CN106520720B - Inhibitor of acetolactate synthetase resistance-associated protein UVALS and its encoding gene and application - Google Patents
Inhibitor of acetolactate synthetase resistance-associated protein UVALS and its encoding gene and application Download PDFInfo
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Abstract
The invention discloses a kind of inhibitor of acetolactate synthetase resistance-associated protein UVALS and its encoding gene and applications.UVALS albumen provided by the invention is following (a) or (b): (a) protein that the amino acid sequence shown in sequence 1 in sequence table forms；(b) by the amino acid sequence of sequence 1 by the substitution and/or deletion and/or addition of one or several amino acid residues and the protein as derived from sequence 1 relevant to resistance of the biology to inhibitor of acetolactate synthetase.The present invention also protects the application of UVALS albumen: (c1) regulates and controls plant to the resistance of inhibitor of acetolactate synthetase；(c2) plant is promoted to increase the resistance of inhibitor of acetolactate synthetase；(c3) resistance of the regulating and controlling microbial to inhibitor of acetolactate synthetase；(c4) microorganism is promoted to increase the resistance of inhibitor of acetolactate synthetase.The present invention has very high application value with resistance new variety of plant to inhibitor of acetolactate synthetase for cultivating.
Description
Technical field
The invention belongs to field of biotechnology, be related to inhibitor of acetolactate synthetase resistance-associated protein UVALS and its
Encoding gene and application.
Background technique
Modern biotechnology has caused the major transformation in several fields in agricultural, wherein most outstanding is to lead foreign gene
Enter the expression in plant, bring revolutionary variation to crop breeding, gets rid of people and cultivated using conventional hybridization technology
The situation of new varieties, freedom of entry construct the new era of inhereditary feature.Transgenic Resistant Herbicide Crops are cultivated by genetic engineering
Kind is with fastest developing speed, most deep, the highest case of peasant's acceptance level of research, and wherein the link of most critical is anti-herbicide gene
Acquisition, from the herbicide resistant plants and microbial body of mutation separate resistant gene be main approach.
Inhibitor of acetolactate synthetase class herbicide, which passes through, inhibits the intracorporal acetolactate synthase activity of plant, thus
The synthesis for preventing branched-chain amino acid, causes the synthesis of protein to be destroyed, the DNA synthesis of block cell division stage, to make
The mitosis of plant cell stops at the M phase of S phase (DNA synthesizes the phase) and G2 stage in Gl stage, disturbs the synthesis of DNA,
Therefore cell cannot complete mitosis, and then plant is made to stop growing, and eventually lead to plant individual death.
Summary of the invention
The object of the present invention is to provide inhibitor of acetolactate synthetase resistance-associated protein UVALS and its encoding gene with
Using.
Protein provided by the invention is named as UVALS albumen, is following (a) or (b):
(a) protein that the amino acid sequence shown in sequence 1 in sequence table forms；
(b) amino acid sequence of sequence 1 by the substitution of one or several amino acid residues and/or missing and/or is added
Add and the protein as derived from sequence 1 relevant to resistance of the biology to inhibitor of acetolactate synthetase.
In order to make protein in (b) convenient for purifying and detection, can in as sequence table amino acid sequence shown in sequence 1
The amino terminal or carboxyl terminal of the protein of composition connect upper label as shown in Table 1.
The sequence of 1 label of table
| Label | Residue | Sequence |
| Poly-Arg | 5-6 (usually 5) | RRRRR |
| Poly-His | 2-10 (usually 6) | HHHHHH |
| FLAG | 8 | DYKDDDDK |
| Strep-tag II | 8 | WSHPQFEK |
| c-myc | 10 | EQKLISEEDL |
Protein in above-mentioned (b) can be artificial synthesized, can also first synthesize its encoding gene, then carries out biological expression and obtain.
The encoding gene of protein in above-mentioned (b) can be one or several by will lack in DNA sequence dna shown in sequence 2 in sequence table
The codon of amino acid residue, and/or the missense mutation of one or several base-pairs is carried out, and/or at its 5 ' end and/or 3 ' ends
The coded sequence for connecting label shown in table 1 obtains.
The gene (UVALS gene) of coding UVALS albumen also belongs to protection scope of the present invention.
UVALS gene is following DNA molecular 1) or 2) or 3):
1) code area DNA molecular as shown in sequence 2 in sequence table；
2) hybridize under strict conditions with the DNA sequence dna 1) limited and encode with biology to inhibitor of acetolactate synthetase
Resistance-associated protein DNA molecular；
3) 1) or 2) with the DNA sequence dna limited there is 90% or more homology and coding with biology to acetolactate synthestase
The DNA molecular of the resistance-associated protein of inhibitor.
Above-mentioned stringent condition can be for 0.1 × SSPE (or 0.1 × SSC), the solution of 0.1%SDS be miscellaneous in DNA or RNA
It hands over and hybridizes at 65 DEG C in experiment and wash film.
Recombinant expression carrier, expression cassette, transgenic cell line, Transgenic plant tissue or recombination containing UVALS gene
Bacterium all belongs to the scope of protection of the present invention.
The recombinant expression carrier of UVALS gene can be contained with existing expression vector establishment.The expression vector includes double
First agrobacterium vector and the carrier etc. that can be used for micropellet bombardment.It, can be in its turn when recombinant expression carrier gene constructed using UVALS
Any enhanced, composing type, organizing specific type or inducible promoter are added before recording initiation nucleotide, they can individually make
It is used in combination with or with other plant promoters；In addition, also can be used when recombinant expression carrier gene constructed using UVALS
Enhancer, including translational enhancer or transcriptional enhancer, these enhancer regions can be ATG initiation codon or neighboring region
Initiation codon etc., but must be identical as the reading frame of coded sequence, to guarantee the correct translation of entire sequence.The translation control
The source of signal processed and initiation codon be it is extensive, can be natural, be also possible to synthesis.Translation initiation region can be with
From transcription initiation region or structural gene.For the ease of genetically modified plants or transgenic microorganism are identified and are screened,
Expression carrier used thereof can be processed, the expression in plant or microorganism, which is such as added, can produce the enzyme or luminousization of color change
Close gene, resistant antibiotic marker or the anti-chemical reagent marker gene etc. of object.Consider from transgenosis safe,
Any selected marker can be not added, plant or microorganism are directly converted with phenotypic screen.
The recombinant expression carrier concretely following recombinant plasmid: pGBKT7 carrier multiple cloning sites (such as
Between BamH I and Sal I restriction enzyme site) it is inserted into the recombinant plasmid that UVALS gene obtains.
The recombinant expression carrier concretely following recombinant plasmid: Super1300 carrier multiple cloning sites (such as
Between I restriction enzyme site of Xba I and Sac) it is inserted into the recombinant plasmid that UVALS gene obtains.
The present invention also protects the application of UVALS albumen, is at least one of following (c1) to (c4):
(c1) resistance of the regulation plant to inhibitor of acetolactate synthetase；
(c2) plant is promoted to increase the resistance of inhibitor of acetolactate synthetase；
(c3) resistance of the regulating and controlling microbial to inhibitor of acetolactate synthetase；
(c4) microorganism is promoted to increase the resistance of inhibitor of acetolactate synthetase.
