CN107217044A - Make plant that there is paddy rice ALS muteins and its application of Herbicid resistant - Google Patents
Make plant that there is paddy rice ALS muteins and its application of Herbicid resistant Download PDFInfo
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- CN107217044A CN107217044A CN201710655418.XA CN201710655418A CN107217044A CN 107217044 A CN107217044 A CN 107217044A CN 201710655418 A CN201710655418 A CN 201710655418A CN 107217044 A CN107217044 A CN 107217044A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G13/00—Protecting plants
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8274—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for herbicide resistance
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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- C12Y202/00—Transferases transferring aldehyde or ketonic groups (2.2)
- C12Y202/01—Transketolases and transaldolases (2.2.1)
- C12Y202/01006—Acetolactate synthase (2.2.1.6)
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/13—Plant traits
Abstract
The invention discloses the paddy rice ALS muteins for making plant have Herbicid resistant.The invention also discloses the nucleic acid for encoding above-mentioned albumen.The albumen source is in the rice mutant plant of anti-ALS inhibitor class herbicide, and compared with the ALS sequences in wild rice genome, its protein sequence is undergone mutation simultaneously in Pro171 and Asp350 sites.The present invention by field spray ALS inhibitor classes herbicide " hundred ridges lead to " test result indicates that, the leaf seedling of paddy rice 34 of the ALS albumen with Herbicid resistant containing the present invention leads to/L water applying the ridges of 3.3mL hundred（10 times recommend concentration）Afterwards, plant still normal growth and development and solid, and the leaf seedling of wild rice 34 apply the ridges of 1 mL hundred it is logical/3L water（1 times recommends concentration）Whole strain is shown as after 30 days dead.
Description
Technical field
The invention belongs to vegetable protein and plant antiweed field, it is related to the paddy rice for making plant that there is Herbicid resistant
ALS muteins, gene and its application；Specifically, the present invention relates to the acetolactate synthestase of paddy rice (ALS) mutation
Albumen, the albumen can assign the characteristic of plant especially Rice Resistance inhibitor of acetolactate synthetase class herbicide.The present invention is disclosed
The sequence of the albumen, and their applications in plant antiweed field.
Background technology
Weeds are the unfavorable factors for restricting agricultural production high and stable yields.With tradition by cultivation step, artificial weeding and machine
The methods such as tool weeding are compared, and the use of chemical herbicide is a kind of efficient, easy and economic improvement weeds method.
The biosynthesis of plant and microorganism branched-chain amino acid (valine, leucine and isoleucine) needs 4 kinds of enzymes to be total to
Same catalytic action, acetolactate synthestase (acetolactate synthase, ALS), keto-alcohol reduction isomerase (ketol-
Acid reductoisomerase), dihydroxylated acid dehydratase (dihydroxyavid dehydratase), branched-chain amino acid turn
Ammonia enzyme (branched-chain amino acidtransaminase).Acetolactate synthestase is in biosynthetic process
The key enzyme in one stage, is catalyzed 2 molecule pyruvic acid generation acetolactic acid and carbon dioxide in the synthesis of valine and leucine,
1 molecule pyruvic acid and 1 molecule alpha batanone acid generation 2- aldehyde-base -2- hydroxybutyric acids and dioxy are catalyzed in the synthesis of isoleucine
Change carbon.ALS inhibitor class herbicides, so as to prevent the synthesis of branched-chain amino acid, are led by suppressing the ALS enzymatic activitys in plant
The synthesis of protein is caused to be destroyed, the DNA synthesis of block cell division stage, so that the mitosis of plant cell is stopped at
The S phases (DNA synthesizes the phase) and the M phases in G2 stages in Gl stages, DNA synthesis is disturbed, therefore cell can not complete mitosis,
And then plant is stopped growing, ultimately result in plant individual dead.
Acetolactate synthestase (ALS) (is also referred to as acetohydroxy acid synthetase, AHAS；EC 4.1.3.18) inhibitor class weeding
Agent causes weeds dead using ALS as target, mainly including sulfonylurea (Sulfonylureas, SU), imidazolone type
(Imidazolinones, IMI), triazolo pyrimidine class (Triazolopyrimidines, TP), pyrimidine oxygen (sulphur) benzoic acids
[Pyrimidinylthio(or oxy)–benzoates,PTB；pyrimidinyl-carboxyherbicides；PCs] and sulphur
The class compound of amide groups carbonyltriazolinone (Sulfonylamino-carbonyltriazolinones, SCT) etc. 13.Second
Acyl lactic acid synzyme, is present in growing process, and it can be catalyzed pyruvic acid with high specificity and high catalytic efficiency
For acetolactic acid, so as to cause the biosynthesis of branched-chain amino acid.
The features such as ALS inhibitor classes herbicide has strong selectivity, broad weed-killing spectrum, efficient low toxicity, large area is pushed away at present
Extensively use.But these herbicides also produce poisoning in itself to the general crops without anti-(resistance to) herbicidal properties, greatly limit
Its use time is made and has used space, crops are just avoided that using herbicide for the previous period if desired in crop seeding
By poisoning.Chemical injury of crops can be reduced, widen the use scope of herbicide by cultivating anti-(resistance to) herbicide crop varieties.
At present, anti-ALS inhibitor mutational site known to paddy rice include Gly 95, Ala 96, Ala 122, Trp 548,
Ser 627, Ser 653 and Gly 654.ALS mutant antiweed levels are relevant with the position of ALS amino acid mutations, also with
The number of amino acid classes and mutating acid after mutation is relevant.
At present, the mechanism of action of ALS inhibitor class herbicide is not yet determined, it is difficult to the other amino of look-ahead ALS albumen
Whether the mutation in sour site can produce Herbicid resistant, rely only on long-term, the arduous practical exploration of scientific research personnel, and rely on
Some fortune are only possible to find the Herbicid resistant site that ALS albumen is new.
The content of the invention
Goal of the invention：First technical problem to be solved by this invention is to provide the egg for making plant have Herbicid resistant
In vain.
The technical problem of the invention also to be solved there is provided the nucleic acid and expression cassette, carrier and cell for encoding the albumen
Deng.
The technical problem of the invention also to be solved there is provided the methods and applications for obtaining the plant with Herbicid resistant.
The technical problem of the invention also to be solved there is provided the identification for judging whether plant uses the inventive method to obtain
Method.
The present inventor, by long-term, arduous research, is indica type conventional rice seed Hefei sterile line to wild rice
EMS mutant plants carry out it is long-term, constantly screen, it was found that a series of ALS mutains, including previously described some
Known ALS mutains and new ALS mutains, it is insensitive to ALS inhibitor class herbicides, so that plant has
ALS inhibitor class Herbicid resistants.Application of the present invention in plant breeding, available for plant of the cultivation with Herbicid resistant
Thing, especially crops, also developed these albumen and its encoding gene in the crop such as transgenosis or non-transgenic such as paddy rice
In application.
Technical scheme：In order to solve the above technical problems, the technical solution adopted by the present invention is as follows：The invention provides cause
Plant has the ALS albumen of Herbicid resistant, and it from proline is changed into histidine in the 171st amino acids of amino acid sequence,
350th amino acids are glutamic acid by Aspartic acid mutations.Show there is presently no report, from rice varieties Hefei infertility
It is the mutant strain of the Pro171 and Asp350 sites simultaneous mutation of ALS albumen.
It is preferred that the albumen of the first aspect of the present invention carries the mutant strain of Pro171 and Asp350 sites simultaneous mutation, except
Outside mutant plant screening or amplification method described in the embodiment that the present invention has, in situation known to protein sequence
Under, those skilled in the art have the ability by the coding gene sequence of protein known to change and are conducted into carrier, it is possible to
Prepare substitution, add or lacked the protein of amino acid residue, these methods are conventional meanses.In the specific of the present invention
In embodiment, the amino acid sequence such as SEQ ID NO of the ALS albumen with Herbicid resistant of the invention:Shown in 2.The egg
The white acetolactate synthestase insensitive to imidazolinone herbicide by confirmation.
The second aspect of the present invention provides nucleic acid, and it encodes the protein of first aspect present invention.In the present invention, core
Acid can be DNA, RNA, wherein preferably DNA.On the premise of coded protein sequence or nucleotide sequence is known, pass through routine
Codon corresponding relation and host expresses frequency, with PCR method, DNA recombination methods or artificial synthesized method, this area skill
Art personnel have the ability to obtain the nucleic acid of the simultaneously protein of Optimized Coding Based first aspect present invention.Once obtain the nucleic acid, it is possible to
Carrier is cloned into, then converts or be transfected into corresponding cell, is then bred by conventional host cell, Cong Zhongfen
From obtaining substantial amounts of nucleic acid.It is preferred that the nucleotide sequence of the nucleic acid of second aspect of the present invention such as SEQ ID NO：Shown in 1.Specifically
, DNA sequence dna of the invention is in the als gene sequence that wild rice is indica type conventional rice seed Hefei sterile line
512nd nucleotides becomes nucleotides A, the 1050th nucleotides by C and becomes nucleotides A by T.
In addition, the wild rice before present invention mutation is indica type conventional rice seed Hefei sterile line, the ALS inhibitor
The wild type Hefei sterile line paddy rice ALS of class herbicide sensitive nucleotide sequence is as shown in SEQ ID NO.3, and amino acid is such as
Shown in SEQ ID NO.4.
The content of the third aspect of the present invention includes expression cassette, recombinant vector or cell, and it contains above-mentioned second aspect
Nucleic acid.
