farmland weed, the innovation that promotes 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 conventionally, 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, as needs are used weedicide for the previous period in crop seeding.
For this reason, people's research and development have the plant (farm crop) of Herbicid resistant (tolerance), can between planting season, use weedicide like this, kill weeds, but do not affect the growth of plant itself, have widened thus the use range of weedicide.Although current, having many bibliographical informations to suddenly change on some gene to import wild-type plant becomes transgenic plant again, 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 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, as we find, in vitro and/or Arabidopis thaliana import the mutator gene that test has Herbicid resistant, after importing corn etc., but there is to have occurred in a large number to reply the situation of losing Herbicid resistant, although some has retained Herbicid resistant, the application at all cannot putting into practice but corn growth is undesired, 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), there is the plant of Herbicid resistant and truly can also not have at present 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 in 21 mutant plants of corn inbred line capital, to find by accident a series of (sudden change) albumen, the basic normal plants physiological function that keeps original albumen under its condition that can exist at weedicide, thus make corn (especially capital 21) there is 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 plant, the especially farm crop with Herbicid resistant, 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.), obtain the methods and applications of the plant with 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 ALS mutein, its aminoacid sequence is as shown in SEQ ID NO:2 or 4.This albumen mass-energy makes plant (as corn, especially 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 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 that the mutain at the aminoacid sequence from corn as shown in SEQ ID NO:2 or 4 can make corresponding plant (especially corn) have Herbicid resistant, more unpredictable when keeping Herbicid resistant, can also keep normal growing state.In this article, capital 21, claims again capital No. 21, the corn selfing strain of Shi You Institute of Crop Science, Chinese Academy of Agricultural Science corn research centre exploitation, the characteristic trait such as it has 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 commercial channel, for example, can buy from Institute of Crop Science, Chinese Academy of Agricultural Science's corn research centre.
Except mutant plant screening and amplification method described in the specific embodiment of the invention, in the situation that protein sequence is known, those skilled in the art have the ability by changing the coding gene sequence of known protein and being imported expression vector, 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, import in (wild-type) capital 21, thereby give Genes For Plant Tolerance weedicide performance.
At present; the weedicide that the inhibition ALS of take is 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, Herbicid resistant refers to comprising that the weedicide of 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, is for No. CAS 81335-77-5).Preferably in the present invention, Herbicid resistant is imidazolinone herbicide resistance, is more preferably 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.Knowing under the prerequisite of coded protein sequence or nucleotide sequence, by conventional codon corresponding relation and host expresses frequency, 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.Once obtain this nucleic acid, just can be cloned into carrier, then be transformed or be transfected into corresponding cell, then by conventional host cell, breed, therefrom separation obtains a large amount of nucleic acid.Preferably the nucleotide sequence of the nucleic acid of second aspect present invention is as shown in SEQ ID NO:1 or 3.
In the third aspect, the invention provides expression cassette (cassette), the nucleic acid that it comprises second aspect present invention.The nucleic acid that the expression cassette of third aspect present invention preferably comprises promoter region, second aspect present invention on 5 '-3 ' transcriptional orientation and the terminator of transcribing and translating.The expression cassette of third aspect present invention is preferably 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 in bacterium, copy required replication orgin, from pBR322 or P15Aori ORI district), 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; 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 leader sequence from 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, the nucleic acid that it comprises second aspect present invention.In this article, carrier refers to bacterial plasmid conventional in this area, clay, phagemid, yeast plasmid, vegetable cell virus, animal virus and other various virus vector.Different according to applied object, carrier can be divided into cloning vector, expression vector and conversion carrier, refers to used object respectively for cloning and verify gene, expressing corresponding gene and corresponding gene is transformed.Carrier available in the present invention includes but not limited to: in bacterium, express use carrier (prokaryotic expression carrier), in yeast, express the carrier (as pichia vector) of use, at the baculovirus vector of expressed in insect cells, the carrier (vaccinia virus vector, retroviral vector, adenovirus carrier, adeno-associated virus carrier etc.) of expressing use in mammalian cell, the various carriers of expressing the plant viral vector of use and expressing use in plant in mammal galactophore.The carrier of fourth aspect present invention is conversion carrier, especially plant conversion carrier, the particularly carrier of applicable Agrobacterium-mediated Transformation plant.The much commercializations of these carriers.
Aspect the 5th, the invention provides cell, the nucleic acid that it comprises 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 certain plants hormone) just developable vegetable cell or cell mass, also can be completely without the vegetable cell or the cell mass that develop into plant ability, generally, be dead vegetable cell or cell mass, 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 being transformed in cell.Preferred cell is bacterial cell, for example, in particular for clone or store nucleic acid or for the bacterial cell of transformed plant cells, 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 comprising 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 by mutating technology (technology as described in the specific embodiment of the invention), the nucleic acid of second aspect present invention to be present in vegetable cell.
