CN106755019A - A kind of application of wheat ALS mutated genes and its albumen in terms of antiweed - Google Patents
A kind of application of wheat ALS mutated genes and its albumen in terms of antiweed Download PDFInfo
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Abstract
The invention discloses a kind of wheat ALS mutated genes, it becomes nucleotides A in the 331st nucleotides of the als gene sequence of the 6BL chromosomes of wheat 1 B gene group by G.The invention also discloses the wheat ALS mutains coded by a kind of wheat ALS mutated genes and its application., in the Wheat Mutant plant of anti-ALS inhibitor class herbicide, compared with wheat wild type ALS sequences, its protein sequence is in Val111 site mutations for the albumen source.Green plants expresses the mutein sequence can be resisted（It is resistance to）Inhibitor of acetolactate synthetase class herbicide, particularly imidazolinone herbicide.The core seedling of 1 leaf of wheat 1 of wheat ALS mutains of the invention is leading to/L water using the ridges of 3mL hundred（9 times recommend concentration）Afterwards, plant still normal growth is developed and solid.
Description
Technical field
The invention belongs to vegetable protein and plant antiweed field, it is related to a kind of wheat ALS mutated genes and its egg
The white application in terms of antiweed.
Background technology
Farmland weed is to cause one of main reason of crop production reduction.With tradition by cultivation step, artificial weeding
Compared with the method such as weeding by machine, the use of chemical herbicide be it has been recognized that to prevent and kill off farmland weed most efficient, easy
With economic administering method.
Acetolactate synthestase (ALS) (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.ALS
Inhibitor class herbicide has that selectivity is strong, broad weed-killing spectrum, low toxicity efficiently, to the low advantage of mammalian toxicity, it is big at present
Area is promoted the use of.But these herbicides also produce poisoning in itself to the general crops without anti-(resistance to) herbicidal properties,
Strongly limit its use time and use space, be just avoided that using herbicide for the previous period if desired in crop seeding
Crops are subjected to poisoning.Therefore, cultivating some anti-(resistance to) herbicide crop varieties can reduce chemical injury of crops, widen making for herbicide
Use scope.
Ripe ALS albumen is about made up of 670 amino acid, and its sequence is highly conserved between different plant species.ALS albumen
In Gly 95, Ala 96, Ala 122, Pro 171, Pro 196, Pro 197, Ala 205, Asp 376, Trp 537, Trp
548、Trp 552、Trp 557、Trp 563、Trp 574、Ser 621、Ser 627、Ser 638、Ser 653、Gly 654、
Undergoing mutation can produce ALS to press down to the grades of Val 669 amino acid sites (the ALS amino acid positions of plant Arabidopsis thaliana are calculated in mode)
Preparation class Herbicid resistant, this intends in multiple kinds of crops (including corn, wheat, wheat, rape, sunflower etc.), model plant
Had been reported that in southern mustard and hundreds of weeds.
The common wheat of plantation is a kind of allohexaploid extensively in production, contains tri- genomes of A, B and D.Wheat is
The anti-ALS inhibitor mutational site known includes Ala 179, Ser 627 (granted patent CN102559646B).Gene order is compared
Analysis finds that the known Ala 179, Ser 627 mutation are on the 6BL chromosomes of wheat 1 B gene group.ALS mutant resists
Herbicide level is relevant with the position of ALS amino acid mutations, also with mutation after amino acid classes and mutating acid number
It is relevant.
At present, the mechanism of action of ALS inhibitor class herbicide not yet determines, it is difficult to look-ahead ALS albumen other amino
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 a kind of wheat ALS mutated genes.
The wheat ALS that the technical problem also to be solved of the invention is to provide coded by above-mentioned wheat ALS mutated genes dashes forward
Become albumen.
The of the invention technical problem also to be solved there is provided the expression cassette containing above-mentioned wheat ALS mutated genes,
Recombinant vector or cell.
The technical problem also to be solved of the invention there is provided above-mentioned wheat ALS mutated genes, mutain, expression
The application of box, recombinant vector or cell in terms of green plants herbicide is prepared.
The technical problem also to be solved of the invention there is provided the method for obtaining the green plants with Herbicid resistant.
The method that the technical problem finally to be solved of the invention there is provided green plants of the identification with Herbicid resistant.
The present inventor (is planted by long-term, arduous research, the EMS mutant plants to Xu's wheat wheat purchased from Jiangsu Province's agricultural
Matter resources protection and utilization platform) for a long time, constantly screen, it was found that a series of ALS mutains, including it is described previously
Some known ALS mutains and new ALS mutains, it is insensitive to ALS inhibitor class herbicides, so that plant
With ALS inhibitor class Herbicid resistants.Application of the present invention in plant breeding, can be used to cultivate with Herbicid resistant
Plant, especially crops, also developed these albumen and its encoding gene and make in transgenosis or non-transgenic such as wheat etc.
Application in thing.
Technical scheme：In order to solve the above technical problems, the technical solution adopted by the present invention is as follows：A kind of wheat ALS mutation
Type gene, it becomes nucleotides A in the 331st nucleotides of the 6BL chromosome als gene sequences of wheat 1 B gene group by G.