Fourth aspect present invention content also includes the above-mentioned ALS albumen with Herbicid resistant, nucleic acid, expression cassette, again
The application of group carrier or cell in terms of green plants antiweed.
Wherein, above-mentioned green plants is paddy rice, tobacco, arabidopsis, cotton etc..
The content of fifth aspect present invention also includes the method for obtaining the plant with Herbicid resistant, including following step
Suddenly：
1) plant is made to include nucleic acid of the present invention；Or
2) albumen for making plant expression described.
Wherein, above-mentioned method, including transgenosis, hybridization, backcrossing or vegetative propagation step.
The content of sixth aspect present invention includes the method for plant identification, and wherein plant is the plant for including described nucleic acid
The plant that the plant of the described albumen of thing, expression or described method are obtained, specifically includes following steps：
1) determine whether the plant includes described nucleic acid；Or,
2) determine whether the plant expresses described albumen.
Beneficial effect：Compared with prior art, the present invention has advantages below：
1) present invention by field spray ALS inhibitor classes herbicide " hundred ridges lead to " test result indicates that, contain this hair
The paddy rice 3-4 leaves seedling of the bright ALS albumen with Herbicid resistant apply 3.3mL hundred ridges it is logical/(10 times recommend L water
Concentration) after, plant still normal growth and development and solid, and wild rice 3-4 leaves seedling apply the ridges of 1mL hundred it is logical/3L water
(1 times recommends concentration) shows as whole strain after 30 days dead.
2) the transgenic paddy rice ALS enzymatic activitys of the als gene containing Hefei sterile line antiweed mutant of the invention
Higher than the ALS enzyme activity of wild type 4.7 times, the als gene of the invention containing Hefei sterile line antiweed mutant turns base
Because the ALS enzyme activity of ALS activity ratio's wild types of tobacco Progeny plants is high 5.2 times.
3) Hefei sterile line mutant of the invention with the patent No. 201610226003.6 CGMCC No.12265 it is anti-
Herbicide mutant plants carry out ALS enzyme activity and are measured, and find the Hefei sterile line mutant ALS enzymes in present patent application
Activity is higher than CGMCC No.12265 by 18%.
Brief description of the drawings
The ridges of Fig. 1 hundred lead to the resistant rice mutant that herbicide screening is obtained；
Fig. 2 PCR expand als gene result figure；1st swimming lane is Marker；Marker molecular weight is followed successively by from top to bottom
5kb, 3kb, 2kb, 1kb, 750bp, 500bp, 250bp, 100bp, the 2nd swimming lane are Hefei sterile line wild rice DNA, the 3rd
Swimming lane is the DNA, purpose fragment length 2091bp of antiweed mutant；
The ridges of Fig. 3 hundred lead to herbicide and the light absorption value of wild type and the ALS inhibition of enzyme activity of antiweed mutant are determined；
The Overexpression vector BamHI/SacI double digestion proof diagrams of the als gene of Fig. 4 antiweed mutant；1st swimming
Road is Marker；Marker molecular weight be followed successively by from top to bottom 5kb, 3kb, 2kb, 1kb, 750bp, 500bp, 250bp,
100bp；2nd swimming lane is the recombinant vector without digestion, and the 3rd swimming lane is that recombinant vector is produced after BamHI/SacI double digestions
Genetic fragment and plasmid fragments DNA, genetic fragment size meets expection, it was demonstrated that vector construction success；
Fig. 5 saltant type als gene paddy rice PCR detection figures；A total of 8 swimming lanes, the 1st swimming lane is Marker；Marker points
Son amount is followed successively by 3kb, 2kb, 1kb, 750bp, 500bp, 250bp from top to bottom；2nd swimming lane is non-transgenic wild type DNA, is made
For negative control, the 3rd swimming lane is that the DNA containing ALS mutant nucleotide sequences is template, and as positive control, the 4th~6 swimming lane is
Convert the transfer-gen plant DNA of antiweed mutant als gene.
The ridges of Fig. 6 hundred lead to transgenic paddy rice of the herbicide to wild rice and containing antiweed mutant als gene
The light absorption value of ALS inhibition of enzyme activity is determined；
The ridges of Fig. 7 hundred lead to transgene tobacco of the herbicide to wild-type tobacco and containing antiweed mutant als gene
The light absorption value of ALS inhibition of enzyme activity is determined.
The ridges of Fig. 8 hundred lead to herbicide to the heterozygous plant in filial generation, antiweed mutant and CGMCC No. 12265
ALS inhibition of enzyme activity light absorption value determine.
Embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.It is unreceipted specific in embodiment
Condition person, the condition advised according to normal condition or manufacturer is carried out.Agents useful for same or the unreceipted production firm person of instrument, be
Can be by the conventional products of acquisition purchased in market.
Embodiment 1：Rice Resistance imidazolinone herbicide mutant acquisition process (hundred ridges lead to)
By indica type conventional rice seed Hefei sterile line (purchased from the agriculture plasm resource protection in Jiangsu Province and utilizing platform) (this
For M0, being soaked 2 hours with clear water) 150kg points to soak 6-9 at room temperature with 0.5-1.0% (w/w) ethyl methane sulfonate (EMS) 6 times small
When, shake a seed within during which every 1 hour；EMS solution is discarded, running water stirs immersion seed 5 times, every time 5 minutes, Ran Houyong
Running water rinses seed and stayed overnight, and next day carries out field sowing, and carries out conventional fertilizer water management (this is M1).After plant is ripe, plant
Son is mixed to be received, dries, preservation of passing the winter.Next year sows field.When paddy rice (this is M2) seedling length to the 3-4 leaf phases, spray for
The ridges of 3.3mL hundred are logical/and (" hundred ridges lead to " is that BASF Aktiengesellschaft produces a kind of water aqua type imidazolinone herbicide to L water, is recommended most
Low concentration is that the ridges of 1mL hundred lead to/1.5~3L water), also in normal green plant it is anti-imidazolinone herbicide after 30 days
Rice mutant (Fig. 1).128 plants of the M2 individual plants of antiweed are obtained altogether, and these resistance individual plants carry out conventional fertilizer water pipe
Reason, has 101 M2 individual plants normally solid, after seed maturity, individual plant sowing, dries, preservation of passing the winter.
Embodiment 2：Anti- imidazolinone herbicide rice mutant mutational site analysis
The Herbicide Resistant Rice mutant plant for taking above-described embodiment 1 to obtain chooses mutant plants blade, extracts genome
DNA, send one of Nanjing bio tech ltd to carry out gene order-checking.Sequencing result and Hefei sterile line wild type ALS bases
Because comparing, it is found that Herbicide Resistant Rice mutant there occurs the mutation in 2 sites, the 512nd, 1050 site alkali on als gene
Base is undergone mutation, and becoming A, T by C respectively becomes A, causes the 171st of the amino acid sequence of its corresponding encoded, 350 sites are by dried meat
Propylhomoserin is changed into the nucleotide sequence such as SEQ that histidine, asparagine are changed into the ALS genes of glutamic acid, i.e. antiweed mutant
Shown in ID NO.1, the amino acid sequence of its ALS albumen encoded is as shown in SEQ ID NO. 2.
Its Classification And Nomenclature of the Herbicide Resistant Rice mutant of the present invention is rice paddy seed HF-407 (Oryza sativa
Indica Group HF-407), the bacterial strain is preserved in China typical culture collection center on June 2nd, 2017
(CCTCC), address：Wuhan City, Hubei Province Wuchang District Wuhan University collection (the first affiliated primary school of Wuhan University opposite), postal
Compile：430072, deposit number is CCTCC No：P201714.
Embodiment 3：Anti- imidazolinone herbicide rice mutant als gene clone
Above-mentioned Herbicide Resistant Rice mutant is taken to extract genomic DNA.According to Hefei sterile line wild type als gene sequence
Design amplification als gene total length special primer be：Forward primer
F5 '-TCGCCCAAACCCAGAAACCC-3 ', reverse primer R 5 '-
CTCTTTATGGGTCATTCAGGTC-3’。
Using Takara PrimerSTAR Max DNA Polymerase polymerases (being purchased from Takara companies) amplification ALS
Gene, its reaction system is as follows：
Pcr amplification reaction program uses two-step method, and annealing and extension are combined into together, using 68 degree.Program is as follows：It is pre- to become
Property：98℃3min；35 circulations：It is denatured 98 DEG C of 10sec；Extend 68 DEG C of 1min；Insulation： 72℃10min.