Aspect the 6th, the invention provides plant, the nucleic acid that it comprises second aspect present invention, and/or it expresses the protein of first aspect present invention.Owing to having introduced the nucleic acid of second aspect present invention and the protein of first aspect present invention, the plant of sixth aspect present invention has Herbicid resistant.In this article, plant refers to by photosynthesis, with inorganicss such as water, carbonic acid gas and inorganic salt, with regard to energy synthetic carbohydrate, protein, maintain single plant, plant group or its reproductive material of existence, comprise the bred part of plant, plant variety, plant, plant event, plant offspring, plant seed or other plant.Wherein, plant offspring itself is exactly plant, the plant offspring who comprise the plant offspring that produces by transgenic technology, produces with 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, preferably the plant of 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 aforementioned aspects of the present invention and there is the application in the plant process of Herbicid resistant in acquisition.In acquisition, have in the plant process of Herbicid resistant, can be applied to aforementioned aspects of the present invention.What seventh aspect present invention provided comprises, the protein of first aspect present invention has the application in the plant process of Herbicid resistant in acquisition, the nucleic acid of second aspect present invention has the application in the plant process of Herbicid resistant in acquisition, the expression cassette of third aspect present invention has the application in the plant process of Herbicid resistant in acquisition, the carrier of fourth aspect present invention has the application in the plant process of Herbicid resistant in acquisition, the cell of fifth aspect present invention has the application in the plant process of Herbicid resistant in acquisition, the plant of sixth aspect present invention has the application in the plant process of Herbicid resistant in acquisition.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 preferably obtaining in 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 and comprise preparation, cultivation, production or otherwise produce and obtain, comprise by transgenic breeding mode and obtaining, as the nucleic acid of second aspect present invention being transformed into the protein that makes it to express first aspect present invention after plant; Also comprise by non-transgenic breeding mode and obtaining, as by hybridizing, backcross, the plant of selfing or vegetative propagation sixth aspect present invention sub-elect the plant that still comprises the nucleic acid of second aspect present invention and express the protein of first aspect present invention.Therefore, the application that a seventh aspect of the present invention provides comprises, in acquisition, has application in the transgenic plant process of Herbicid resistant and has the application in the non-transgenic plant process of Herbicid resistant in acquisition.
First independently aspect, the invention provides (wild-type) capital 21 acquisition have Herbicid resistant plant (as, corn) application in, the application in the corn with Herbicid resistant of the protein of the nucleic acid that preferably comprises second aspect present invention in acquisition and/or expression first aspect present invention.Capital 21 itself has good proterties, but the more important thing is, the inventor finds, to its weedicide for seed (as, imidazolinone herbicide, preferably Imazethapyr) carry out mutagenesis, can obtain the corn with Herbicid resistant.
In eight aspect, the invention provides the method that obtains the plant with Herbicid resistant, it comprises the steps:
(1) nucleic acid that makes plant comprise 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 aforementioned or that those skilled in the art can be known and non-transgenic method, the nucleic acid that makes plant comprise second aspect present invention, or 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, for the technician in transgenosis or non-transgenic breeding field, all can be known and be implemented.
Aspect the 9th, the invention provides the method for the plant of identifying that the plant of sixth aspect present invention or the method for eighth aspect present invention obtain, 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, whether belongs to the plant of sixth aspect present invention, or whether belong to the plant of the method acquisition of eighth aspect present invention.The step of measuring can be undertaken by conventional detection of nucleic acids and/or protein detection method, 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 the plant variety with 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 in and carried out reference herein, just looks like that repeated description is excessively the same in this article for their full text.For the ease of understanding, below the drawings and Examples by concrete are described in detail the present invention.It needs to be noted, these descriptions are only exemplary descriptions, do not form 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.
Accompanying drawing explanation
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 conventional molecular biology, tissue culture technique and agronomy handbook are recorded.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 purchased from Institute of Crop Science, Chinese Academy of Agricultural Science's corn research centre by corn inbred line capital 21() sowing, before sowing, by imidazolinone herbicide Imazethapyr ((RS) 5-ethyl-2-(4-isopropyl-4-methyl-5-oxo-3-tetrahydroglyoxaline-2-yl) nicotinic acid for various corn seeds, CAS 81335-77-5) (concentration is 0.5% to aqua, purchased from Shandong Cynda Chemical Co., Ltd) soak 18 hours, after draining away the water, be seeded in experimental plot, after 10 days, observe Course of Corn Seed Germination situation.Through great many of experiments screening, finally chance on, wherein there is green rice shoot to continue growth (Fig. 1), retain 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 San Bo polygala root Bioisystech Co., Ltd check order and carry out sequence comparison, find: mutant plant 1 is with respect to No. 21, wild-type capital, undergo mutation in the 518th site at acetolactate synthestase (ALS) gene, from C, become T, cause the 173rd of corresponding encoded albumen from L-Ala, to become α-amino-isovaleric acid, 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, from G, become T, cause the 542nd of corresponding encoded albumen from tryptophane, to become halfcystine, 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. referring to Han WN etc., compile .The Proceedings of the18th Asia-Pacific Weed Science Society Conference.515-522.May28-June2,2001.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 in brief, is got respectively after each plant seedling 5g chopping, adds the 50mmol/LK of 10mL pH7
2hPO
4-KH
2pO
4damping fluid (wherein containing 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, by 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 containing 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 to 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, in 60 ℃, place decarboxylation in 15 minutes.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 wild-type in No. 21, capital and the active impact of each sudden change ALS enzyme.