Wherein, its nucleotide sequence of above-mentioned wheat ALS mutated genes such as SEQ ID NO:Shown in 1.
Wherein, the wheat ALS mutains coded by above-mentioned wheat ALS mutated genes.The mutagenic origin is in wheat B
The 6BL chromosome als genes of genome, compared with to the wild-type wheat ALS of ALS inhibitor class herbicide sensitives, it is small
331st nucleotides of the 6BL chromosome als gene sequences of wheat 1 B gene group becomes nucleotides A by G, causes its corresponding encoded
111st amino acids of amino acid sequence are changed into isoleucine from valine.Show there is presently no report, from wheat B
The Val111 site mutations of the 6BL chromosome ALS albumen of genome, can cause that wheat has ALS inhibitor class Herbicid resistants.
Wherein, above-mentioned wheat ALS albumen, its amino acid sequence such as SEQ ID NO:Shown in 2.
Present invention also includes expression cassette, recombinant vector or cell, and it contains above-mentioned wheat ALS mutated genes.
Present invention also includes above-mentioned wheat ALS mutated genes, mutain, expression cassette, recombinant vector or thin
Application of the born of the same parents in terms of green plants antiweed.
Wherein, above-mentioned green plants is wheat, tobacco, arabidopsis etc..
Present invention also includes the method for obtaining the green plants with Herbicid resistant, comprises the following steps：
1) green plants is made to include described wheat ALS mutated genes；Or
2) green plants is made to express described wheat ALS mutains.
Above-mentioned method, including transgenosis, hybridization, backcrossing or vegetative propagation step.
The method for identifying the green plants that the method for preceding claim is obtained, comprises the following steps：
1) determine whether the green plants includes above-mentioned wheat ALS mutated genes；Or,
2) determine whether the green plants expresses above-mentioned wheat ALS mutains.
Present invention also includes a kind of protective agent of herbicide, and the protective agent is by above-mentioned wheat ALS mutain systems
Into.
Beneficial effect：By field spray ALS inhibitor classes herbicide " hundred ridges lead to " test result indicate that, contain this
The core seedling of 1 leaf of wheat 1 of the wheat ALS mutains of invention apply 3mL hundred ridges it is logical/L water (9 times recommend concentration) after,
Plant still normal growth development and solid, and wild-type wheat seedling apply 1mL hundred ridges it is logical/3L water (1 times recommend it is dense
Degree) after, plant strain growth is tapered off, and gradually chlorosis, change are light yellow for blade, and plant is unable to jointing, and final whole strain is dead；Further
Ground, wheat ALS mutators of the invention convert Ben Shi cigarette leaf dish, and after obtaining transfer-gen plant sowing, Progeny plants are long to 3-4
During the leaf phase, after being positive through PCR identifications, spray the ridges of 4mL hundred and lead to/L water (12 times recommend concentration), transgenosis is found after 30 days
Tobacco growing is in good condition, and non-transgenic tobacco is then all withered；Further, wheat ALS mutators of the invention turn
After the arabidopsis maturation sowing of change, sow immediately, the Arabidopsis thaliana Seedlings of non-bolting are sprayed the ridges of 3mL hundred it is logical/(9 times of recommendations make L water
With concentration), find that non-transgenic arabidopsis is withered after 30 days, and transgenic arabidopsis growth conditions are good.
Brief description of the drawings
The resistant wheat mutant of the invention that the logical herbicide screening in the ridges of Fig. 1 hundred is obtained；
Fig. 2 PCR expand wheat als gene total length result figure；1st swimming lane is Marker；Marker molecular weight is from top to bottom
5kb, 3kb, 2kb, 1kb, 750bp, 500bp are followed successively by, the 2nd swimming lane is the DNA of antiweed mutant plant；Purpose fragment length
2104bp；
BamHI the and SacI double digestions checking electrophoretogram of Fig. 3 recombinant plasmids pCAMBIA1301-ALS；Swimming lane 1 for
Marker；Marker molecular weight is followed successively by 8kb, 5kb, 3kb, 2kb, 1kb, 750bp, 500bp, 250bp from top to bottom；Swimming lane 2
It is the fragment after recombinant plasmid pCAMBIA1301-ALS digestions.
Specific 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, are
Can by city available from conventional products.