2 μ l PCR primers are taken to be detected through 1% agarose gel electrophoresis, discovery has after the fragment of expected size (Fig. 2), it is remaining
PCR primer reclaimed through PCR cleaning agents box (be purchased from Axygen companies) cleaning after, be cloned into pMD19-T carriers and (be purchased from
Takara companies), then convert Escherichia coli.Each convert random 12 Escherichia coli clones of picking and enter performing PCR detection, take
6 monoclonals that PCR results are positive, send Nanjing one of bio tech ltd's sequencing, obtain mutation als gene sequence,
The genetic fragment length is 2091bp, and the sequencing result of Herbicide Resistant Rice mutant 1ALS genetic fragments is as follows：
CTGCATCGCGTGTCGCCCTTTCGCCCAAACCCAGAAACCCTCGCCGCCGCCGCCGCCGCCATC
ACCCACCATGGCTACGACCGCCGCGGCCGCGGCCGCCACCTTGTCCGCCGCCGCGACGGCCAA
GACCGGCCGTAAGAACCACCAGCGACACCACGTCCTTCCCGCTCGAGGCCGGGTGGGGGCGG
CGGCGGTCAGGTGCTCGGCGGTGTCCCCGGTCACCCCGCCGTCCCCGGCGCCGCCGGCCACGC
CGCTCCGGCCGTGGGGGACGGCCGAGCCCCGCAAGGGCGCGGACATCCTCGTGGAGGCGCTG
GAGCGGTGCGGCGTCAGCGACGTGTTCGCCTACCCGGGCGGCGCGTCCATGGAGATCCACCAG
GCGCTGACGCGCTCCCCGGTCATCACCAACCACCTCTTCCGCCACGAGCAGGGCGAGGCGTTC
GCGGCGTCCGGGTACGCGCGCGCGTCCGGCCGCGTCGGGGTCTGCGTCGCCACCTCCGGCCCC
GGGGCAACCAACCTCGTGTCCGCGCTCGCCGACGCGCTGCTCGACTCCGTCCCGATGGTCGCC
ATCACGGGCCAGGTCACCCGCCGCATGATCGGCACCGACGCCTTCCAGGAGACGCCCATAGTC
GAGGTCACCCGCTCCATCACCAAGCACAATTACCTTGTCCTTGATGTGGAGGACATCCCCCGCG
TCATACAGGAAGCCTTCTTCCTCGCGTCCTCGGGCCGTCCTGGCCCGGTGCTGGTCGACATCCC
CAAGGACATCCAGCAGCAGATGGCTGTGCCAGTCTGGGACACCTCGATGAATCTACCGGGGTA
CATTGCACGCCTGCCCAAGCCACCCGCGACAGAATTGCTTGAGCAGGTCTTGCGTCTGGTTGG
CGAGTCACGGCGCCCGATTCTCTATGTCGGTGGTGGCTGCTCTGCATCTGGTGATGAATTGCGC
CGGTTTGTTGAGCTGACCGGCATCCCAGTTACAACCACTCTGATGGGCCTCGGCAATTTCCCCA
GTGATGATCCGTTGTCCCTGCGCATGCTTGGGATGCATGGCACGGTGTACGCAAATTATGCGGT
GGATAAGGCTGACCTGTTGCTTGCATTTGGCGTGCGGTTTGATGAACGTGTGACAGGGAAAATT
GAGGCTTTTGCAAGCAGGGCCAAGATTGTGCACATTGACATTGATCCAGCGGAGATTGGAAAG
AACAAGCAACCACATGTGTCAATTTGCGCAGATGTTAAGCTTGCTTTACAGGGCTTGAATGCTC
TGCTAGACCAGAGCACAACAAAGACAAGTTCTGATTTTAGTGCATGGCACAATGAGTTGGACC
AGCAGAAGAGGGAGTTTCCTCTGGGGTACAAGACTTTTGGTGAAGAGATCCCACCGCAATATG
CTATTCAGGTGCTGGATGAGCTGACGAAAGGGGAGGCAATCATCGCTACTGGTGTTGGACAGC
ACCAGATGTGGGCGGCACAATATTACACCTACAAGCGGCCACGGCAGTGGCTGTCTTCGGCTG
GTCTGGGCGCAATGGGATTTGGGCTGCCTGCTGCAGCTGGTGCTTCTGTGGCTAACCCAGGTGT
CACAGTTGTTGATATTGATGGGGATGGTAGCTTCCTCATGAACATTCAGGAGTTGGCATTGATCC
GCATTGAGAACCTCCCGGTGAAGGTGATGGTGTTGAACAACCAACATTTGGGTATGGTTGTGC
AATGGGAGGATAGGTTTTACAAGGCAAATAGGGCGCATACATACTTGGGCAACCCAGAATGTGA
GAGCGAGATATATCCAGATTTTGTGACTATTGCTAAAGGGTTCAATATTCCTGCAGTCCGTGTAA
CAAAGAAGAGTGAAGTCCGTGCCGCCATCAAGAAGATGCTCGAGACCCCAGGGCCATACTTGT
TGGATATCATCGTCCCACACCAGGAGCATGTGCTGCCTATGATCCCAAGTGGGGGCGCATTCAA
GGACATGATCCTGGATGGTGATGGCAGGACTATGTATTAATCTATAATCTGTATGTTGGCAAAGC
ACCAGCCCGGCCTATGTTTGACCTGAATGACCCATAAAGAGAAGGGCGACACGCGATGCAG
Embodiment 4：The anti-imidazolinone herbicide of paddy rice M3 mutant (hundred ridges lead to)
The seed insemination and emergence (this is M3) that Hefei sterile line antiweed mutant is harvested, treats M3 rice seedlings length extremely
During 3-4 leaf phases, spray the ridges of 3.3mL hundred it is logical/L water (recommend minimum concentration for the ridges of 1mL hundred it is logical/3L water, it is dense equivalent to 10 times
Degree).After herbicide spraying 15 days, resistance seedling M3 is in normal green, can continue long up to 20-30cm, and non-resistance seedling leaf loses
Go green even part it is withered and yellow, plant does not grow tall, only 5-9cm.After 30 days, M3 resistant strains are in normal green plant, and are sprayed
The wild rice for applying same concentration herbicidal agent is all withered, shows that rice mutant at least resists hundred ridges of 10 times of concentration to lead to
Herbicide.
Embodiment 5：The ALS enzyme activity determinations of paddy rice M3 antiweed mutant
In order to verify rice mutant Herbicid resistant whether by ALS be mutated caused by, present inventor has performed ALS enzyme activity
Property determine.Method (Singh B.K., Stidham M.A., Shaner D.L.Assay of of the assay method with reference to Singh etc.
acetohydroxyacid synthase.Analytical Biochemistry,1988,171:173-179.).Specifically,
Wild type, the M3 plant leaf 0.2g of Hefei sterile line antiweed mutant are taken respectively, are crushed in mortar with liquid nitrogen grinding,
Add 2mL extract solutions (100mM K2HPO4, pH 7.5,10mM Sodium Pyruvates, 5mM EDTA, 1mM valine, 1mM leucines,
10mM cysteines, 0.1mM flavin adenine dinucleotide (FAD)s, 5mM magnesium chlorides, 10% (V/V) glycerine, 1% (w/v) polyethylene
Pyrrolidones), lyolysis to be extracted continues to grind 1min or so after freezing.12000rpm, 4 DEG C of centrifugation 30min, Aspirate supernatant, plus
Enter ammonium sulfate and make up to 50% saturation degree, in placing on ice half an hour, 12000rpm, 4 DEG C of centrifugation 30min abandon supernatant, will be heavy
Shallow lake is dissolved in 0.2mL reaction buffers (100 mM K2HPO4, pH 7.0,1mM EDTA, 10mM magnesium chloride, 100mM pyruvic acid
Sodium, 1mM diphosphothiamines, 0.1mM flavin adenine dinucleotide (FAD)s), respectively each plant ALS extract solutions.
10 μ L herbicides " hundred ridges lead to " (aqua, active ingredient 240g/L) are separately added into ALS extract solutions are obtained, are mixed
It is even, 37 DEG C of incubation 1h, plus 0.1ml 3M sulfuric acid terminating reactions, reactant mixture is incubated in 60 DEG C of reactions is easy to decarboxylation in 30 minutes.
Plus 0.4mL nitrite ions (0.09g/L 1- naphthols and 0.009g/L creatines, with 2.5M NaOH dissolve) then.Mixed liquor is at 37 DEG C
Incubation developed the color within 30 minutes (ALS is catalyzed 2 pyruvic acid formation acetolactic acids, acetolactic acid decarboxylation formation 3-Hydroxybutanone, then
Pink compound is formed with creatine and 1- naphthols, the compound has obtained the maximum absorption at 530nm), then determine it
530nm absorbance, ALS activity represents that the height of A530 light absorption values reflects the height of ALS activity with A530 light absorption values.Experiment
Using water as control, wild type, Hefei sterile line antiweed mutant respectively survey 5 individual plants.
A530 light absorption values measurement result is found, when in wild type, the ALS extract solutions of Hefei sterile line antiweed mutant
When not having the ridge of ALS inhibitor hundred to lead to, their A530 light absorption values show the ALS enzymes of wild type and mutant between 1.2-1.4
There was no significant difference (Fig. 3) for activity；And add the ridge of ALS inhibitor hundred it is logical after, the A530 light absorption values of wild type are only 0.25, are closed
The A530 light absorption values of fertile sterile line antiweed mutant are only control for the ALS enzymatic activitys of 1.21, i.e. wild type
18.9%, and the ALS enzymatic activitys of Hefei sterile line antiweed mutant still have more than 89% (Fig. 3), show Hefei sterile line
The ALS of antiweed mutant leads to insensitive to hundred ridges, so as to impart resistance.