The plant is monocotyledon or dicotyledon, the dicotyledon concretely arabidopsis, such as brother
Rival Asia Arabidopsis thaliana ecotype.
The microorganism can be yeast, concretely yeast AH109R.
The present invention also protects UVALS gene cultivating the application in genetically modified plants；The genetically modified plants are to acetyl
The increased plant of the resistance of lactic acid synthetase inhibitors.The plant be monocotyledon or dicotyledon, it is described dicotyledonous
Plant concretely arabidopsis, such as Columbia ecotype arabidopsis.
The present invention also protects a kind of method for cultivating genetically modified plants, is obtained in UVALS channel genes purpose plant
Genetically modified plants；The genetically modified plants are higher than the purpose plant to the resistance of inhibitor of acetolactate synthetase.The mesh
Plant be monocotyledon or dicotyledon, the dicotyledon concretely arabidopsis, such as Colombia's ecology
Type arabidopsis.
The UVALS gene can specifically pass through the plant that sets out described in the importing of any description above recombinant expression carrier.It carries
There is the recombinant expression carrier of UVALS gene can be by Ti-plasmids, Ri plasmid, plant viral vector, directly delivered DNA, micro- note
It penetrates, the conventional biology methods such as conductance, mediated by agriculture bacillus are transformed into plant.
The present invention also protects a kind of method for cultivating recombinant microorganism, is obtained in UVALS channel genes purpose microorganism
To recombinant microorganism；The recombinant microorganism is higher than the purpose microorganism to the resistance of inhibitor of acetolactate synthetase.Institute
Stating UVALS gene specifically can be by the microorganism that sets out described in the importing of any description above recombinant expression carrier.The microorganism can
For yeast, concretely yeast AH109R.
The present invention also protects the application of UVALS albumen or UVALS gene or any description above method in plant breeding.
The purpose of the breeding is to cultivate the plant increased to the resistance of inhibitor of acetolactate synthetase.The plant is unifacial leaf plant
Object or dicotyledon, the dicotyledon concretely arabidopsis, such as Columbia ecotype arabidopsis.
Any description above inhibitor of acetolactate synthetase concretely chlorimuronethyl.
The present invention also protects UVALS albumen as the application of acetolactate synthestase.
The albumen for being significantly higher than existing albumen to the resistance of inhibitor of acetolactate synthetase present invention finds one is dashed forward
Variant has very inhibitor of acetolactate synthetase with resistance new variety of plant or recombinant microorganism for cultivating
High application value.
Specific embodiment
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method is unless otherwise specified conventional method.Test material as used in the following examples is unless otherwise specified certainly
What routine biochemistry reagent shop was commercially available.Quantitative test in following embodiment is respectively provided with three repeated experiments, as a result makes even
Mean value.Chlorimuronethyl used in embodiment is 98% chlorimuronethyl raw medicine, is produced by Ruize Farm-Chemicals Co., Ltd., Dalian.
The discovery of embodiment 1, UVALS albumen and UVALS gene
One, the determination of best mutation time
1, aspergillus niger TR-H is transferred on PDA slant medium, 28 DEG C of culture 3-5d.
2, the aspergillus niger spore on lower inclined plane is washed with sterile saline, pours into and sterilized 20~30 diameters 4 is housed
In the triangular flask of the bead of~5mm, 220rpm shaken cultivation 1h makes spore dispersion sufficiently, then with 4 layers of sterile lens paper mistake
Mycelia is filtered out, it is 10 that concentration, which is made,5The spore suspension of a/ml.
3, it takes 5ml spore suspension in the plate of diameter 9cm, puts a sterilized magnetic stirrer postposition magnetic stirring apparatus
On, open ware lid, away from the vertical place 30cm of ultraviolet lamp fluorescent tube (power 30W), irradiate 30 under stiring respectively, 60,90,120,150,
180,210,240,270,300 or 600s closes ultraviolet lamp, wraps culture dish with black cloth, be immediately placed in 1-2h in refrigerator, prevents
Only light reparation.
4, different irradiation times are taken respectively treated suspension, be coated on PDA culture medium plate after gradient dilution, 30
It DEG C is protected from light culture 72h, viable count is recorded, calculates the lethality under each irradiation time.
When ultraviolet irradiation time be 60s when, lethality 71%, further increase irradiation time then lethality on
Rise, upon irradiation between be 600s when, lethality 97%.Therefore, the dose,optimum of ultraviolet mutagenesis be using 30W ultraviolet lamp,
Irradiate 60s.
Two, the acquisition of aspergillus niger UVTR-H
2 spore suspensions prepared of step 1 are taken to be carried out continuously six mutagenesis, the method for each mutagenesis is as follows: by spore
Sub- suspension is placed in the plate of diameter 9cm, is put on a sterilized magnetic stirrer postposition magnetic stirring apparatus, ware lid is opened, away from purple
At the vertical 30cm of outer lamp fluorescent tube (power 30W), 60s is irradiated under stiring, ultraviolet lamp is closed, is wrapped culture dish with black cloth, is stood
It is put into 1-2h in refrigerator, prevents light reparation.
After each mutagenesis, screening the single colonie with obvious colony characteristics, (speed of growth is fast, colony colour is deep, bacterium colony is all
Side is neat), after flat lining out, 30 DEG C of inversion culture 3d, thallus is accessed in liquid czapek's medium, 28 DEG C, 150rpm vibration
Culture 3d is swung, filters out mycelium with clean gauze, measures the activity of acetolactate synthestase, selects acetolactate synthase activity
Highest single colonie carries out next mutagenesis.
One plant of new strains with genetic stability is obtained, the acetolactate synthestase of the new strains is living under parallel condition
Property is significantly higher than aspergillus niger TR-H, and by the new strains, it is named as aspergillus niger UVTR-H.
Three, the discovery of UVALS albumen and UVALS gene
1, it extracts the total serum IgE of aspergillus niger TR-H and reverse transcription is cDNA.
2, the cDNA obtained using step 1 carries out PCR amplification using the primer pair that AncDR and AncDF is formed as template.
AncDF:5'-ATGATGCCTATGAGACCTTC-3'；
AncDR:5'-TTAGAAACCGGGAACTTTC-3'.
3, the pcr amplification product for obtaining step 2 is sequenced, the results showed that the sequence 2 of pcr amplification product such as sequence table
It is shown, protein shown in the sequence 1 of polynucleotide.
Protein shown in sequence 1 by sequence table is named as UVALS albumen.The theoretical molecular weight of UVALS albumen is
75.6kDa, isoelectric point 8.0.It is UVALS gene, open reading frame such as sequence table by the unnamed gene for encoding UVALS albumen
Sequence 2 shown in.
Homologous protein (ALS1 albumen) in aspergillus niger TR-H is as shown in the sequence 3 of sequence table, encoding gene (ALS1 base
Cause) as shown in the sequence 4 of sequence table.