Result is as shown in the table, the ALS enzyme that respectively suddenlys change that No. 21, 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, the ALS endonuclease capable that shows to suddenly change 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 white mutant gene of embodiment 3 turning egg(s)
Entrust Weimingkaituo Agro-Biological Technology Co., Ltd., Beijing, with conventional agrobacterium-mediated transformation, the encoding gene of the sudden change ALS enzyme in No. 21, above-mentioned capital is proceeded to respectively to wild-type Arabidopis thaliana plant.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, als gene by nucleotide sequence as shown in SEQ ID NO:1 and 3 is cloned in pCAMBIA1303 plasmid (purchased from Cambia company), select positive colony and transform Agrobacterium AGL0, cultivate thalline arabidopsis thaliana transformation, PCR obtains the T1 transgenic seed in generation after identifying and transforming positive Arabidopis thaliana sowing, during plantation, with the spraying of imidazolinone herbicide Imazethapyr, process, discovery growth conditions is good, and not genetically modified Arabidopis thaliana seedling is all withered.
Claims (26)
1. make plant have the ALS mutein of Imazethapyr resistance, its aminoacid sequence is as shown in SEQ IDNO:2 or 4.
2. nucleic acid, its protein claimed in claim 1 of encoding.
3. nucleic acid claimed in claim 2, its nucleotide sequence is as shown in SEQ ID NO:1 or 3.
4. expression cassette, it comprises the nucleic acid described in claim 2 or 3.
5. carrier, it comprises the nucleic acid described in claim 2 or 3.
6. protein claimed in claim 1 has the application in the plant of Imazethapyr resistance in acquisition.
7. application claimed in claim 6, wherein said plant is corn.
8. the nucleic acid described in claim 2 or 3 has the application in the plant of Imazethapyr resistance in acquisition.
9. application claimed in claim 8, wherein said plant is corn.
10. expression cassette claimed in claim 4 has the application in the plant of Imazethapyr resistance in acquisition.
11. application claimed in claim 10, wherein said plant is corn.
12. carriers claimed in claim 5 have the application in the plant of Imazethapyr resistance in acquisition.
Application described in 13. claims 12, wherein said plant is corn.
14. cells that comprise the nucleic acid described in claim 2 or 3 have the application in the plant of Imazethapyr resistance in acquisition.
Application described in 15. claims 14, wherein said plant is corn.
16. plants that comprise the nucleic acid described in claim 2 or 3 have the application in the plant of Imazethapyr resistance in acquisition.
Application described in 17. claims 16, wherein said plant is corn.
18. plants of expressing protein claimed in claim 1 have the application in the plant of Imazethapyr resistance in acquisition.
Application described in 19. claims 18, wherein said plant is corn.
20. obtain the method for the plant with Imazethapyr resistance, and it comprises the steps:
(1) make plant comprise the nucleic acid described in claim 2 or 3; Or,
(2) make expression of plants protein claimed in claim 1.
Method described in 21. claims 20, it comprises transgenosis, hybridizes, backcrosses, selfing or vegetative propagation step.
Method described in 22. claims 20, wherein said plant is corn.
The method of 23. plant identifications, wherein plant is the corn that comprises the nucleic acid described in claim 2 or 3, it comprises the steps:
(1) measure described plant and whether comprise the nucleic acid described in claim 2 or 3; Or,
(2) measure described plant and whether express protein claimed in claim 1.
The method of 24. plant identifications, wherein plant is the corn of expressing protein claimed in claim 1, it comprises the steps:
(1) measure described plant and whether comprise the nucleic acid described in claim 2 or 3; Or,
(2) measure described plant and whether express protein claimed in claim 1.
The method of 25. plant identifications, wherein plant is the plant of arbitrary described method acquisition of claim 20-22, it comprises the steps:
(1) measure described plant and whether comprise the nucleic acid described in claim 2 or 3; Or,
(2) measure described plant and whether express protein claimed in claim 1.
Method described in 26. claims 25, wherein said plant is corn.
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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 |
| 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 |
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| Insights into corn genes derived from large-scale cDNA sequencing;Nickolai N. Alexandrov等;《Plant Mol Biol.》;20081021;第69卷(第1-2期);序列EU972128 * |
| Nickolai N. Alexandrov等.Insights into corn genes derived from large-scale cDNA sequencing.《Plant Mol Biol.》.2008,第69卷(第1-2期),序列EU972128. |
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Effective date of registration: 20200623 Address after: 518035 Shenzhen, Guangming New District, Guangdong, Guangming Street, Guangming Road, No. 3009, China Merchants science and Technology Park, Guangming science and Technology Park, B6 building, C 2. Patentee after: SHENZHEN JIETIAN MODEL BIOTECHNOLOGY Co.,Ltd. Address before: 102206, Changping District, Zhongguancun Beijing Life Science Park medical industry park, No. 9, building 4, 405 Patentee before: Weimingwang system crop Design Frontier Laboratory (Beijing) Co.,Ltd. |