Embodiment 6：The ridge of transgenosis ALS Rice Resistances hundred leads to
Design special primer 5 '-CGCGGATCCATCCGAGCCACACATCGCCTC-3 ' and 5 '-
TCCCCGCGGCCTACGGAAAACAACACAC-3 ', it 5 ' is separately added into BamHI and SacI digestion decorating sites.Reference implementation
The method of example 3, is amplified by PCR from the genomic DNA of above-mentioned rice mutant Hefei sterile line antiweed mutant
Als gene is mutated, after sequencing is correct, with BamHI and SacI difference double digestion mutation als gene fragments and plant expression vector
PCAMBIA1301 plasmids (are purchased from pcambia companies), digestion products T4-DNA enzymes (being purchased from TaKaRa companies) connection, connection
Product converts Escherichia coli.Recombinant plasmid extracts DNA, is verified with BamHI and SacI double digestions, can produce big plasmid piece
Section and small genetic fragment (Fig. 4), it was demonstrated that als gene of the nucleotide sequence as shown in SEQ ID NO.1 is cloned into plant
In expression vector pCAMBIA1301 plasmids (being purchased from pcambia companies).The plasmid vector built is converted into Agrobacterium
EHA105, cultivates thalline.Using conventional Agrobacterium-mediated transformation Japonica rice Nipponbare (purchased from the agriculture germplasm money in Jiangsu Province
Source protection is with utilizing platform), obtain after transfer-gen plant sowing, during Progeny plants length to the 3-4 leaf phases, PCR detection transgenosis is planted
Strain (Fig. 5).PCR detection primers be forward primer 35SF 5 '-ATGGTTAGAGAGGCTTACGC-3 ', reverse primer 5R 5 '-
AGCAACAGGTCAGCCTTATCCAC-3 ', amplified fragments include 5 ' terminal sequences of CaMV35S promoters and als gene, size
About 2kb.Pcr amplification reaction system reference implementation example 3, pcr amplification reaction program uses two-step method, and annealing and extension are combined into one
Rise, using 68 degree.Amplification program is as follows：Pre-degeneration：98℃ 3min；30 circulations：It is denatured 98 DEG C of 10sec；68 DEG C of extension
2min；Insulation：72℃10min.After being positive through PCR identifications, spray the ridges of 3.3mL hundred and lead to/L water (10 times recommend concentration), 7
After it, ALS enzymatic activitys are determined with reference to embodiment 6 methods described, it is found that the ALS enzymatic activitys of transgenic paddy rice are significantly higher than and non-turns base
Because of paddy rice；Find that transgenic paddy rice growth conditions are good after 30 days, and non-transgenic Nipponbare paddy rice is then all withered.Compare and turn
The ALS enzymatic activitys of the Transgenic Rice plant of the als gene of chemical combination fertilizer sterile line antiweed mutant, find containing conjunction
The ALS enzyme activity of transfer-gen plant ALS activity ratio's wild types of the als gene of fertile sterile line antiweed mutant is high 4.7 times
(Fig. 6).
Embodiment 7：Transgenosis ALS tobaccos resist hundred ridges to lead to
Design special primer 5 '-CGCGGATCCATCCGAGCCACACATCGCCTC-3 ' and 5 '-
TCCCCGCGGCCTACGGAAAACAACACAC-3 ', it 5 ' is separately added into BamHI and SacI digestion decorating sites.By PCR from
Mutation ALS genes are amplified in the genomic DNA of Hefei sterile line antiweed mutant, after sequencing is correct, with reference to embodiment
Als gene of the nucleotide sequence as shown in SEQ ID NO.1 is cloned into plant expression vector pCAMBIA2301 plasmids by 7 methods
In (being purchased from pcambia companies).Positive colony conversion Agrobacterium EHA105 is selected, using conventional Agrobacterium-mediated transformation sheet
Family name's tobacco leaf disk, is obtained after transfer-gen plant sowing, during Progeny plants length to the 3-4 leaf phases, after being positive through PCR identifications, is sprayed
The ridges of 3.3mL hundred lead to/L water (10 times recommend concentration), after 7 days, and ALS enzymatic activitys are determined with reference to the methods described of embodiment 6, find
The ALS enzymatic activitys of transgene tobacco are significantly higher than non-transgenic tobacco；Find that transgene tobacco growth conditions are good after 30 days,
And non-transgenic tobacco is then all withered.Compare the transgenosis cigarette of the als gene of conversion Hefei sterile line antiweed mutant
The ALS enzymatic activitys of careless Progeny plants, find the transfer-gen plant of the als gene containing Hefei sterile line antiweed mutant
The ALS enzyme activity of ALS activity ratio's wild types is high 5.2 times (Fig. 7).
Embodiment 8：The comparison and identification of resistant material
In order to detect whether the patent application than before of the resistant plant with 2 mutational sites mentioned in this patent
(201610226003.6) the weeding tolerance for the resistant plant mentioned in is stronger, and we are sterile by the Hefei in embodiment 2
It is the antiweed mutant plants progress ALS enzyme activity of mutant and CGMCC No.12265 in the patent No. 201610226003.6
It is measured, finds the Hefei sterile line mutant ALS activity ratio CGMCC No. 12265 high by 18% (Fig. 8) in the present invention.
Although the embodiment of the present invention has been described in detail, it will be understood to those of skill in the art that.According to
Those details can be carried out various modifications and replacement, these are in protection scope of the present invention by disclosed all teachings
It is interior.The four corner of the present invention is provided by appended claims and its any equivalent.
SEQUENCE LISTING
<110>Jiangsu Province Agriculture Science Institute
<120>Make plant that there is paddy rice ALS muteins and its application of Herbicid resistant
<130> SG20170801001
<160> 7
<170> PatentIn version 3.3
<210> 1
<211> 1935
<212> DNA
<213>Rice paddy seed Hefei sterile line antiweed mutant als gene
<220>
<221> CDS
<222> (1)..(1935)
<400> 1
atg gct acg acc gcc gcg gcc gcg gcc gcc acc ttg tcc gcc gcc gcg 48
Met Ala Thr Thr Ala Ala Ala Ala Ala Ala Thr Leu Ser Ala Ala Ala
1 5 10 15
acg gcc aag acc ggc cgt aag aac cac cag cga cac cac gtc ctt ccc 96
Thr Ala Lys Thr Gly Arg Lys Asn His Gln Arg His His Val Leu Pro
20 25 30
gct cga ggc cgg gtg ggg gcg gcg gcg gtc agg tgc tcg gcg gtg tcc 144
Ala Arg Gly Arg Val Gly Ala Ala Ala Val Arg Cys Ser Ala Val Ser
35 40 45
ccg gtc acc ccg ccg tcc ccg gcg ccg ccg gcc acg ccg ctc cgg ccg 192
Pro Val Thr Pro Pro Ser Pro Ala Pro Pro Ala Thr Pro Leu Arg Pro
50 55 60
tgg ggg acg gcc gag ccc cgc aag ggc gcg gac atc ctc gtg gag gcg 240
Trp Gly Thr Ala Glu Pro Arg Lys Gly Ala Asp Ile Leu Val Glu Ala
65 70 75 80
ctg gag cgg tgc ggc gtc agc gac gtg ttc gcc tac ccg ggc ggc gcg 288