Embodiment 1：The anti-imidazolinone herbicide mutant acquisition process of wheat (hundred ridges lead to)
By Xu's Wheat Seeds (be purchased from Jiangsu Province's agricultural plasm resource protection and utilize platform), (this is M0, and it is small to soak 2 with clear water
When) 120kg soaks 6-9 hour points for 6 times at room temperature with 0.4-0.6% (w/w) ethyl methane sulfonate (EMS), every 1 hour of period shook
Seed；EMS solution is discarded, running water stirs immersion seed 5 times, 5 minutes every time, then rinses seed mistake with running water
Night, next day carries out field sowing, and carries out conventional fertilizer water management (this is M1).After plant maturation, seed is mixed to be received, dries, and is passed the winter
Preserve.Next year sows field.When wheat (this is M2) seedling 1 core phase to 1 leaf long, spray as the ridges of 3mL hundred lead to/L water (" hundred ridges
It is logical " it is that BASF Aktiengesellschaft produces a kind of water aqua type imidazolinone herbicide, it is the ridges of 1mL hundred to recommend minimum concentration
Logical/1.5~3L water), also it is anti-imidazolinone herbicide in the plant of normal green and the normal jointing of energy after 3 months
Wheat mutant plant (Fig. 1).141 plants of the M2 individual plants of antiweed are obtained altogether, and these resistance individual plants carry out conventional rich water quality management,
Can be normally solid, after seed maturity, individual plant sowing, dry, preservation of passing the winter.Our 141 plants of M2 to above-mentioned acquisition carry out list
Strain is harvested, and carries out planting M3 seeds, and the logical herbicide screening identification in further hundred ridge, screening concentration found No. 75 as before
The growth of mutant does not influence completely, and the then complete yellow of the blade of WT lines.We further to the mutant with
The ALS albumen of wild type carries out enzyme activity determination, and when hair there are currently no addition herbicide, two kinds of enzyme activity of ALS albumen are in 1.2-1.4
Between, but after adding herbicide hundred ridge logical, the ALS protease activities of mutant are significantly higher than wild type ALS albumen.
Embodiment 2：Analyze in antiweed Wheat Mutant mutational site
In the antiweed wheat mutant plant that above-described embodiment 1 is obtained, the blade of No. 75 mutant plants and wild is chosen
The blade of type plant, extracts genomic DNA, and serving Hai Han spaces bio tech ltd carries out gene order-checking.Sequencing result
It was found that：Compared with WT lines, the 6BL chromosome ALS bases of No. 75 Wheat Mutants of above-mentioned antiweed in its 1 B gene group
Because the 331st nucleotides of sequence there occurs single base mutation, A is become by G, cause the of the amino acid sequence of its corresponding encoded
111 amino acid are changed into the 6BL chromosomes of the 1 B gene group of isoleucine, i.e., No. 75 endowing wheat with herbicide resistance mutant plant from valine
The nucleotide sequence of als gene as shown in SEQ ID NO.1, its coding ALS albumen amino acid sequence such as SEQ ID NO.2
It is shown.Its Classification And Nomenclature of No. 75 wheat mutant plants of the invention is common wheat seed Xu wheat M3 (Triticum aestivum
L.Xumai-M3), the bacterial strain is preserved in China typical culture collection center (CCTCC), address on the 27th in September in 2016：
Wuhan City, Hubei Province Wuchang District Wuhan University collection (the first affiliated primary school of Wuhan University opposite), postcode：430072, preservation
Numbering is CCTCC No：P201622.
The anti-imidazolinone herbicide Wheat Mutant als gene clone of embodiment 3
No. 75 blades of Wheat Mutant of above-mentioned antiweed are taken, genomic DNA is extracted.Designed according to sequencing sequence and expanded
Increase als gene full-length gene special primer be：- the CCATCACCCCTCCCCAATTCC-3 ' of forward primer 5 ', reverse primer 5 '-
CACTTGTAGGTCTTGTAGGTCG-3’.Using Takara PrimerSTAR Max DNA Polymerase polymerases (10U/
μ l) (be purchased from Takara companies) amplification als gene full length sequence, its reaction system is as follows：
Pcr amplification reaction program uses two-step method, and annealing and extension are combined into together, using 68 DEG C.
Program is as follows：Predegeneration：98℃3min；35 circulations；98 DEG C of 10sec of denaturation；Extend 68 DEG C of 3min；Insulation：72
℃10min。
Take 2 μ l PCR primers 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 random 12 Escherichia coli clones of picking of conversion enters performing PCR detection, takes
6 monoclonals that PCR results are positive, send Jin Sirui bio tech ltd to be sequenced, and obtain mutation als gene sequence.
The ALS enzyme activity determinations of the logical herbicide Wheat Mutant in anti-hundred ridges of embodiment 4
In order to verify the Herbicid resistant of Wheat Mutant whether caused by ALS is mutated, 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,
The blade 0.2g of wild type Xu wheat wheat and No. 75 Wheat Mutants is taken respectively, is crushed with liquid nitrogen grinding in mortar, add 2mL
Extract solution (100mM K2HPO4, pH 7.5,10mM Sodium Pyruvates, 5mM EDTA, 1mM valine, 1mM leucines, the Guangs of 10mM half
Propylhomoserin, 0.1mM flavin adenine dinucleotide (FAD)s, 5mM magnesium chlorides, 10% (V/V) glycerine, 1% (w/v) polyvinylpyrrolidone),
Lyolysis to be extracted continues to grind 1min or so after freezing.12000rpm, 4 DEG C of centrifugation 30min, Aspirate supernatant add ammonium sulfate to make
Reach 50% saturation degree, in placing on ice half an hour, 12000rpm, 4 DEG C of centrifugation 30min abandon supernatant, will be precipitated and dissolved in
0.2mL reaction buffers (100mM K2HPO4, pH 7.0,1mM EDTA, 10mM magnesium chloride, 100mM Sodium Pyruvates, 1mM Jiao's phosphorus
Allithiamine element, 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 proteins carries pyruvic acid formed acetolactic acid, acetolactic acid decarboxylation formed 3-Hydroxybutanone,
Pink compound is formed with creatine and 1- naphthols again, the compound has obtained the maximum absorption at 530nm), then determine it
The absorbance of 530nm, ALS protein actives represent that the height of A530 light absorption values reflects the height of ALS protein actives with A530 light absorption values
It is low.Experiment is to compare with water, if 3 repetitions.