The functional verification of embodiment 2, UVALS albumen
One, construction recombination plasmid
1, double chain DNA molecule shown in the sequence 2 of composition sequence table.
2, the DNA molecular synthesized using step 1 carries out PCR expansion using the primer pair that AncDF1 and AncDF1 is formed as template
Increase, obtains pcr amplification product.
AncDF1:5'-GAGGATCCGTATGATGCCTATGAGACCTTC-3'；
AncDF1:5'-TCGTCGACTTAGAAACCGGGAACTTTCC-3'。
3, the pcr amplification product for taking step 2 to obtain carries out double digestion using restriction enzyme BamH I and Sal I, returns
Receive digestion products.
4, take pGBKT7 carrier (purchased from Clontech company, catalog number are as follows: 630443), using restriction enzyme
BamH I and Sal I carry out double digestion, recycle the carrier framework of about 7.3kb.
5, the digestion products that step 3 obtains are connected with the carrier framework that step 4 obtains, obtains recombinant plasmid pGBKT7-
UVALS.According to sequencing result, structure is carried out to recombination plasmid pGBKT7-UVALS and is described as follows: by the BamH of pGBKT7 carrier
Small fragment between I and Sal I restriction enzyme site replaces double chain DNA molecule shown in the sequence 5 for sequence table.The sequence of sequence table
3-2075 nucleotide and the sequence 2 of sequence table are consistent in column 5.
6, double chain DNA molecule shown in the sequence 4 of composition sequence table.
7, the DNA molecular synthesized using step 6 carries out PCR expansion using the primer pair that AncDF1 and AncDF1 is formed as template
Increase, obtains pcr amplification product.
8, the pcr amplification product for taking step 7 to obtain carries out double digestion using restriction enzyme BamH I and Sal I, returns
Receive digestion products.
9, the digestion products that step 8 obtains are connected with the carrier framework that step 4 obtains, obtains recombinant plasmid pGBKT7-
ALS1.According to sequencing result, structure is carried out to recombination plasmid pGBKT7-ALS1 and is described as follows: by the BamH I of pGBKT7 carrier
Small fragment between Sal I restriction enzyme site replaces double chain DNA molecule shown in the sequence 6 for sequence table.The sequence of sequence table
3-2075 nucleotide and the sequence 4 of sequence table are consistent in column 6.
Two, preparation and reorganization yeast
By recombinant plasmid pGBKT7-UVALS import yeast AH109R (be purchased from Clontech company, catalog number are as follows:
630444) recombination yeast, is obtained, yeast pGB-UVALS is named as.
Recombinant plasmid pGBKT7-ALS1 is imported into yeast AH109R, recombination yeast is obtained, is named as yeast pGB-
ALS1。
By pGBKT7 vector introduction yeast AH109R, recombination yeast is obtained, yeast pGB is named as.
Three, the chlorimuronethyl Resistance Identification of recombination yeast
Test yeast is respectively as follows: yeast pGB-UVALS, yeast pGB-ALS1, yeast pGB or yeast AH109R.
By on the solid YPD culture medium flat plate of test yeast streak inoculation to the chlorimuronethyl containing various concentration, 35 DEG C are stood
Then strain growth situation is observed in culture 72 hours.Chlorimuronethyl concentration be respectively set to 2000mg/L, 3000mg/L,
4000mg/L, 5000mg/L, 6000mg/L, 7000mg/L, 8000mg/L, 9000mg/L, 10g/L, each concentration are arranged five
Processing.Three control treatments for being added without chlorimuronethyl are set.
In control treatment, each equal well-grown of test strain.
Chlorimuronethyl concentration is used to be cultivated for the culture medium of 2000mg/L, faint growth (the five processing tables of yeast pGB
It is now consistent), the faint growth of yeast AH109R (five processing performances are consistent), yeast pGB-UVALS normal growth (five processing tables
It is now consistent), yeast pGB-ALS1 normal growth (five processing performances are consistent).
Chlorimuronethyl concentration is used to be cultivated for the culture medium of 3000mg/L, yeast pGB cannot be grown completely (at five
Reason performance is consistent), yeast AH109R cannot grow (five processing performances are consistent), yeast pGB-UVALS normal growth (five completely
A processing performance is consistent), yeast pGB-ALS1 normal growth (five processing performances are consistent).
Chlorimuronethyl concentration is used to be cultivated for the culture medium of 4000mg/L, yeast pGB cannot be grown completely (at five
Reason performance is consistent), yeast AH109R cannot grow (five processing performances are consistent), yeast pGB-UVALS normal growth (five completely
A processing performance is consistent), yeast pGB-ALS1 normal growth (five processing performances are consistent).
Chlorimuronethyl concentration is used to be cultivated for the culture medium of 5000mg/L, yeast pGB cannot be grown completely (at five
Reason performance is consistent), yeast AH109R cannot grow (five processing performances are consistent), yeast pGB-UVALS normal growth (five completely
A processing performance is consistent), yeast pGB-ALS1 normal growth (five processing performances are consistent).
Chlorimuronethyl concentration is used to be cultivated for the culture medium of 6000mg/L, yeast pGB cannot be grown completely (at five
Reason performance is consistent), yeast AH109R cannot grow (five processing performances are consistent), yeast pGB-UVALS normal growth (five completely
A processing performance is consistent), yeast pGB-ALS1 growth is inhibited (five processing performances are consistent) by certain.
Chlorimuronethyl concentration is used to be cultivated for the culture medium of 7000mg/L, yeast pGB cannot be grown completely (at five
Reason performance is consistent), yeast AH109R cannot grow (five processing performances are consistent), yeast pGB-UVALS normal growth (five completely
A processing performance is consistent), the faint growth of yeast pGB-ALS1 (five processing performances are consistent).
Chlorimuronethyl concentration is used to be cultivated for the culture medium of 8000mg/L, yeast pGB cannot be grown completely (at five
Reason performance is consistent), yeast AH109R cannot grow (five processing performances are consistent), yeast pGB-UVALS normal growth (five completely
A processing performance is consistent), yeast pGB-ALS1 cannot grow (five processing performances are consistent) completely.
Chlorimuronethyl concentration is used to be cultivated for the culture medium of 9000mg/L, yeast pGB cannot be grown completely (at five
Reason performance is consistent), yeast AH109R cannot grow (five processing performances are consistent), yeast pGB-UVALS normal growth (five completely
A processing performance is consistent), yeast pGB-ALS1 cannot grow (five processing performances are consistent) completely.
Chlorimuronethyl concentration is used to be cultivated for the culture medium of 10g/L, yeast pGB cannot grow (five processing completely
Performance is consistent), yeast AH109R cannot grow (five processing performances are consistent), yeast pGB-UVALS normal growth (five completely
Processing performance is consistent), yeast pGB-ALS1 cannot grow (five processing performances are consistent) completely.
The result shows that UVALS albumen can significantly improve yeast to the resistance of chlorimuronethyl, and relative to ALS1 albumen pair
The resistance of chlorimuronethyl improves 2 times or more.
The acquisition and identification of embodiment 3, genetically modified plants
One, the building of recombinant plasmid
1, double chain DNA molecule shown in the sequence 2 of composition sequence table.