Leu Glu Arg Cys Gly Val Ser Asp Val Phe Ala Tyr Pro Gly Gly Ala
85 90 95
tcc atg gag atc cac cag gcg ctg acg cgc tcc ccg gtc atc acc aac 336
Ser Met Glu Ile His Gln Ala Leu Thr Arg Ser Pro Val Ile Thr Asn
100 105 110
cac ctc ttc cgc cac gag cag ggc gag gcg ttc gcg gcg tcc ggg tac 384
His Leu Phe Arg His Glu Gln Gly Glu Ala Phe Ala Ala Ser Gly Tyr
115 120 125
gcg cgc gcg tcc ggc cgc gtc ggg gtc tgc gtc gcc acc tcc ggc ccc 432
Ala Arg Ala Ser Gly Arg Val Gly Val Cys Val Ala Thr Ser Gly Pro
130 135 140
ggg gca acc aac ctc gtg tcc gcg ctc gcc gac gcg ctg ctc gac tcc 480
Gly Ala Thr Asn Leu Val Ser Ala Leu Ala Asp Ala Leu Leu Asp Ser
145 150 155 160
gtc ccg atg gtc gcc atc acg ggc cag gtc cac cgc cgc atg atc ggc 528
Val Pro Met Val Ala Ile Thr Gly Gln Val His Arg Arg Met Ile Gly
165 170 175
acc gac gcc ttc cag gag acg ccc ata gtc gag gtc acc cgc tcc atc 576
Thr Asp Ala Phe Gln Glu Thr Pro Ile Val Glu Val Thr Arg Ser Ile
180 185 190
acc aag cac aat tac ctt gtc ctt gat gtg gag gac atc ccc cgc gtc 624
Thr Lys His Asn Tyr Leu Val Leu Asp Val Glu Asp Ile Pro Arg Val
195 200 205
ata cag gaa gcc ttc ttc ctc gcg tcc tcg ggc cgt cct ggc ccg gtg 672
Ile Gln Glu Ala Phe Phe Leu Ala Ser Ser Gly Arg Pro Gly Pro Val
210 215 220
ctg gtc gac atc ccc aag gac atc cag cag cag atg gct gtg cca gtc 720
Leu Val Asp Ile Pro Lys Asp Ile Gln Gln Gln Met Ala Val Pro Val
225 230 235 240
tgg gac acc tcg atg aat cta ccg ggg tac att gca cgc ctg ccc aag 768
Trp Asp Thr Ser Met Asn Leu Pro Gly Tyr Ile Ala Arg Leu Pro Lys
245 250 255
cca ccc gcg aca gaa ttg ctt gag cag gtc ttg cgt ctg gtt ggc gag 816
Pro Pro Ala Thr Glu Leu Leu Glu Gln Val Leu Arg Leu Val Gly Glu
260 265 270
tca cgg cgc ccg att ctc tat gtc ggt ggt ggc tgc tct gca tct ggt 864
Ser Arg Arg Pro Ile Leu Tyr Val Gly Gly Gly Cys Ser Ala Ser Gly
275 280 285
gat gaa ttg cgc cgg ttt gtt gag ctg acc ggc atc cca gtt aca acc 912
Asp Glu Leu Arg Arg Phe Val Glu Leu Thr Gly Ile Pro Val Thr Thr
290 295 300
act ctg atg ggc ctc ggc aat ttc ccc agt gat gat ccg ttg tcc ctg 960
Thr Leu Met Gly Leu Gly Asn Phe Pro Ser Asp Asp Pro Leu Ser Leu
305 310 315 320
cgc atg ctt ggg atg cat ggc acg gtg tac gca aat tat gcg gtg gat 1008
Arg Met Leu Gly Met His Gly Thr Val Tyr Ala Asn Tyr Ala Val Asp
325 330 335
aag gct gac ctg ttg ctt gca ttt ggc gtg cgg ttt gat gaa cgt gtg 1056
Lys Ala Asp Leu Leu Leu Ala Phe Gly Val Arg Phe Asp Glu Arg Val
340 345 350
aca ggg aaa att gag gct ttt gca agc agg gcc aag att gtg cac att 1104
Thr Gly Lys Ile Glu Ala Phe Ala Ser Arg Ala Lys Ile Val His Ile
355 360 365
gac att gat cca gcg gag att gga aag aac aag caa cca cat gtg tca 1152
Asp Ile Asp Pro Ala Glu Ile Gly Lys Asn Lys Gln Pro His Val Ser
370 375 380
att tgc gca gat gtt aag ctt gct tta cag ggc ttg aat gct ctg cta 1200
Ile Cys Ala Asp Val Lys Leu Ala Leu Gln Gly Leu Asn Ala Leu Leu
385 390 395 400
gac cag agc aca aca aag aca agt tct gat ttt agt gca tgg cac aat 1248
Asp Gln Ser Thr Thr Lys Thr Ser Ser Asp Phe Ser Ala Trp His Asn
405 410 415
gag ttg gac cag cag aag agg gag ttt cct ctg ggg tac aag act ttt 1296
Glu Leu Asp Gln Gln Lys Arg Glu Phe Pro Leu Gly Tyr Lys Thr Phe
420 425 430
ggt gaa gag atc cca ccg caa tat gct att cag gtg ctg gat gag ctg 1344
Gly Glu Glu Ile Pro Pro Gln Tyr Ala Ile Gln Val Leu Asp Glu Leu
435 440 445
acg aaa ggg gag gca atc atc gct act ggt gtt gga cag cac cag atg 1392
Thr Lys Gly Glu Ala Ile Ile Ala Thr Gly Val Gly Gln His Gln Met
450 455 460
tgg gcg gca caa tat tac acc tac aag cgg cca cgg cag tgg ctg tct 1440
Trp Ala Ala Gln Tyr Tyr Thr Tyr Lys Arg Pro Arg Gln Trp Leu Ser
465 470 475 480
tcg gct ggt ctg ggc gca atg gga ttt ggg ctg cct gct gca gct ggt 1488
Ser Ala Gly Leu Gly Ala Met Gly Phe Gly Leu Pro Ala Ala Ala Gly
485 490 495
gct tct gtg gct aac cca ggt gtc aca gtt gtt gat att gat ggg gat 1536
Ala Ser Val Ala Asn Pro Gly Val Thr Val Val Asp Ile Asp Gly Asp
500 505 510
ggt agc ttc ctc atg aac att cag gag ttg gca ttg atc cgc att gag 1584
Gly Ser Phe Leu Met Asn Ile Gln Glu Leu Ala Leu Ile Arg Ile Glu
515 520 525
aac ctc ccg gtg aag gtg atg gtg ttg aac aac caa cat ttg ggt atg 1632
Asn Leu Pro Val Lys Val Met Val Leu Asn Asn Gln His Leu Gly Met
530 535 540
gtt gtg caa tgg gag gat agg ttt tac aag gca aat agg gcg cat aca 1680
Val Val Gln Trp Glu Asp Arg Phe Tyr Lys Ala Asn Arg Ala His Thr
545 550 555 560
tac ttg ggc aac cca gaa tgt gag agc gag ata tat cca gat ttt gtg 1728
Tyr Leu Gly Asn Pro Glu Cys Glu Ser Glu Ile Tyr Pro Asp Phe Val
565 570 575
act att gct aaa ggg ttc aat att cct gca gtc cgt gta aca aag aag 1776
Thr Ile Ala Lys Gly Phe Asn Ile Pro Ala Val Arg Val Thr Lys Lys
580 585 590
agt gaa gtc cgt gcc gcc atc aag aag atg ctc gag acc cca ggg cca 1824
Ser Glu Val Arg Ala Ala Ile Lys Lys Met Leu Glu Thr Pro Gly Pro
595 600 605
tac ttg ttg gat atc atc gtc cca cac cag gag cat gtg ctg cct atg 1872
Tyr Leu Leu Asp Ile Ile Val Pro His Gln Glu His Val Leu Pro Met
610 615 620
atc cca agt ggg ggc gca ttc aag gac atg atc ctg gat ggt gat ggc 1920
Ile Pro Ser Gly Gly Ala Phe Lys Asp Met Ile Leu Asp Gly Asp Gly
625 630 635 640
agg act atg tat taa 1935
Arg Thr Met Tyr
<210> 2
<211> 644
<212> PRT
<213>Rice paddy seed Hefei sterile line antiweed mutant als gene
<400> 2
Met Ala Thr Thr Ala Ala Ala Ala Ala Ala Thr Leu Ser Ala Ala Ala
1 5 10 15
Thr Ala Lys Thr Gly Arg Lys Asn His Gln Arg His His Val Leu Pro
20 25 30
Ala Arg Gly Arg Val Gly Ala Ala Ala Val Arg Cys Ser Ala Val Ser
35 40 45
Pro Val Thr Pro Pro Ser Pro Ala Pro Pro Ala Thr Pro Leu Arg Pro
50 55 60
Trp Gly Thr Ala Glu Pro Arg Lys Gly Ala Asp Ile Leu Val Glu Ala
65 70 75 80
Leu Glu Arg Cys Gly Val Ser Asp Val Phe Ala Tyr Pro Gly Gly Ala
85 90 95
Ser Met Glu Ile His Gln Ala Leu Thr Arg Ser Pro Val Ile Thr Asn
100 105 110
His Leu Phe Arg His Glu Gln Gly Glu Ala Phe Ala Ala Ser Gly Tyr
115 120 125
Ala Arg Ala Ser Gly Arg Val Gly Val Cys Val Ala Thr Ser Gly Pro
130 135 140
Gly Ala Thr Asn Leu Val Ser Ala Leu Ala Asp Ala Leu Leu Asp Ser
145 150 155 160
Val Pro Met Val Ala Ile Thr Gly Gln Val His Arg Arg Met Ile Gly
165 170 175
Thr Asp Ala Phe Gln Glu Thr Pro Ile Val Glu Val Thr Arg Ser Ile
180 185 190
Thr Lys His Asn Tyr Leu Val Leu Asp Val Glu Asp Ile Pro Arg Val
195 200 205
Ile Gln Glu Ala Phe Phe Leu Ala Ser Ser Gly Arg Pro Gly Pro Val
210 215 220
Leu Val Asp Ile Pro Lys Asp Ile Gln Gln Gln Met Ala Val Pro Val
225 230 235 240
Trp Asp Thr Ser Met Asn Leu Pro Gly Tyr Ile Ala Arg Leu Pro Lys
245 250 255
Pro Pro Ala Thr Glu Leu Leu Glu Gln Val Leu Arg Leu Val Gly Glu
260 265 270
Ser Arg Arg Pro Ile Leu Tyr Val Gly Gly Gly Cys Ser Ala Ser Gly
275 280 285
Asp Glu Leu Arg Arg Phe Val Glu Leu Thr Gly Ile Pro Val Thr Thr
290 295 300
Thr Leu Met Gly Leu Gly Asn Phe Pro Ser Asp Asp Pro Leu Ser Leu
305 310 315 320
Arg Met Leu Gly Met His Gly Thr Val Tyr Ala Asn Tyr Ala Val Asp