A530 light absorption values measurement result finds, when not having hundred ridges in wild type and No. 75 ALS extract solutions of Wheat Mutant
When logical, their A530 light absorption values show that the ALS enzymatic activitys of wild type and mutant are poor without conspicuousness between 1.2-1.5
Different (table 1)；And after adding hundred ridges logical, the A530 light absorption values of WT lines are only 0.31, the A530 of mutant wheat plant inhales
Light value is only the 25.6% of control for the ALS enzymatic activitys of 0.85 or so, i.e. WT lines, and the ALS of mutant wheat plant
Enzymatic activity still has 61.6% (table 1), shows that the mutation ALS of No. 75 Wheat Mutants leads to insensitive to hundred ridges, anti-so as to impart
Property.
Table 1
The transgene tobacco of embodiment 5 is obtained
Design special primer 5 '-CGCGGATCCCCATCACCCCTCCCCAATTCC-3 ' and 5 '-
TCCCCGCGGCACTTGTAGGTCTTGTAGGTCG-3 ', it 5 ' is separately added into BamHI and SacI digestion decorating sites.Pass through
PCR amplifies mutation als gene from the genomic DNA of above-mentioned No. 75 Wheat Mutants, after sequencing is correct, with BamHI and
SacI difference double digestion mutation als gene fragments and plant expression vector pCAMBIA1301 plasmids (being purchased from pcambia companies),
Digestion products T4-DNA enzymes (being purchased from TaKaRa companies) connection, connection product conversion Escherichia coli obtain recombinant plasmid
pCAMBIA1301-ALS.Recombinant plasmid pCAMBIA1301-ALS extracts DNA, is verified with BamHI and SacI double digestions, can produce
The big plasmid fragments of life and small genetic fragment (Fig. 3), it was demonstrated that by nucleotide sequence such as SEQ ID NO：ALS bases shown in 1
Because being cloned into plant expression vector pCAMBIA1301 plasmids (purchased from pcambia companies).The plasmid vector conversion that will be built
Agrobacterium EHA105, selects positive colony, cultivates thalline.Using conventional Agrobacterium-mediated transformation Ben Shi cigarette leaf dish, obtain
After transfer-gen plant sowing, during Progeny plants leaf phase to 3-4 long, after being positive through PCR identifications, spray the ridges of 4mL hundred it is logical/L water (12
Concentration is recommended again), after 7 days, ALS enzymatic activitys are determined with reference to the methods described of embodiment 4, find the ALS enzymes of transgene tobacco
Activity is significantly higher than non-transgenic tobacco；Find that transgene tobacco growth conditions are good after 30 days, and non-transgenic tobacco is then complete
Portion is withered.
The transgenic arabidopsis of embodiment 6 are obtained
Design special primer 5 '-CGCGGATCCCCATCACCCCTCCCCAATTCC-3 ' and 5 '-
TCCCCGCGGCACTTGTAGGTCTTGTAGGTCG-3 ', it 5 ' is separately added into BamHI and SacI digestion decorating sites.Pass through
PCR amplifies mutation als gene from the genomic DNA of above-mentioned No. 75 Wheat Mutants, after sequencing is correct, with reference to embodiment 5
Method by nucleotide sequence such as SEQ ID NO：Als gene shown in 1 is cloned into plant expression vector pCAMBIA2301 plasmids
In (being purchased from pcambia companies), positive colony conversion Agrobacterium EHA105 is selected, thalline is cultivated, by agriculture bacillus mediated method
Arabidopsis thaliana transformation (Clough S, Bent A.Floral dip:a simplified method for Agrobacterium-
mediated transformation of Arabidopsis thaliana.Plant Journal,1998,16(6):735-
743.), after the arabidopsis maturation sowing of conversion, sow immediately, the Arabidopsis thaliana Seedlings of non-bolting are sprayed the ridges of 3mL hundred it is logical/L water (9
Concentration is recommended again), find that non-transgenic arabidopsis is withered after 30 days, and transgenic arabidopsis growth conditions are good.
Although specific embodiment of the 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 by disclosed all teachings, and these are in protection scope of the present invention
It is interior.Four corner of the invention is given by extremely any equivalent of appended patent requirements.