2, the double chain DNA molecule obtained using step 1 is carried out as template using the primer pair that Primer1 and Primer2 is formed
PCR amplification obtains pcr amplification product, recycles the DNA fragmentation of about 2000bp.
Primer1:5'-GCTCTAGAATGATGCCTATGAGACCTTC-3'；
Primer2:5'-TCGAGCTCTTAGAAACCGGGAACTTTCC-3'。
3, the DNA fragmentation obtained with restriction enzyme Xba I and I double digestion step 2 of Sac recycles digestion products.
4, with I double digestion Super1300 carrier of restriction enzyme Xba I and Sac, the carrier of about 10000bp or so is recycled
Skeleton.Super1300 carrier: being purchased from Shanghai North Connaught biological technology CO., LTD., and article No. is addgene 0595.
5, the digestion products of step 3 are connected with the carrier framework of step 4, obtains recombinant plasmid first.According to sequencing result,
Structure is carried out to recombinant plasmid first to be described as follows: the small fragment between I restriction enzyme site of Super1300 carrier Xba I and Sac is taken
Double chain DNA molecule shown in sequence 2 on behalf of sequence table.
6, double chain DNA molecule shown in the sequence 4 of composition sequence table.
7, the double chain DNA molecule obtained using step 6 is carried out as template using the primer pair that Primer1 and Primer2 is formed
PCR amplification obtains pcr amplification product, recycles the DNA fragmentation of about 2000bp.
8, the DNA fragmentation obtained with restriction enzyme Xba I and I double digestion step 7 of Sac recycles digestion products.
9, the digestion products of step 8 are connected with the carrier framework of step 4, obtains recombinant plasmid second.According to sequencing result,
Structure is carried out to recombinant plasmid second to be described as follows: the small fragment between Super1300 carrier Xba I and Sac I is replaced for sequence
Double chain DNA molecule shown in the sequence 4 of list.
Two, genetically modified plants are cultivated
1, recombinant plasmid first is imported into agrobacterium strains GV3101, obtains recombinational agrobacterium.
2, using bud infusion method (Clough and Bent, Floral dip:a simplified method for
Agrobacterium-mediated transformation of Arabidopsis thaliana.Plant
Journal1998,16:735-743.), Columbia ecotype arabidopsis is infected with the recombinational agrobacterium that step 1 obtains, is harvested
T1For seed.T2T is shown in representative1The generation selfing seed generated and the plant grown up to by it, T3T is shown in representative2The kind that generation selfing generates
Son and the plant grown up to by it.T is screened in the MS solid medium tablets containing 50 μ g/L hygromycin1For plant and carry out T2
Generation and T3The segregation ratio in generation counts, in T3In generation, obtains transgenic arabidopsis list copy homozygous lines, takes two strains at random, names
For strain 1 and strain 2.
Three, genetically modified plants are cultivated
1, recombinant plasmid second is imported into agrobacterium strains GV3101, obtains recombinational agrobacterium.
2, using bud infusion method (Clough and Bent, Floral dip:a simplified method for
Agrobacterium-mediated transformation of Arabidopsis thaliana.Plant
Journal1998,16:735-743.), Columbia ecotype arabidopsis is infected with the recombinational agrobacterium that step 1 obtains, is harvested
T1For seed.T2T is shown in representative1The generation selfing seed generated and the plant grown up to by it, T3T is shown in representative2The kind that generation selfing generates
Son and the plant grown up to by it.T is screened in the MS solid medium tablets containing 50 μ g/L hygromycin1For plant and carry out T2
Generation and T3The segregation ratio in generation counts, in T3In generation, obtains transgenic arabidopsis list copy homozygous lines, takes two strains at random, names
For strain 3 and strain 4.
Four, Resistance detecting
Seed to be measured is respectively as follows: the T of strain 13For seed, the T of strain 23For seed, the T of strain 33For seed, strain 4
T3For seed, the seed of Columbia ecotype arabidopsis.
Every 1g chlorimuronethyl is mixed with 5ml water, obtains chlorimuronethyl solution.
1, seed to be measured is seeded in MS solid medium, continues culture 2 weeks after sprouting.
2, the plant for completing step 1 sprays chlorimuronethyl solution (spraying twice, be spaced 3 days).
3, after completing step 2, continue culture 1 week, count survival rate.
Condition of culture: 22 DEG C, (the 60 μm of ol/m of light intensity of illumination in 24 hours2/s)。
Every kind of seed to be measured carries out repeating to test three times, repeats every kind of seed to be measured in test every time and takes 200 seeds.
The average viability of strain 1 is 95%.The average viability of strain 2 is 98%.The average viability of strain 3 is
38%.The average viability of strain 4 is 42%.The average viability of Columbia ecotype arabidopsis is 0%.
The result shows that plant can be significantly improved and resisted to chlorimuronethyl UVALS gene or ALS1 gene transfered plant
Property, the significant effect of UVALS gene is better than ALS1 gene.