325 330 335
Lys Ala Asp Leu Leu Leu Ala Phe Gly Val Arg Phe Asp Glu Arg Val
340 345 350
Thr Gly Lys Ile Glu Ala Phe Ala Ser Arg Ala Lys Ile Val His Ile
355 360 365
Asp Ile Asp Pro Ala Glu Ile Gly Lys Asn Lys Gln Pro His Val Ser
370 375 380
Ile Cys Ala Asp Val Lys Leu Ala Leu Gln Gly Leu Asn Ala Leu Leu
385 390 395 400
Asp Gln Ser Thr Thr Lys Thr Ser Ser Asp Phe Ser Ala Trp His Asn
405 410 415
Glu Leu Asp Gln Gln Lys Arg Glu Phe Pro Leu Gly Tyr Lys Thr Phe
420 425 430
Gly Glu Glu Ile Pro Pro Gln Tyr Ala Ile Gln Val Leu Asp Glu Leu
435 440 445
Thr Lys Gly Glu Ala Ile Ile Ala Thr Gly Val Gly Gln His Gln Met
450 455 460
Trp Ala Ala Gln Tyr Tyr Thr Tyr Lys Arg Pro Arg Gln Trp Leu Ser
465 470 475 480
Ser Ala Gly Leu Gly Ala Met Gly Phe Gly Leu Pro Ala Ala Ala Gly
485 490 495
Ala Ser Val Ala Asn Pro Gly Val Thr Val Val Asp Ile Asp Gly Asp
500 505 510
Gly Ser Phe Leu Met Asn Ile Gln Glu Leu Ala Leu Ile Arg Ile Glu
515 520 525
Asn Leu Pro Val Lys Val Met Val Leu Asn Asn Gln His Leu Gly Met
530 535 540
Val Val Gln Trp Glu Asp Arg Phe Tyr Lys Ala Asn Arg Ala His Thr
545 550 555 560
Tyr Leu Gly Asn Pro Glu Cys Glu Ser Glu Ile Tyr Pro Asp Phe Val
565 570 575
Thr Ile Ala Lys Gly Phe Asn Ile Pro Ala Val Arg Val Thr Lys Lys
580 585 590
Ser Glu Val Arg Ala Ala Ile Lys Lys Met Leu Glu Thr Pro Gly Pro
595 600 605
Tyr Leu Leu Asp Ile Ile Val Pro His Gln Glu His Val Leu Pro Met
610 615 620
Ile Pro Ser Gly Gly Ala Phe Lys Asp Met Ile Leu Asp Gly Asp Gly
625 630 635 640
Arg Thr Met Tyr
<210> 3
<211> 1935
<212> DNA
<213>Rice paddy seed Hefei sterile line wild type als gene
<220>
<221> CDS
<222> (1)..(1935)
<400> 3
atg gct acg acc gcc gcg gcc gcg gcc gcc acc ttg tcc gcc gcc gcg 48
Met Ala Thr Thr Ala Ala Ala Ala Ala Ala Thr Leu Ser Ala Ala Ala
1 5 10 15
acg gcc aag acc ggc cgt aag aac cac cag cga cac cac gtc ctt ccc 96
Thr Ala Lys Thr Gly Arg Lys Asn His Gln Arg His His Val Leu Pro
20 25 30
gct cga ggc cgg gtg ggg gcg gcg gcg gtc agg tgc tcg gcg gtg tcc 144
Ala Arg Gly Arg Val Gly Ala Ala Ala Val Arg Cys Ser Ala Val Ser
35 40 45
ccg gtc acc ccg ccg tcc ccg gcg ccg ccg gcc acg ccg ctc cgg ccg 192
Pro Val Thr Pro Pro Ser Pro Ala Pro Pro Ala Thr Pro Leu Arg Pro
50 55 60
tgg ggg acg gcc gag ccc cgc aag ggc gcg gac atc ctc gtg gag gcg 240
Trp Gly Thr Ala Glu Pro Arg Lys Gly Ala Asp Ile Leu Val Glu Ala
65 70 75 80
ctg gag cgg tgc ggc gtc agc gac gtg ttc gcc tac ccg ggc ggc gcg 288
Leu Glu Arg Cys Gly Val Ser Asp Val Phe Ala Tyr Pro Gly Gly Ala
85 90 95
tcc atg gag atc cac cag gcg ctg acg cgc tcc ccg gtc atc acc aac 336
Ser Met Glu Ile His Gln Ala Leu Thr Arg Ser Pro Val Ile Thr Asn
100 105 110
cac ctc ttc cgc cac gag cag ggc gag gcg ttc gcg gcg tcc ggg tac 384
His Leu Phe Arg His Glu Gln Gly Glu Ala Phe Ala Ala Ser Gly Tyr
115 120 125
gcg cgc gcg tcc ggc cgc gtc ggg gtc tgc gtc gcc acc tcc ggc ccc 432
Ala Arg Ala Ser Gly Arg Val Gly Val Cys Val Ala Thr Ser Gly Pro
130 135 140
ggg gca acc aac ctc gtg tcc gcg ctc gcc gac gcg ctg ctc gac tcc 480
Gly Ala Thr Asn Leu Val Ser Ala Leu Ala Asp Ala Leu Leu Asp Ser
145 150 155 160
gtc ccg atg gtc gcc atc acg ggc cag gtc ccc cgc cgc atg atc ggc 528
Val Pro Met Val Ala Ile Thr Gly Gln Val Pro Arg Arg Met Ile Gly
165 170 175
acc gac gcc ttc cag gag acg ccc ata gtc gag gtc acc cgc tcc atc 576
Thr Asp Ala Phe Gln Glu Thr Pro Ile Val Glu Val Thr Arg Ser Ile
180 185 190
acc aag cac aat tac ctt gtc ctt gat gtg gag gac atc ccc cgc gtc 624
Thr Lys His Asn Tyr Leu Val Leu Asp Val Glu Asp Ile Pro Arg Val
195 200 205
ata cag gaa gcc ttc ttc ctc gcg tcc tcg ggc cgt cct ggc ccg gtg 672
Ile Gln Glu Ala Phe Phe Leu Ala Ser Ser Gly Arg Pro Gly Pro Val
210 215 220
ctg gtc gac atc ccc aag gac atc cag cag cag atg gct gtg cca gtc 720
Leu Val Asp Ile Pro Lys Asp Ile Gln Gln Gln Met Ala Val Pro Val
225 230 235 240
tgg gac acc tcg atg aat cta ccg ggg tac att gca cgc ctg ccc aag 768
Trp Asp Thr Ser Met Asn Leu Pro Gly Tyr Ile Ala Arg Leu Pro Lys
245 250 255
cca ccc gcg aca gaa ttg ctt gag cag gtc ttg cgt ctg gtt ggc gag 816
Pro Pro Ala Thr Glu Leu Leu Glu Gln Val Leu Arg Leu Val Gly Glu
260 265 270
tca cgg cgc ccg att ctc tat gtc ggt ggt ggc tgc tct gca tct ggt 864
Ser Arg Arg Pro Ile Leu Tyr Val Gly Gly Gly Cys Ser Ala Ser Gly
275 280 285
gat gaa ttg cgc cgg ttt gtt gag ctg acc ggc atc cca gtt aca acc 912
Asp Glu Leu Arg Arg Phe Val Glu Leu Thr Gly Ile Pro Val Thr Thr
290 295 300
act ctg atg ggc ctc ggc aat ttc ccc agt gat gat ccg ttg tcc ctg 960
Thr Leu Met Gly Leu Gly Asn Phe Pro Ser Asp Asp Pro Leu Ser Leu
305 310 315 320
cgc atg ctt ggg atg cat ggc acg gtg tac gca aat tat gcg gtg gat 1008
Arg Met Leu Gly Met His Gly Thr Val Tyr Ala Asn Tyr Ala Val Asp
325 330 335
aag gct gac ctg ttg ctt gca ttt ggc gtg cgg ttt gat gat cgt gtg 1056
Lys Ala Asp Leu Leu Leu Ala Phe Gly Val Arg Phe Asp Asp Arg Val
340 345 350
aca ggg aaa att gag gct ttt gca agc agg gcc aag att gtg cac att 1104
Thr Gly Lys Ile Glu Ala Phe Ala Ser Arg Ala Lys Ile Val His Ile
355 360 365
gac att gat cca gcg gag att gga aag aac aag caa cca cat gtg tca 1152
Asp Ile Asp Pro Ala Glu Ile Gly Lys Asn Lys Gln Pro His Val Ser
370 375 380
att tgc gca gat gtt aag ctt gct tta cag ggc ttg aat gct ctg cta 1200
Ile Cys Ala Asp Val Lys Leu Ala Leu Gln Gly Leu Asn Ala Leu Leu
385 390 395 400
gac cag agc aca aca aag aca agt tct gat ttt agt gca tgg cac aat 1248
Asp Gln Ser Thr Thr Lys Thr Ser Ser Asp Phe Ser Ala Trp His Asn
405 410 415
gag ttg gac cag cag aag agg gag ttt cct ctg ggg tac aag act ttt 1296
Glu Leu Asp Gln Gln Lys Arg Glu Phe Pro Leu Gly Tyr Lys Thr Phe
420 425 430
ggt gaa gag atc cca ccg caa tat gct att cag gtg ctg gat gag ctg 1344
Gly Glu Glu Ile Pro Pro Gln Tyr Ala Ile Gln Val Leu Asp Glu Leu
435 440 445
acg aaa ggg gag gca atc atc gct act ggt gtt gga cag cac cag atg 1392
Thr Lys Gly Glu Ala Ile Ile Ala Thr Gly Val Gly Gln His Gln Met
450 455 460
tgg gcg gca caa tat tac acc tac aag cgg cca cgg cag tgg ctg tct 1440
Trp Ala Ala Gln Tyr Tyr Thr Tyr Lys Arg Pro Arg Gln Trp Leu Ser
465 470 475 480
tcg gct ggt ctg ggc gca atg gga ttt ggg ctg cct gct gca gct ggt 1488
Ser Ala Gly Leu Gly Ala Met Gly Phe Gly Leu Pro Ala Ala Ala Gly
485 490 495
gct tct gtg gct aac cca ggt gtc aca gtt gtt gat att gat ggg gat 1536
Ala Ser Val Ala Asn Pro Gly Val Thr Val Val Asp Ile Asp Gly Asp
500 505 510
ggt agc ttc ctc atg aac att cag gag ttg gca ttg atc cgc att gag 1584
Gly Ser Phe Leu Met Asn Ile Gln Glu Leu Ala Leu Ile Arg Ile Glu
515 520 525
aac ctc ccg gtg aag gtg atg gtg ttg aac aac caa cat ttg ggt atg 1632
Asn Leu Pro Val Lys Val Met Val Leu Asn Asn Gln His Leu Gly Met
530 535 540
gtt gtg caa tgg gag gat agg ttt tac aag gca aat agg gcg cat aca 1680
Val Val Gln Trp Glu Asp Arg Phe Tyr Lys Ala Asn Arg Ala His Thr
545 550 555 560
tac ttg ggc aac cca gaa tgt gag agc gag ata tat cca gat ttt gtg 1728