SEQUENCE LISTING
<110>Jiangsu Province Agriculture Science Institute
<120>A kind of application of wheat ALS mutated genes and its albumen in terms of antiweed
<130> SG20161219002
<160>6
<170> PatentIn version 3.3
<210> 1
<211> 1935
<212> DNA
<213>Wheat ALS mutated genes
<220>
<220>
<221> CDS
<222> (1)..(1941)
<400> 1
atg gcc gca gcc acc tcc ccc gcc gtc gca ttc tcg ggc gcc gcc gcc 48
Met Ala Ala Ala Thr Ser Pro Ala Val Ala Phe Ser Gly Ala Ala Ala
1 5 10 15
gcc gcc gcc gcc ata ccc aaa ccc gcc cgc cag cct ctc ccg cgc cac 96
Ala Ala Ala Ala Ile Pro Lys Pro Ala Arg Gln Pro Leu Pro Arg His
20 25 30
cag ccc gcc tcg cgc cgc gcg ctc ccc gcc cgc atc gtc agg tgc tgc 144
Gln Pro Ala Ser Arg Arg Ala Leu Pro Ala Arg Ile Val Arg Cys Cys
35 40 45
gcc gcg tcc ccc gcc gcc acc tcc gtc gcg cct ccc gcc acc gcg ctc 192
Ala Ala Ser Pro Ala Ala Thr Ser Val Ala Pro Pro Ala Thr Ala Leu
50 55 60
cgg ccg tgg ggc ccc tcc gag ccc cgc aag ggc gcc gac atc ctc gtc 240
Arg Pro Trp Gly Pro Ser Glu Pro Arg Lys Gly Ala Asp Ile Leu Val
65 70 75 80
gag gcg ctg gag cgc tgc ggc atc gtc gac gtc ttc gcc tac cct ggc 288
Glu Ala Leu Glu Arg Cys Gly Ile Val Asp Val Phe Ala Tyr Pro Gly
85 90 95
ggc gcg tcc atg gag atc cac cag gcg ctg acg cgc tcg cca atc atc 336
Gly Ala Ser Met Glu Ile His Gln Ala Leu Thr Arg Ser Pro Ile Ile
100 105 110
acc aac cac ctc ttc cgc cac gag cag ggg gag gcg ttc gcg gcg tcc 384
Thr Asn His Leu Phe Arg His Glu Gln Gly Glu Ala Phe Ala Ala Ser
115 120 125
ggg tac gcc cgc gcg tcc ggc cgc gtc ggc gtc tgc gtc gcc acc tcc 432
Gly Tyr Ala Arg Ala Ser Gly Arg Val Gly Val Cys Val Ala Thr Ser
130 135 140
ggc ccg ggg gcc acc aac ctc gtc tcc gcg ctc gcc gac gct ctc ctc 480
Gly Pro Gly Ala Thr Asn Leu Val Ser Ala Leu Ala Asp Ala Leu Leu
145 150 155 160
gac tcc atc ccc atg gtc gcc atc acg ggc cag gtc ccc cgc cgc atg 528
Asp Ser Ile Pro Met Val Ala Ile Thr Gly Gln Val Pro Arg Arg Met
165 170 175
atc ggc acg gat gcg ttc cag gag acg ccc atc gtg gag gtc acg cgc 576
Ile Gly Thr Asp Ala Phe Gln Glu Thr Pro Ile Val Glu Val Thr Arg
180 185 190
tcc atc acc aag cac aac tac ctg gtc ctt gac gtg gag gat atc ccc 624
Ser Ile Thr Lys His Asn Tyr Leu Val Leu Asp Val Glu Asp Ile Pro
195 200 205
cgc gtc atc cag gaa gcc ttc ttc ctc gca tcc tct ggc cgc ccg ggg 672
Arg Val Ile Gln Glu Ala Phe Phe Leu Ala Ser Ser Gly Arg Pro Gly
210 215 220
ccg gtg ctg gtt gat atc ccc aag gac atc cag cag cag atg gct gtg 720
Pro Val Leu Val Asp Ile Pro Lys Asp Ile Gln Gln Gln Met Ala Val
225 230 235 240
cct gtc tgg gac acg ccg atg agt ttg cca ggg tac atc gcc cgc ctg 768
Pro Val Trp Asp Thr Pro Met Ser Leu Pro Gly Tyr Ile Ala Arg Leu
245 250 255
ccc aag cca cca tct act gaa tcg ctt gag cag gtc ctg cgt ctg gtt 816
Pro Lys Pro Pro Ser Thr Glu Ser Leu Glu Gln Val Leu Arg Leu Val
260 265 270
ggc gag tca cgg cgc cca att ctg tat gtt ggt ggt ggc tgc gct gca 864
Gly Glu Ser Arg Arg Pro Ile Leu Tyr Val Gly Gly Gly Cys Ala Ala
275 280 285
tct ggt gag gag ttg cgc cgc ttt gtt gag ctc act ggg att cca gtt 912
Ser Gly Glu Glu Leu Arg Arg Phe Val Glu Leu Thr Gly Ile Pro Val
290 295 300
aca act act ctt atg ggc ctt ggc aac ttc ccc agt gac gac cca ctg 960
Thr Thr Thr Leu Met Gly Leu Gly Asn Phe Pro Ser Asp Asp Pro Leu
305 310 315 320
tct ctg cgc atg ctg ggg atg cat ggc act gtg tat gca aat tat gca 1008
Ser Leu Arg Met Leu Gly Met His Gly Thr Val Tyr Ala Asn Tyr Ala