SEQUENCE LISTING
<110>Northeast Agricultural University
<120>inhibitor of acetolactate synthetase resistance-associated protein UVALS and its encoding gene and application
<130> GNCYX162311
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 690
<212> PRT
<213>artificial sequence
<400> 1
Met Met Pro Met Arg Pro Ser Lys Ser Ala Met Arg Ala Leu His Tyr
1 5 10 15
Gln Arg Tyr Ile Ala Ser Gly Arg Met Ser Phe Thr Thr Ala Ser Val
20 25 30
Ala Ala Thr Ala Pro His Arg Phe Ser Ala Gln Lys Arg Phe Gln Ser
35 40 45
Thr Ala Ser Ala Pro Ala Glu Asn Thr Arg Pro Ile Pro Ser Pro Ala
50 55 60
Phe Asn Gln Glu Pro His Arg Asn Glu Val Ser Pro Leu Gln Asn Arg
65 70 75 80
Gln Val Pro Glu Leu Asp Asp Ser Phe Val Gly Leu Ser Gly Gly Glu
85 90 95
Ile Phe His Glu Met Met Leu Arg Leu Gly Val Lys His Val Phe Gly
100 105 110
Tyr Pro Gly Gly Ala Ile Leu Pro Val Phe Asp Ala Ile Tyr Asn Ser
115 120 125
Lys His Phe Asp Phe Val Leu Pro Arg His Glu Gln Gly Ala Gly His
130 135 140
Met Ala Glu Gly Tyr Ala Arg Ala Ser Gly Lys Pro Gly Val Val Leu
145 150 155 160
Val Thr Ser Gly Pro Gly Ala Thr Asn Val Ile Thr Pro Met Gln Asp
165 170 175
Ala Leu Ser Asp Gly Thr Pro Met Val Val Phe Cys Gly Gln Val Pro
180 185 190
Thr Ser Leu Ile Gly Thr Asp Ser Phe Gln Glu Ala Asp Val Ile Gly
195 200 205
Ile Ser Arg Ala Cys Thr Lys Trp Asn Val Met Val Lys Ser Val Ala
210 215 220
Glu Leu Pro Arg Arg Ile Gln Glu Ala Phe Glu Ile Ala Thr Ser Gly
225 230 235 240
Arg Pro Gly Pro Val Leu Val Asp Leu Pro Lys Asp Ile Thr Ala Gly
245 250 255
Ile Leu Arg Lys Pro Ile Pro Met Asn Ser Thr Leu Pro Ser Leu Pro
260 265 270
Ser Ala Ala Thr Met Ala Ala Arg Glu Leu Ser Met Gln Gln Leu Lys
275 280 285
Gly Thr Ile Asn Arg Val Ala Arg Leu Val Asn Ile Ser Lys Lys Pro
290 295 300
Ile Leu Tyr Val Gly Gln Gly Leu Leu Ala Arg Pro Asp Gly Pro Gln
305 310 315 320
Ile Leu Lys Glu Leu Ala Asp Lys Ala Cys Ile Pro Val Thr Thr Thr
325 330 335
Leu Gln Gly Leu Gly Gly Phe Asp Glu Thr Asp Pro Lys Ala Leu His
340 345 350
Met Leu Gly Met His Gly Ser Ala Tyr Ala Asn Met Ala Met Gln Glu
355 360 365
Ala Asp Leu Ile Ile Ala Ile Gly Ala Arg Phe Asp Asp Arg Val Thr
370 375 380
Gly Asn Ile Ser Lys Phe Ala Pro Gln Ala Lys Leu Ala Ala Ser Glu
385 390 395 400
Asn Arg Gly Gly Ile Val His Phe Glu Ile Met Pro Lys Asn Ile Asn
405 410 415
Lys Val Val Gln Ala Asn Glu Ala Val Glu Gly Asp Cys Ala Glu Asn
420 425 430
Ile Arg Leu Leu Leu Pro His Ala Glu Ala Val Ala Glu Arg Lys Glu
435 440 445
Trp Phe Asp Gln Ile Asn Asp Trp Lys Ala Arg Phe Pro Phe Ser Leu
450 455 460
Tyr Glu Arg Glu Thr Ala Glu Gly Pro Ile Lys Pro Gln Ala Val Ile
465 470 475 480
Glu Lys Leu Ser Asp Leu Thr Ala His Met Lys Asp Arg Thr Ala Ile
485 490 495
Thr Thr Gly Val Gly Gln His Gln Met Trp Ala Ala Gln His Phe Arg
500 505 510
Trp Arg His Pro Arg Thr Met Ile Thr Ser Gly Gly Leu Gly Thr Met
515 520 525
Gly Tyr Gly Leu Pro Ala Ala Ile Gly Ala Lys Val Ala Arg Pro Asp
530 535 540
Ala Leu Val Val Asp Ile Asp Gly Asp Ala Ser Phe Asn Met Thr Leu
545 550 555 560
Thr Glu Leu Thr Thr Ala Ala Gln Phe Asn Ile Gly Val Lys Val Leu
565 570 575
Leu Leu Asn Asn Glu Glu Gln Gly Met Val Thr Gln Trp Gln Asn Leu
580 585 590
Phe Tyr Glu Asp Arg Tyr Ser His Thr His Gln Lys Asn Pro Asp Phe
595 600 605
Val Pro Met Ala Gln Ala Met Gly Val Ala Ala Asp Arg Cys Thr Lys
610 615 620
Pro Ser Glu Val Glu Glu Lys Leu Lys Trp Leu Ile Glu Gln Asp Gly
625 630 635 640
Pro Ala Leu Pro Glu Val Phe Thr Asp Arg Lys Val Pro Val Leu Pro
645 650 655
Met Val Pro Ala Gly Cys Ala Leu His Glu Phe Phe Val Tyr Asp Glu
660 665 670
Ala Lys Glu Lys Glu Arg Lys Ala Leu Met Lys Lys Arg Lys Val Pro
675 680 685
Gly Phe
690
<210> 2
<211> 2073
<212> DNA
<213>artificial sequence
<400> 2
atgatgccta tgagaccttc gaaaagcgcc atgcgcgctc tgcattacca gaggtacatt 60
gcctctggca gaatgagttt tactactgct tccgtcgctg cgaccgcgcc ccaccgcttt 120
tcagctcaga agagattcca gagcaccgca agtgccccgg ctgaaaacac gagaccgatc 180
cccagccctg ccttcaacca ggaacctcac cgcaatgagg tctcgccact gcaaaatcgc 240
caggtccccg aactggacga ctcttttgtc ggactcagtg gaggagagat ctttcatgag 300
atgatgctgc gtcttggcgt caagcatgtc ttcggttacc ctggaggtgc cattcttccc 360
gttttcgatg ccatctacaa ctccaaacac ttcgacttcg tcctcccgag acatgaacag 420
ggcgccggac acatggccga aggctacgcc cgtgcctctg gaaagcccgg tgtcgtcctc 480
gtcacctccg gccctggagc caccaacgtt atcaccccca tgcaggatgc cctttcggac 540
ggtactccca tggtcgtctt ctgcggtcag gtgcccacca gcctgatcgg taccgactct 600
ttccaggaag ccgatgttat cggtatctcc cgtgcttgca ccaagtggaa cgtcatggtc 660
aagtccgtcg ctgaactccc ccgtcgcatc caggaggctt tcgaaatcgc caccagcggc 720
cgccccggac ctgtcctcgt cgatctgccc aaggatatca ccgccggtat cctccgtaaa 780
cccatcccga tgaacagcac cctgccctcc ctccccagcg ctgcgaccat ggctgcccgt 840
gaactgagca tgcagcagct caagggtacc atcaaccgtg tggctcgcct tgtgaacatc 900
tccaagaagc ccatcctgta tgtgggtcag ggtctccttg ctcgccccga tggcccccag 960
atcttgaagg agctggccga caaggcttgc attccggtca ccacgaccct ccagggtctg 1020
ggtggatttg acgagacgga ccccaaggcc ctgcacatgc tgggcatgca cggatctgcc 1080
tatgccaaca tggccatgca ggaggctgat ctcatcatcg ctatcggtgc tcgcttcgat 1140