Tyr Leu Gly Asn Pro Glu Cys Glu Ser Glu Ile Tyr Pro Asp Phe Val
565 570 575
act att gct aaa ggg ttc aat att cct gca gtc cgt gta aca aag aag 1776
Thr Ile Ala Lys Gly Phe Asn Ile Pro Ala Val Arg Val Thr Lys Lys
580 585 590
agt gaa gtc cgt gcc gcc atc aag aag atg ctc gag acc cca ggg cca 1824
Ser Glu Val Arg Ala Ala Ile Lys Lys Met Leu Glu Thr Pro Gly Pro
595 600 605
tac ttg ttg gat atc atc gtc cca cac cag gag cat gtg ctg cct atg 1872
Tyr Leu Leu Asp Ile Ile Val Pro His Gln Glu His Val Leu Pro Met
610 615 620
atc cca agt ggg ggc gca ttc aag gac atg atc ctg gat ggt gat ggc 1920
Ile Pro Ser Gly Gly Ala Phe Lys Asp Met Ile Leu Asp Gly Asp Gly
625 630 635 640
agg act atg tat taa 1935
Arg Thr Met Tyr
<210> 4
<211> 644
<212> PRT
<213>Rice paddy seed Hefei sterile line wild type als gene
<400> 4
Met Ala Thr Thr Ala Ala Ala Ala Ala Ala Thr Leu Ser Ala Ala Ala
1 5 10 15
Thr Ala Lys Thr Gly Arg Lys Asn His Gln Arg His His Val Leu Pro
20 25 30
Ala Arg Gly Arg Val Gly Ala Ala Ala Val Arg Cys Ser Ala Val Ser
35 40 45
Pro Val Thr Pro Pro Ser Pro Ala Pro Pro Ala Thr Pro Leu Arg Pro
50 55 60
Trp Gly Thr Ala Glu Pro Arg Lys Gly Ala Asp Ile Leu Val Glu Ala
65 70 75 80
Leu Glu Arg Cys Gly Val Ser Asp Val Phe Ala Tyr Pro Gly Gly Ala
85 90 95
Ser Met Glu Ile His Gln Ala Leu Thr Arg Ser Pro Val Ile Thr Asn
100 105 110
His Leu Phe Arg His Glu Gln Gly Glu Ala Phe Ala Ala Ser Gly Tyr
115 120 125
Ala Arg Ala Ser Gly Arg Val Gly Val Cys Val Ala Thr Ser Gly Pro
130 135 140
Gly Ala Thr Asn Leu Val Ser Ala Leu Ala Asp Ala Leu Leu Asp Ser
145 150 155 160
Val Pro Met Val Ala Ile Thr Gly Gln Val Pro Arg Arg Met Ile Gly
165 170 175
Thr Asp Ala Phe Gln Glu Thr Pro Ile Val Glu Val Thr Arg Ser Ile
180 185 190
Thr Lys His Asn Tyr Leu Val Leu Asp Val Glu Asp Ile Pro Arg Val
195 200 205
Ile Gln Glu Ala Phe Phe Leu Ala Ser Ser Gly Arg Pro Gly Pro Val
210 215 220
Leu Val Asp Ile Pro Lys Asp Ile Gln Gln Gln Met Ala Val Pro Val
225 230 235 240
Trp Asp Thr Ser Met Asn Leu Pro Gly Tyr Ile Ala Arg Leu Pro Lys
245 250 255
Pro Pro Ala Thr Glu Leu Leu Glu Gln Val Leu Arg Leu Val Gly Glu
260 265 270
Ser Arg Arg Pro Ile Leu Tyr Val Gly Gly Gly Cys Ser Ala Ser Gly
275 280 285
Asp Glu Leu Arg Arg Phe Val Glu Leu Thr Gly Ile Pro Val Thr Thr
290 295 300
Thr Leu Met Gly Leu Gly Asn Phe Pro Ser Asp Asp Pro Leu Ser Leu
305 310 315 320
Arg Met Leu Gly Met His Gly Thr Val Tyr Ala Asn Tyr Ala Val Asp
325 330 335
Lys Ala Asp Leu Leu Leu Ala Phe Gly Val Arg Phe Asp Asp Arg Val
340 345 350
Thr Gly Lys Ile Glu Ala Phe Ala Ser Arg Ala Lys Ile Val His Ile
355 360 365
Asp Ile Asp Pro Ala Glu Ile Gly Lys Asn Lys Gln Pro His Val Ser
370 375 380
Ile Cys Ala Asp Val Lys Leu Ala Leu Gln Gly Leu Asn Ala Leu Leu
385 390 395 400
Asp Gln Ser Thr Thr Lys Thr Ser Ser Asp Phe Ser Ala Trp His Asn
405 410 415
Glu Leu Asp Gln Gln Lys Arg Glu Phe Pro Leu Gly Tyr Lys Thr Phe
420 425 430
Gly Glu Glu Ile Pro Pro Gln Tyr Ala Ile Gln Val Leu Asp Glu Leu
435 440 445
Thr Lys Gly Glu Ala Ile Ile Ala Thr Gly Val Gly Gln His Gln Met
450 455 460
Trp Ala Ala Gln Tyr Tyr Thr Tyr Lys Arg Pro Arg Gln Trp Leu Ser
465 470 475 480
Ser Ala Gly Leu Gly Ala Met Gly Phe Gly Leu Pro Ala Ala Ala Gly
485 490 495
Ala Ser Val Ala Asn Pro Gly Val Thr Val Val Asp Ile Asp Gly Asp
500 505 510
Gly Ser Phe Leu Met Asn Ile Gln Glu Leu Ala Leu Ile Arg Ile Glu
515 520 525
Asn Leu Pro Val Lys Val Met Val Leu Asn Asn Gln His Leu Gly Met
530 535 540
Val Val Gln Trp Glu Asp Arg Phe Tyr Lys Ala Asn Arg Ala His Thr
545 550 555 560
Tyr Leu Gly Asn Pro Glu Cys Glu Ser Glu Ile Tyr Pro Asp Phe Val
565 570 575
Thr Ile Ala Lys Gly Phe Asn Ile Pro Ala Val Arg Val Thr Lys Lys
580 585 590
Ser Glu Val Arg Ala Ala Ile Lys Lys Met Leu Glu Thr Pro Gly Pro
595 600 605
Tyr Leu Leu Asp Ile Ile Val Pro His Gln Glu His Val Leu Pro Met
610 615 620
Ile Pro Ser Gly Gly Ala Phe Lys Asp Met Ile Leu Asp Gly Asp Gly
625 630 635 640
Arg Thr Met Tyr
<210> 5
<211> 30
<212> DNA
<213>Sense primer
<400> 5
cgcggatcca tccgagccac acatcgcctc 30
<210> 6
<211> 28
<212> DNA
<213>Anti-sense primer
<400> 6
tccccgcggc ctacggaaaa caacacac 28
<210> 7
<211> 2091
<212> DNA
<213>Herbicide Resistant Rice mutant als gene fragment
<400> 7
ctgcatcgcg tgtcgccctt tcgcccaaac ccagaaaccc tcgccgccgc cgccgccgcc 60
atcacccacc atggctacga ccgccgcggc cgcggccgcc accttgtccg ccgccgcgac 120
ggccaagacc ggccgtaaga accaccagcg acaccacgtc cttcccgctc gaggccgggt 180
gggggcggcg gcggtcaggt gctcggcggt gtccccggtc accccgccgt ccccggcgcc 240
gccggccacg ccgctccggc cgtgggggac ggccgagccc cgcaagggcg cggacatcct 300
cgtggaggcg ctggagcggt gcggcgtcag cgacgtgttc gcctacccgg gcggcgcgtc 360
catggagatc caccaggcgc tgacgcgctc cccggtcatc accaaccacc tcttccgcca 420
cgagcagggc gaggcgttcg cggcgtccgg gtacgcgcgc gcgtccggcc gcgtcggggt 480
ctgcgtcgcc acctccggcc ccggggcaac caacctcgtg tccgcgctcg ccgacgcgct 540
gctcgactcc gtcccgatgg tcgccatcac gggccaggtc caccgccgca tgatcggcac 600
cgacgccttc caggagacgc ccatagtcga ggtcacccgc tccatcacca agcacaatta 660
ccttgtcctt gatgtggagg acatcccccg cgtcatacag gaagccttct tcctcgcgtc 720
ctcgggccgt cctggcccgg tgctggtcga catccccaag gacatccagc agcagatggc 780
tgtgccagtc tgggacacct cgatgaatct accggggtac attgcacgcc tgcccaagcc 840
acccgcgaca gaattgcttg agcaggtctt gcgtctggtt ggcgagtcac ggcgcccgat 900
tctctatgtc ggtggtggct gctctgcatc tggtgatgaa ttgcgccggt ttgttgagct 960
gaccggcatc ccagttacaa ccactctgat gggcctcggc aatttcccca gtgatgatcc 1020
gttgtccctg cgcatgcttg ggatgcatgg cacggtgtac gcaaattatg cggtggataa 1080
ggctgacctg ttgcttgcat ttggcgtgcg gtttgatgaa cgtgtgacag ggaaaattga 1140
ggcttttgca agcagggcca agattgtgca cattgacatt gatccagcgg agattggaaa 1200
gaacaagcaa ccacatgtgt caatttgcgc agatgttaag cttgctttac agggcttgaa 1260
tgctctgcta gaccagagca caacaaagac aagttctgat tttagtgcat ggcacaatga 1320
gttggaccag cagaagaggg agtttcctct ggggtacaag acttttggtg aagagatccc 1380
accgcaatat gctattcagg tgctggatga gctgacgaaa ggggaggcaa tcatcgctac 1440
tggtgttgga cagcaccaga tgtgggcggc acaatattac acctacaagc ggccacggca 1500
gtggctgtct tcggctggtc tgggcgcaat gggatttggg ctgcctgctg cagctggtgc 1560
ttctgtggct aacccaggtg tcacagttgt tgatattgat ggggatggta gcttcctcat 1620
gaacattcag gagttggcat tgatccgcat tgagaacctc ccggtgaagg tgatggtgtt 1680
gaacaaccaa catttgggta tggttgtgca atgggaggat aggttttaca aggcaaatag 1740
ggcgcataca tacttgggca acccagaatg tgagagcgag atatatccag attttgtgac 1800
tattgctaaa gggttcaata ttcctgcagt ccgtgtaaca aagaagagtg aagtccgtgc 1860
cgccatcaag aagatgctcg agaccccagg gccatacttg ttggatatca tcgtcccaca 1920
ccaggagcat gtgctgccta tgatcccaag tgggggcgca ttcaaggaca tgatcctgga 1980
tggtgatggc aggactatgt attaatctat aatctgtatg ttggcaaagc accagcccgg 2040
cctatgtttg acctgaatga cccataaaga gaagggcgac acgcgatgca g 2091
Claims (11)
1. make plant that there is the paddy rice ALS muteins of Herbicid resistant, its amino acid sequence such as SEQ ID NO：Shown in 2.