325 330 335
gta gat aag gct gac ctg ttg ctt gca ttt ggt gtg cgg ttt gat gat 1056
Val Asp Lys Ala Asp Leu Leu Leu Ala Phe Gly Val Arg Phe Asp Asp
340 345 350
cgt gtg acc ggg aaa atc gag gct ttt gca agc agg tcc aag att gtg 1104
Arg Val Thr Gly Lys Ile Glu Ala Phe Ala Ser Arg Ser Lys Ile Val
355 360 365
cac att gac att gac cca gct gag att ggc aag aac aag cag cca cat 1152
His Ile Asp Ile Asp Pro Ala Glu Ile Gly Lys Asn Lys Gln Pro His
370 375 380
gtc tcc att tgt gca gat gtt aag ctt gct tta cag ggg ttg aat gct 1200
Val Ser Ile Cys Ala Asp Val Lys Leu Ala Leu Gln Gly Leu Asn Ala
385 390 395 400
cta tta aat ggg agc aaa gca caa cag ggt ctg gat ttt ggt cca tgg 1248
Leu Leu Asn Gly Ser Lys Ala Gln Gln Gly Leu Asp Phe Gly Pro Trp
405 410 415
cac aag gag ttg gat cag cag aag agg gag ttt cct cta gga ttc aag 1296
His Lys Glu Leu Asp Gln Gln Lys Arg Glu Phe Pro Leu Gly Phe Lys
420 425 430
act ttt ggt gag gcc atc ccg ccg caa tat gct atc cag gta ctg gat 1344
Thr Phe Gly Glu Ala Ile Pro Pro Gln Tyr Ala Ile Gln Val Leu Asp
435 440 445
gag ctg aca aaa ggg gag gcg atc att gcc acc ggt gtt ggg cag cat 1392
Glu Leu Thr Lys Gly Glu Ala Ile Ile Ala Thr Gly Val Gly Gln His
450 455 460
cag atg tgg gcg gct cag tat tac act tac aag cgg cca cgg cag tgg 1440
Gln Met Trp Ala Ala Gln Tyr Tyr Thr Tyr Lys Arg Pro Arg Gln Trp
465 470 475 480
ctg tct tca tcc ggt ttg ggt gca atg gga ttt ggg ttg cca gct gca 1488
Leu Ser Ser Ser Gly Leu Gly Ala Met Gly Phe Gly Leu Pro Ala Ala
485 490 495
gct ggc gct gct gtg gcc aac cca ggt gtt aca gtt gtt gac att gat 1536
Ala Gly Ala Ala Val Ala Asn Pro Gly Val Thr Val Val Asp Ile Asp
500 505 510
ggg gat ggt agt ttc ctc atg aac att cag gag ttg gcg ttg atc cgt 1584
Gly Asp Gly Ser Phe Leu Met Asn Ile Gln Glu Leu Ala Leu Ile Arg
515 520 525
att gag aac ctc cca gtg aag gtg atg ata ttg aac aac cag cat ctg 1632
Ile Glu Asn Leu Pro Val Lys Val Met Ile Leu Asn Asn Gln His Leu
530 535 540
gga atg gtg gtg cag tgg gag gat agg ttt tac aag gcc aac cgg gcg 1680
Gly Met Val Val Gln Trp Glu Asp Arg Phe Tyr Lys Ala Asn Arg Ala
545 550 555 560
cac aca tac ctt ggc aac cca gaa aat gag agt gag ata tat cca gat 1728
His Thr Tyr Leu Gly Asn Pro Glu Asn Glu Ser Glu Ile Tyr Pro Asp
565 570 575
ttt gtg acg att gct aaa gga ttc aac gtt ccg gca gtt cgt gtg acg 1776
Phe Val Thr Ile Ala Lys Gly Phe Asn Val Pro Ala Val Arg Val Thr
580 585 590
aag aag agc gaa gtc act gca gca atc aag aag atg ctt gag acc cca 1824
Lys Lys Ser Glu Val Thr Ala Ala Ile Lys Lys Met Leu Glu Thr Pro
595 600 605
ggg cca tac ttg ttg gat atc att gtc ccg cat cag gag cac gtg ctg 1872
Gly Pro Tyr Leu Leu Asp Ile Ile Val Pro His Gln Glu His Val Leu
610 615 620
cct atg atc cca agc ggt ggt gct ttt aag gac atg atc atg gag ggt 1920
Pro Met Ile Pro Ser Gly Gly Ala Phe Lys Asp Met Ile Met Glu Gly
625 630 635 640
gat ggc agg acc tcg tac tga 1941
Asp Gly Arg Thr Ser Tyr
645
<210> 2
<211> 646
<212> PRT
<213>Wheat ALS mutated genes
<400> 2
Met Ala Ala Ala Thr Ser Pro Ala Val Ala Phe Ser Gly Ala Ala Ala
1 5 10 15
Ala Ala Ala Ala Ile Pro Lys Pro Ala Arg Gln Pro Leu Pro Arg His
20 25 30
Gln Pro Ala Ser Arg Arg Ala Leu Pro Ala Arg Ile Val Arg Cys Cys
35 40 45
Ala Ala Ser Pro Ala Ala Thr Ser Val Ala Pro Pro Ala Thr Ala Leu
50 55 60
Arg Pro Trp Gly Pro Ser Glu Pro Arg Lys Gly Ala Asp Ile Leu Val
65 70 75 80
Glu Ala Leu Glu Arg Cys Gly Ile Val Asp Val Phe Ala Tyr Pro Gly