gaccgtgtga ccggtaacat ctccaagttc gcccctcagg ccaagcttgc tgcctcggag 1200
aaccgtggtg gtatcgtcca cttcgaaatc atgcccaaga acatcaacaa ggttgtccag 1260
gccaacgagg ctgttgaggg tgactgtgct gagaacatcc gtctcctcct gccccacgcc 1320
gaggctgttg ctgagcgtaa ggagtggttc gaccagatca acgactggaa ggctcggttc 1380
cccttctccc tctatgagag ggagactgct gagggaccca tcaagcccca ggctgtcatt 1440
gagaaactca gcgatctcac tgctcacatg aaggaccgta ctgccatcac taccggtgtc 1500
ggtcagcacc agatgtgggc tgctcagcac ttccgctggc gccacccccg taccatgatc 1560
acctctggtg gtcttggaac tatgggatac ggtctccccg ctgctatcgg tgccaaggtt 1620
gctcgccctg acgctcttgt cgtcgatatc gacggtgatg cctcgttcaa catgaccctg 1680
accgagctca ccactgctgc tcagttcaac attggtgtca aggttctcct cctgaacaac 1740
gaggaacagg gtatggtgac gcaatggcag aacctgttct acgaggaccg ctactcgcac 1800
actcaccaga agaaccccga ctttgtcccg atggcccagg ccatgggtgt tgccgcggac 1860
cgctgcacca agccctcgga ggttgaggag aagctgaagt ggctgatcga gcaggacggc 1920
cctgcccttc cggaagtgtt cactgatcgc aaggtgcctg tgctccccat ggtgcccgct 1980
ggctgtgccc tgcacgaatt ctttgtttat gacgaagcca aggagaagga gcgcaaggct 2040
ctgatgaaga agcggaaagt tcccggtttc taa 2073
<210> 3
<211> 690
<212> PRT
<213>artificial sequence
<400> 3
Met Met Pro Met Arg Pro Ser Lys Ser Ala Met Arg Ala Leu His Tyr
1 5 10 15
Gln Arg Tyr Ile Ala Ser Gly Arg Met Ser Phe Thr Thr Ala Ser Val
20 25 30
Ala Ala Thr Ala Pro His Arg Phe Ser Ala Gln Lys Arg Phe Gln Ser
35 40 45
Thr Ala Ser Ala Pro Ala Glu Asn Thr Arg Pro Ile Pro Ser Pro Ala
50 55 60
Phe Asn Gln Glu Pro His Arg Asn Glu Val Ser Pro Leu Gln Asn Arg
65 70 75 80
Gln Val Pro Glu Leu Asp Asp Ser Phe Val Gly Leu Ser Gly Gly Glu
85 90 95
Ile Phe His Glu Met Met Leu Arg Leu Gly Val Lys His Val Phe Gly
100 105 110
Tyr Pro Gly Gly Ala Ile Leu Pro Val Phe Asp Ala Ile Tyr Asn Ser
115 120 125
Lys His Phe Asp Phe Val Leu Pro Arg His Glu Gln Gly Ala Gly His
130 135 140
Met Ala Glu Gly Tyr Ala Arg Ala Ser Gly Lys Pro Gly Val Val Leu
145 150 155 160
Val Thr Ser Gly Pro Gly Ala Thr Asn Val Ile Thr Pro Met Gln Asp
165 170 175
Ala Leu Ser Asp Gly Thr Pro Met Val Val Phe Cys Gly Gln Val Pro
180 185 190
Thr Ser Leu Ile Gly Thr Asp Ser Phe Gln Glu Ala Asp Val Ile Gly
195 200 205
Ile Ser Arg Ala Cys Thr Lys Trp Asn Val Met Val Lys Ser Val Ala
210 215 220
Glu Leu Pro Arg Arg Ile Gln Glu Ala Phe Glu Ile Ala Thr Ser Gly
225 230 235 240
Arg Pro Gly Pro Val Leu Val Asp Leu Pro Lys Asp Ile Thr Ala Gly
245 250 255
Ile Leu Arg Lys Pro Ile Pro Met Asn Ser Thr Leu Pro Ser Leu Pro
260 265 270
Ser Ala Ala Thr Met Ala Ala Arg Glu Leu Ser Met Gln Gln Leu Lys
275 280 285
Gly Thr Ile Asn Arg Val Ala Arg Leu Val Asn Ile Ser Lys Lys Pro
290 295 300
Ile Leu Tyr Val Gly Gln Gly Leu Leu Ala Arg Pro Asp Gly Pro Gln
305 310 315 320
Ile Leu Lys Glu Leu Ala Asp Lys Ala Cys Ile Pro Val Thr Thr Thr
325 330 335
Leu Gln Gly Leu Gly Gly Phe Asp Glu Thr Asp Pro Lys Ala Leu His
340 345 350
Met Leu Gly Met His Gly Ser Ala Tyr Ala Asn Met Ala Met Gln Glu
355 360 365
Ala Asp Leu Ile Ile Ala Ile Gly Ala Arg Phe Asp Asp Arg Val Thr
370 375 380
Gly Asn Ile Ser Lys Phe Ala Pro Gln Ala Lys Leu Ala Ala Ser Glu
385 390 395 400
Asn Arg Gly Gly Ile Val His Phe Glu Ile Met Pro Lys Asn Ile Asn
405 410 415
Lys Val Val Gln Ala Asn Glu Ala Val Glu Gly Asp Cys Ala Glu Asn
420 425 430
Ile Arg Leu Leu Pro Pro His Val Glu Ala Val Ala Glu Arg Lys Glu
435 440 445
Trp Phe Asp Gln Ile Asn Asp Trp Lys Ala Arg Phe Pro Phe Ser Leu
450 455 460
Tyr Glu Arg Glu Thr Ala Glu Gly Pro Ile Lys Pro Gln Ala Val Ile
465 470 475 480
Glu Lys Leu Ser Asp Leu Thr Ala His Met Lys Asp Arg Thr Val Ile
485 490 495
Thr Thr Gly Val Gly Gln His Gln Met Trp Ala Ala Gln His Phe Arg
500 505 510
Trp Arg His Pro Arg Thr Met Ile Thr Ser Gly Gly Leu Gly Thr Met
515 520 525
Gly Tyr Gly Leu Pro Ala Ala Ile Gly Ala Lys Val Ala Arg Pro Asp
530 535 540
Ala Leu Val Val Asp Ile Asp Gly Asp Ala Ser Phe Asn Met Thr Leu
545 550 555 560
Thr Glu Leu Thr Thr Ala Ala Gln Phe Asn Ile Gly Val Lys Val Leu
565 570 575
Leu Leu Asn Asn Glu Glu Gln Gly Met Val Thr Gln Trp Gln Asn Leu
580 585 590
Phe Tyr Glu Asp Arg Tyr Ser His Thr His Gln Lys Asn Pro Asp Phe
595 600 605
Val Pro Met Ala Gln Ala Met Gly Val Ala Ala Asp Arg Cys Thr Lys
610 615 620
Pro Ser Glu Val Glu Glu Lys Leu Lys Trp Leu Ile Glu Gln Asp Gly
625 630 635 640
Pro Ala Leu Leu Glu Val Phe Thr Asp Arg Lys Val Pro Val Leu Pro
645 650 655
Met Val Pro Ala Gly Cys Ala Leu His Glu Phe Leu Val Tyr Asp Glu
660 665 670
Ala Lys Glu Lys Glu Arg Lys Ala Leu Met Lys Lys Arg Lys Val Pro
675 680 685
Gly Phe
690
<210> 4
<211> 2073
<212> DNA
<213>artificial sequence
<400> 4
atgatgccta tgagaccttc gaaaagcgcc atgcgcgctc tgcattacca gaggtacatt 60
gcctctggca gaatgagttt tactactgct tccgtcgctg cgaccgcgcc ccaccgcttt 120
tcagctcaga agagattcca gagcaccgca agtgccccgg ctgaaaacac gagaccgatc 180
cccagccctg ccttcaacca ggaacctcac cgcaatgagg tctcgccact gcaaaatcgc 240
caggtccccg aactggacga ctcttttgtc ggactcagtg gaggagagat ctttcatgag 300
atgatgctgc gtcttggcgt caagcatgtc ttcggttacc ctggaggtgc cattcttccc 360