2. the albumen described in claim 1, wherein herbicide are imidazolinone herbicides.
Lead to 3. the albumen described in claim 2, wherein imidazolinone herbicide are hundred ridges.
4. nucleic acid, it encodes the albumen described in any one of claim 1 ~ 3.
5. nucleic acid according to claim 4, its nucleotide sequence such as SEQ ID NO:Shown in 1.
6. expression cassette, recombinant vector or cell, it contains the nucleic acid described in claim 4 or 5.
7. the albumen described in any one of claim 1 ~ 3, the nucleic acid described in claim 4 or 5, the expression described in claim 6
The application of box, recombinant vector or cell in terms of plant antiweed.
8. application according to claim 7, it is characterised in that the plant is paddy rice, tobacco, arabidopsis or cotton.
9. obtain the method for the plant with Herbicid resistant, it is characterised in that comprise the following steps：
1）Plant is set to include the nucleic acid described in claim 4 or 5；Or
2）Plant is set to express any described albumen of claim 1 ~ 3.
10. method according to claim 9, it is characterised in that it includes transgenosis, hybridization, backcrossing or vegetative propagation step
Suddenly.
11. the method for plant identification, wherein plant are the plant comprising the nucleic acid described in claim 4 or 5, expression claim
The plant that the plant of 1 ~ 3 any described albumen or any described method of claim 9 ~ 10 are obtained, it is characterised in that
Comprise the following steps：
1) whether the plant is determined comprising the nucleic acid described in claim 4 or 5；Or,
2) any described the albumen whether plant expresses claim 1 ~ 3 is determined.
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710655418.XA CN107217044A (en) | 2017-08-03 | 2017-08-03 | Make plant that there is paddy rice ALS muteins and its application of Herbicid resistant |
| CN201711477560.6A CN108004224B (en) | 2017-08-03 | 2017-12-29 | Make plant that there is rice ALS mutein and its application of Herbicid resistant |
| PCT/CN2018/082758 WO2019024534A1 (en) | 2017-08-03 | 2018-04-12 | Rice als mutant protein for conferring herbicide resistance to plants, and use thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710655418.XA CN107217044A (en) | 2017-08-03 | 2017-08-03 | Make plant that there is paddy rice ALS muteins and its application of Herbicid resistant |
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| CN107217044A true CN107217044A (en) | 2017-09-29 |
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| CN201710655418.XA Pending CN107217044A (en) | 2017-08-03 | 2017-08-03 | Make plant that there is paddy rice ALS muteins and its application of Herbicid resistant |
| CN201711477560.6A Active CN108004224B (en) | 2017-08-03 | 2017-12-29 | Make plant that there is rice ALS mutein and its application of Herbicid resistant |
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| CN201711477560.6A Active CN108004224B (en) | 2017-08-03 | 2017-12-29 | Make plant that there is rice ALS mutein and its application of Herbicid resistant |
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| WO (1) | WO2019024534A1 (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2019024534A1 (en) * | 2017-08-03 | 2019-02-07 | 江苏省农业科学院 | Rice als mutant protein for conferring herbicide resistance to plants, and use thereof |
| CN111100852A (en) * | 2019-12-16 | 2020-05-05 | 中国农业科学院植物保护研究所 | Directional mutation method of OsALS1 and crop endogenous gene directed evolution method |
| CN112029742B (en) * | 2020-08-28 | 2022-03-15 | 天津市农业科学院 | ALS mutant protein of plant and application thereof |
| CN115873088A (en) * | 2022-11-24 | 2023-03-31 | 北京大学现代农业研究院 | ALS-resistant muteins and use thereof |
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| CN109097346B (en) * | 2018-09-06 | 2021-07-13 | 江苏省农业科学院 | ALS mutant protein based on gene editing technology and application of ALS mutant protein gene in plant breeding |
| CN113005105B (en) * | 2019-12-21 | 2022-07-29 | 科稷达隆(北京)生物技术有限公司 | Herbicide-resistant protein and application thereof in plant breeding |
| CN111593031B (en) * | 2020-05-07 | 2022-04-08 | 海南波莲水稻基因科技有限公司 | Rice ALS mutant gene, plant transgenic screening vector pCALSm3 containing gene and application thereof |
| CN112410308B (en) * | 2020-11-20 | 2023-11-10 | 江苏省农业科学院 | Rice ACCase mutant gene and application of rice ACCase mutant gene protein in herbicide resistance of plants |
| CN112813045B (en) * | 2021-02-09 | 2023-02-28 | 江苏省农业科学院 | Cotton GhALS mutant protein, gene, molecular marker and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1280928B1 (en) * | 2000-05-10 | 2016-11-30 | Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College | Resistance to acetohydroxyacid synthase-inhibiting herbicides |
| US20050283855A1 (en) * | 2002-03-29 | 2005-12-22 | Koichiro Kaku | Genes encoding acetolactate synthase |
| CN101297040A (en) * | 2005-08-24 | 2008-10-29 | 先锋高级育种国际公司 | Compositions providing tolerance to multiple herbicides and methods of use thereof |
| CN102559646B (en) * | 2011-11-24 | 2013-01-23 | 未名兴旺系统作物设计前沿实验室（北京）有限公司 | Protein for endowing wheat with herbicide resistance and application of protein in plant breeding |
| CN105779479B (en) * | 2016-04-12 | 2017-08-25 | 江苏省农业科学院 | A kind of ALS mutated genes and its application in terms of antiweed |
| 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 |
| CN106867977B (en) * | 2017-01-20 | 2018-06-05 | 湖南杂交水稻研究中心 | A kind of rice herbicide resistant protein and gene and its application |
| CN107217044A (en) * | 2017-08-03 | 2017-09-29 | 江苏省农业科学院 | Make plant that there is paddy rice ALS muteins and its application of Herbicid resistant |
-
2017
- 2017-08-03 CN CN201710655418.XA patent/CN107217044A/en active Pending
- 2017-12-29 CN CN201711477560.6A patent/CN108004224B/en active Active
-
2018
- 2018-04-12 WO PCT/CN2018/082758 patent/WO2019024534A1/en active Application Filing
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019024534A1 (en) * | 2017-08-03 | 2019-02-07 | 江苏省农业科学院 | Rice als mutant protein for conferring herbicide resistance to plants, and use thereof |
| CN111100852A (en) * | 2019-12-16 | 2020-05-05 | 中国农业科学院植物保护研究所 | Directional mutation method of OsALS1 and crop endogenous gene directed evolution method |
| CN111100852B (en) * | 2019-12-16 | 2021-04-13 | 中国农业科学院植物保护研究所 | Directional mutation method of OsALS1 and crop endogenous gene directed evolution method |
| CN112029742B (en) * | 2020-08-28 | 2022-03-15 | 天津市农业科学院 | ALS mutant protein of plant and application thereof |
| CN115873088A (en) * | 2022-11-24 | 2023-03-31 | 北京大学现代农业研究院 | ALS-resistant muteins and use thereof |
| CN115873088B (en) * | 2022-11-24 | 2023-06-27 | 北京大学现代农业研究院 | ALS resistant muteins and uses thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108004224B (en) | 2019-01-18 |
| WO2019024534A1 (en) | 2019-02-07 |
| CN108004224A (en) | 2018-05-08 |
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Application publication date: 20170929 |