85 90 95
Gly Ala Ser Met Glu Ile His Gln Ala Leu Thr Arg Ser Pro Ile Ile
100 105 110
Thr Asn His Leu Phe Arg His Glu Gln Gly Glu Ala Phe Ala Ala Ser
115 120 125
Gly Tyr Ala Arg Ala Ser Gly Arg Val Gly Val Cys Val Ala Thr Ser
130 135 140
Gly Pro Gly Ala Thr Asn Leu Val Ser Ala Leu Ala Asp Ala Leu Leu
145 150 155 160
Asp Ser Ile Pro Met Val Ala Ile Thr Gly Gln Val Pro Arg Arg Met
165 170 175
Ile Gly Thr Asp Ala Phe Gln Glu Thr Pro Ile Val Glu Val Thr Arg
180 185 190
Ser Ile Thr Lys His Asn Tyr Leu Val Leu Asp Val Glu Asp Ile Pro
195 200 205
Arg Val Ile Gln Glu Ala Phe Phe Leu Ala Ser Ser Gly Arg Pro Gly
210 215 220
Pro Val Leu Val Asp Ile Pro Lys Asp Ile Gln Gln Gln Met Ala Val
225 230 235 240
Pro Val Trp Asp Thr Pro Met Ser Leu Pro Gly Tyr Ile Ala Arg Leu
245 250 255
Pro Lys Pro Pro Ser Thr Glu Ser Leu Glu Gln Val Leu Arg Leu Val
260 265 270
Gly Glu Ser Arg Arg Pro Ile Leu Tyr Val Gly Gly Gly Cys Ala Ala
275 280 285
Ser Gly Glu Glu Leu Arg Arg Phe Val Glu Leu Thr Gly Ile Pro Val
290 295 300
Thr Thr Thr Leu Met Gly Leu Gly Asn Phe Pro Ser Asp Asp Pro Leu
305 310 315 320
Ser Leu Arg Met Leu Gly Met His Gly Thr Val Tyr Ala Asn Tyr Ala
325 330 335
Val Asp Lys Ala Asp Leu Leu Leu Ala Phe Gly Val Arg Phe Asp Asp
340 345 350
Arg Val Thr Gly Lys Ile Glu Ala Phe Ala Ser Arg Ser Lys Ile Val
355 360 365
His Ile Asp Ile Asp Pro Ala Glu Ile Gly Lys Asn Lys Gln Pro His
370 375 380
Val Ser Ile Cys Ala Asp Val Lys Leu Ala Leu Gln Gly Leu Asn Ala
385 390 395 400
Leu Leu Asn Gly Ser Lys Ala Gln Gln Gly Leu Asp Phe Gly Pro Trp
405 410 415
His Lys Glu Leu Asp Gln Gln Lys Arg Glu Phe Pro Leu Gly Phe Lys
420 425 430
Thr Phe Gly Glu Ala Ile Pro Pro Gln Tyr Ala Ile Gln Val Leu Asp
435 440 445
Glu Leu Thr Lys Gly Glu Ala Ile Ile Ala Thr Gly Val Gly Gln His
450 455 460
Gln Met Trp Ala Ala Gln Tyr Tyr Thr Tyr Lys Arg Pro Arg Gln Trp
465 470 475 480
Leu Ser Ser Ser Gly Leu Gly Ala Met Gly Phe Gly Leu Pro Ala Ala
485 490 495
Ala Gly Ala Ala Val Ala Asn Pro Gly Val Thr Val Val Asp Ile Asp
500 505 510
Gly Asp Gly Ser Phe Leu Met Asn Ile Gln Glu Leu Ala Leu Ile Arg
515 520 525
Ile Glu Asn Leu Pro Val Lys Val Met Ile Leu Asn Asn Gln His Leu
530 535 540
Gly Met Val Val Gln Trp Glu Asp Arg Phe Tyr Lys Ala Asn Arg Ala
545 550 555 560
His Thr Tyr Leu Gly Asn Pro Glu Asn Glu Ser Glu Ile Tyr Pro Asp
565 570 575
Phe Val Thr Ile Ala Lys Gly Phe Asn Val Pro Ala Val Arg Val Thr
580 585 590
Lys Lys Ser Glu Val Thr Ala Ala Ile Lys Lys Met Leu Glu Thr Pro
595 600 605
Gly Pro Tyr Leu Leu Asp Ile Ile Val Pro His Gln Glu His Val Leu
610 615 620
Pro Met Ile Pro Ser Gly Gly Ala Phe Lys Asp Met Ile Met Glu Gly
625 630 635 640
Asp Gly Arg Thr Ser Tyr
645
<210> 3
<211> 21
<212> DNA
<213>Forward primer
<400> 3
ccatcacccc tccccaattc c 21
<210> 4
<211> 22
<212> DNA
<213>Reverse primer
<400> 4
cacttgtagg tcttgtaggt cg 22
<210> 5
<211> 30
<212> DNA
<213>Special sense primer
<400> 5
cgcggatccc catcacccct ccccaattcc 30
<210> 6
<211> 31
<212> DNA
<213>Special anti-sense primer
<400> 6
tccccgcggc acttgtaggt cttgtaggtc g 31
Claims (11)
1. a kind of wheat ALS mutated genes, it becomes nucleotides in the 331st nucleotides of the als gene sequence of wheat by G
A。
2. wheat ALS mutated genes according to claim 1, it is characterised in that described its nucleosides of ALS mutated genes
Acid sequence such as SEQ ID No：Shown in 1.