gttttcgatg ccatctacaa ctccaaacac ttcgacttcg tcctcccgag acatgaacag 420
ggcgccggac acatggccga aggctacgcc cgtgcctctg gaaagcccgg tgtcgtcctc 480
gtcacctccg gccctggagc caccaacgtt atcaccccca tgcaggatgc cctttcggac 540
ggtactccca tggtcgtctt ctgcggtcag gtgcccacca gcctgatcgg taccgactct 600
ttccaggaag ccgatgttat cggtatctcc cgtgcttgca ccaagtggaa cgtcatggtc 660
aagtccgtcg ctgaactccc ccgtcgcatc caggaggctt tcgaaatcgc caccagcggc 720
cgccccggac ctgtcctcgt cgatctgccc aaggatatca ccgccggtat cctccgtaaa 780
cccatcccga tgaacagcac cctgccctcc ctccccagcg ctgcgaccat ggctgcccgt 840
gaactgagca tgcagcagct caagggtacc atcaaccgtg tggctcgcct tgtgaacatc 900
tccaagaagc ccatcctgta tgtgggtcag ggtctccttg ctcgccccga tggcccccag 960
atcttgaagg agctggccga caaggcttgc attccggtca ccacgaccct ccagggtctg 1020
ggtggatttg acgagacgga ccccaaggcc ctgcacatgc tgggcatgca cggatctgcc 1080
tatgccaaca tggccatgca ggaggctgat ctcatcatcg ctatcggtgc tcgcttcgat 1140
gaccgtgtga ccggtaacat ctccaagttc gcccctcagg ccaagcttgc tgcctcggag 1200
aaccgtggtg gtatcgtcca cttcgaaatc atgcccaaga acatcaacaa ggttgtccag 1260
gccaacgagg ctgttgaggg tgactgtgct gagaacatcc gtctcctccc gccccacgtc 1320
gaggctgttg ctgagcgtaa ggagtggttc gaccagatca acgactggaa ggctcggttc 1380
cccttctccc tctatgagag ggagactgct gagggaccca tcaagcccca ggctgtcatt 1440
gagaaactca gcgatctcac tgctcacatg aaggaccgta ctgtcatcac taccggtgtc 1500
ggtcagcacc agatgtgggc tgctcagcac ttccgctggc gccacccccg taccatgatc 1560
acctctggtg gtcttggaac tatgggatac ggtctccccg ctgctatcgg tgccaaggtt 1620
gctcgccctg acgctcttgt cgtcgatatc gacggtgatg cctcgttcaa catgaccctg 1680
accgagctca ccactgctgc tcagttcaac attggtgtca aggttctcct cctgaacaac 1740
gaggaacagg gtatggtgac gcaatggcag aacctgttct acgaggaccg ctactcgcac 1800
actcaccaga agaaccccga ctttgtcccg atggcccagg ccatgggtgt tgccgcggac 1860
cgctgcacca agccctcgga ggttgaggag aagctgaagt ggctgatcga gcaggacggc 1920
cctgcccttc tggaagtgtt cactgatcgc aaggtgcctg tgctccccat ggtgcccgct 1980
ggctgtgccc tgcacgaatt ccttgtttat gacgaagcca aggagaagga gcgcaaggct 2040
ctgatgaaga agcggaaagt tcccggtttc taa 2073
<210> 5
<211> 2075
<212> DNA
<213>artificial sequence
<400> 5
gtatgatgcc tatgagacct tcgaaaagcg ccatgcgcgc tctgcattac cagaggtaca 60
ttgcctctgg cagaatgagt tttactactg cttccgtcgc tgcgaccgcg ccccaccgct 120
tttcagctca gaagagattc cagagcaccg caagtgcccc ggctgaaaac acgagaccga 180
tccccagccc tgccttcaac caggaacctc accgcaatga ggtctcgcca ctgcaaaatc 240
gccaggtccc cgaactggac gactcttttg tcggactcag tggaggagag atctttcatg 300
agatgatgct gcgtcttggc gtcaagcatg tcttcggtta ccctggaggt gccattcttc 360
ccgttttcga tgccatctac aactccaaac acttcgactt cgtcctcccg agacatgaac 420
agggcgccgg acacatggcc gaaggctacg cccgtgcctc tggaaagccc ggtgtcgtcc 480
tcgtcacctc cggccctgga gccaccaacg ttatcacccc catgcaggat gccctttcgg 540
acggtactcc catggtcgtc ttctgcggtc aggtgcccac cagcctgatc ggtaccgact 600
ctttccagga agccgatgtt atcggtatct cccgtgcttg caccaagtgg aacgtcatgg 660
tcaagtccgt cgctgaactc ccccgtcgca tccaggaggc tttcgaaatc gccaccagcg 720
gccgccccgg acctgtcctc gtcgatctgc ccaaggatat caccgccggt atcctccgta 780
aacccatccc gatgaacagc accctgccct ccctccccag cgctgcgacc atggctgccc 840
gtgaactgag catgcagcag ctcaagggta ccatcaaccg tgtggctcgc cttgtgaaca 900
tctccaagaa gcccatcctg tatgtgggtc agggtctcct tgctcgcccc gatggccccc 960
agatcttgaa ggagctggcc gacaaggctt gcattccggt caccacgacc ctccagggtc 1020
tgggtggatt tgacgagacg gaccccaagg ccctgcacat gctgggcatg cacggatctg 1080
cctatgccaa catggccatg caggaggctg atctcatcat cgctatcggt gctcgcttcg 1140
atgaccgtgt gaccggtaac atctccaagt tcgcccctca ggccaagctt gctgcctcgg 1200
agaaccgtgg tggtatcgtc cacttcgaaa tcatgcccaa gaacatcaac aaggttgtcc 1260
aggccaacga ggctgttgag ggtgactgtg ctgagaacat ccgtctcctc ctgccccacg 1320
ccgaggctgt tgctgagcgt aaggagtggt tcgaccagat caacgactgg aaggctcggt 1380
tccccttctc cctctatgag agggagactg ctgagggacc catcaagccc caggctgtca 1440
ttgagaaact cagcgatctc actgctcaca tgaaggaccg tactgccatc actaccggtg 1500
tcggtcagca ccagatgtgg gctgctcagc acttccgctg gcgccacccc cgtaccatga 1560
tcacctctgg tggtcttgga actatgggat acggtctccc cgctgctatc ggtgccaagg 1620
ttgctcgccc tgacgctctt gtcgtcgata tcgacggtga tgcctcgttc aacatgaccc 1680
tgaccgagct caccactgct gctcagttca acattggtgt caaggttctc ctcctgaaca 1740
acgaggaaca gggtatggtg acgcaatggc agaacctgtt ctacgaggac cgctactcgc 1800
acactcacca gaagaacccc gactttgtcc cgatggccca ggccatgggt gttgccgcgg 1860
accgctgcac caagccctcg gaggttgagg agaagctgaa gtggctgatc gagcaggacg 1920
gccctgccct tccggaagtg ttcactgatc gcaaggtgcc tgtgctcccc atggtgcccg 1980
ctggctgtgc cctgcacgaa ttctttgttt atgacgaagc caaggagaag gagcgcaagg 2040
ctctgatgaa gaagcggaaa gttcccggtt tctaa 2075
<210> 6
<211> 2075
<212> DNA
<213>artificial sequence
<400> 6
gtatgatgcc tatgagacct tcgaaaagcg ccatgcgcgc tctgcattac cagaggtaca 60
ttgcctctgg cagaatgagt tttactactg cttccgtcgc tgcgaccgcg ccccaccgct 120
tttcagctca gaagagattc cagagcaccg caagtgcccc ggctgaaaac acgagaccga 180
tccccagccc tgccttcaac caggaacctc accgcaatga ggtctcgcca ctgcaaaatc 240
gccaggtccc cgaactggac gactcttttg tcggactcag tggaggagag atctttcatg 300