3. wheat ALS mutains coded by the wheat ALS mutated genes described in claim 1 or 2.
4. ALS mutains according to claim 3, it is characterised in that its amino acid sequence such as SEQ ID NO：2 institutes
Show.
5. expression cassette, recombinant vector or cell, it contains the wheat ALS mutated genes described in claim 1 or 2.
6. the wheat ALS mutated genes described in claim 1 or 2, the wheat ALS mutains described in claim 3 or 4,
The application of expression cassette, recombinant vector or cell described in claim 5 in terms of green plants antiweed.
7. application according to claim 6, it is characterised in that the green plants is wheat, tobacco or arabidopsis.
8. the method for obtaining the green plants with Herbicid resistant, it is characterised in that comprise the following steps：
1）Green plants is set to include the wheat ALS mutated genes described in claim 1 or 2；Or
2）Green plants is set to express any described wheat ALS mutains of claim 3 or 4.
9. method according to claim 8, it is characterised in that it includes transgenosis, hybridization, backcrossing or vegetative propagation step
Suddenly.
10. the method for identifying the green plants that the method described in claim 8 or 9 is obtained, it is characterised in that including following step
Suddenly：
1) whether the green plants is determined comprising the wheat ALS mutated genes described in claim 1 or 2；Or,
2) any described wheat ALS the mutains whether green plants expresses claim 3 or 4 are determined.
A kind of 11. protective agents of herbicide, it is characterised in that the protective agent by claim 3 or 4 any described wheat
ALS mutains are made.
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| CN201611178950.9A CN106755019B (en) | 2016-12-19 | 2016-12-19 | A kind of application of wheat ALS mutated genes and its albumen in terms of antiweed |
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| CN106755019B CN106755019B (en) | 2019-11-08 |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107354139A (en) * | 2017-09-18 | 2017-11-17 | 江苏省农业科学院 | Wheat ALS muteins, nucleic acid and its application |
| CN109090107A (en) * | 2018-10-25 | 2018-12-28 | 江苏省农业科学院 | Plant protein source film forming insecticides adjuvant and its preparation method and application |
| CN112813045A (en) * | 2021-02-09 | 2021-05-18 | 江苏省农业科学院 | Cotton GhALS mutant protein, gene, molecular marker and application thereof |
| CN114561409A (en) * | 2022-03-03 | 2022-05-31 | 山西农谷稼祺种业有限公司 | Quinoa CqALS gene mutant and molecular identification method and application thereof |
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| CN102559646A (en) * | 2011-11-24 | 2012-07-11 | 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 |
| CN105779479A (en) * | 2016-04-12 | 2016-07-20 | 江苏省农业科学院 | ALS mutant type gene and application thereof in herbicide resistance |
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| CN102559646A (en) * | 2011-11-24 | 2012-07-11 | 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 |
| CN105779479A (en) * | 2016-04-12 | 2016-07-20 | 江苏省农业科学院 | ALS mutant type gene and application thereof in herbicide resistance |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107354139A (en) * | 2017-09-18 | 2017-11-17 | 江苏省农业科学院 | Wheat ALS muteins, nucleic acid and its application |
| CN109090107A (en) * | 2018-10-25 | 2018-12-28 | 江苏省农业科学院 | Plant protein source film forming insecticides adjuvant and its preparation method and application |
| CN112813045A (en) * | 2021-02-09 | 2021-05-18 | 江苏省农业科学院 | Cotton GhALS mutant protein, gene, molecular marker and application thereof |
| CN114561409A (en) * | 2022-03-03 | 2022-05-31 | 山西农谷稼祺种业有限公司 | Quinoa CqALS gene mutant and molecular identification method and application thereof |
| CN114561409B (en) * | 2022-03-03 | 2023-06-20 | 山西农谷稼祺种业有限公司 | Quinoa CqALS gene mutant and molecular identification method and application |
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| Publication number | Publication date |
|---|---|
| CN106755019B (en) | 2019-11-08 |
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