agatgatgct gcgtcttggc gtcaagcatg tcttcggtta ccctggaggt gccattcttc 360
ccgttttcga tgccatctac aactccaaac acttcgactt cgtcctcccg agacatgaac 420
agggcgccgg acacatggcc gaaggctacg cccgtgcctc tggaaagccc ggtgtcgtcc 480
tcgtcacctc cggccctgga gccaccaacg ttatcacccc catgcaggat gccctttcgg 540
acggtactcc catggtcgtc ttctgcggtc aggtgcccac cagcctgatc ggtaccgact 600
ctttccagga agccgatgtt atcggtatct cccgtgcttg caccaagtgg aacgtcatgg 660
tcaagtccgt cgctgaactc ccccgtcgca tccaggaggc tttcgaaatc gccaccagcg 720
gccgccccgg acctgtcctc gtcgatctgc ccaaggatat caccgccggt atcctccgta 780
aacccatccc gatgaacagc accctgccct ccctccccag cgctgcgacc atggctgccc 840
gtgaactgag catgcagcag ctcaagggta ccatcaaccg tgtggctcgc cttgtgaaca 900
tctccaagaa gcccatcctg tatgtgggtc agggtctcct tgctcgcccc gatggccccc 960
agatcttgaa ggagctggcc gacaaggctt gcattccggt caccacgacc ctccagggtc 1020
tgggtggatt tgacgagacg gaccccaagg ccctgcacat gctgggcatg cacggatctg 1080
cctatgccaa catggccatg caggaggctg atctcatcat cgctatcggt gctcgcttcg 1140
atgaccgtgt gaccggtaac atctccaagt tcgcccctca ggccaagctt gctgcctcgg 1200
agaaccgtgg tggtatcgtc cacttcgaaa tcatgcccaa gaacatcaac aaggttgtcc 1260
aggccaacga ggctgttgag ggtgactgtg ctgagaacat ccgtctcctc ccgccccacg 1320
tcgaggctgt tgctgagcgt aaggagtggt tcgaccagat caacgactgg aaggctcggt 1380
tccccttctc cctctatgag agggagactg ctgagggacc catcaagccc caggctgtca 1440
ttgagaaact cagcgatctc actgctcaca tgaaggaccg tactgtcatc actaccggtg 1500
tcggtcagca ccagatgtgg gctgctcagc acttccgctg gcgccacccc cgtaccatga 1560
tcacctctgg tggtcttgga actatgggat acggtctccc cgctgctatc ggtgccaagg 1620
ttgctcgccc tgacgctctt gtcgtcgata tcgacggtga tgcctcgttc aacatgaccc 1680
tgaccgagct caccactgct gctcagttca acattggtgt caaggttctc ctcctgaaca 1740
acgaggaaca gggtatggtg acgcaatggc agaacctgtt ctacgaggac cgctactcgc 1800
acactcacca gaagaacccc gactttgtcc cgatggccca ggccatgggt gttgccgcgg 1860
accgctgcac caagccctcg gaggttgagg agaagctgaa gtggctgatc gagcaggacg 1920
gccctgccct tctggaagtg ttcactgatc gcaaggtgcc tgtgctcccc atggtgcccg 1980
ctggctgtgc cctgcacgaa ttccttgttt atgacgaagc caaggagaag gagcgcaagg 2040
ctctgatgaa gaagcggaaa gttcccggtt tctaa 2075
Claims (10)
1. a kind of protein, the protein that the amino acid sequence shown in sequence 1 in sequence table forms.
2. encoding the gene of protein described in claim 1.
3. gene as claimed in claim 2, it is characterised in that: the gene is DNA molecular shown in sequence 2 in sequence table.
4. recombinant expression carrier, expression cassette or recombinant bacterium containing gene described in Claims 2 or 3.
5. the application of protein described in claim 1, for as follows (c1) or/and (c2):
(c1) plant is promoted to increase the resistance of inhibitor of acetolactate synthetase；
(c2) microorganism is promoted to increase the resistance of inhibitor of acetolactate synthetase；
The plant is arabidopsis；
The microorganism is yeast；
The inhibitor of acetolactate synthetase is chlorimuronethyl.
6. gene described in Claims 2 or 3 is cultivating the application in genetically modified plants；The genetically modified plants are to acetolactic acid
The increased plant of the resistance of synthetase inhibitors；
The plant is arabidopsis；
The inhibitor of acetolactate synthetase is chlorimuronethyl.
7. a kind of method for cultivating genetically modified plants, is turned in channel genes purpose plant described in Claims 2 or 3
Gene plant；The genetically modified plants are higher than the purpose plant to the resistance of inhibitor of acetolactate synthetase；
The plant is arabidopsis；
The inhibitor of acetolactate synthetase is chlorimuronethyl.
8. a kind of method for cultivating recombinant microorganism, is obtained in channel genes purpose microorganism described in Claims 2 or 3
Recombinant microorganism；The recombinant microorganism is higher than the purpose microorganism to the resistance of inhibitor of acetolactate synthetase；
The microorganism is yeast；
The inhibitor of acetolactate synthetase is chlorimuronethyl.
9. protein described in claim 1, or, gene described in Claims 2 or 3, or, claim 7 the method, in plant
Application in breeding.
10. application of the protein described in claim 1 as acetolactate synthestase.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611191772.3A CN106520720B (en) | 2016-12-21 | 2016-12-21 | Inhibitor of acetolactate synthetase resistance-associated protein UVALS and its encoding gene and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611191772.3A CN106520720B (en) | 2016-12-21 | 2016-12-21 | Inhibitor of acetolactate synthetase resistance-associated protein UVALS and its encoding gene and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106520720A CN106520720A (en) | 2017-03-22 |
| CN106520720B true CN106520720B (en) | 2019-09-06 |
Family
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Non-Patent Citations (4)
| Title |
|---|
| acetolactate synthase, large subunit [Aspergillus kawachii IFO 4308];Futagami T.，等;《GenBank: GAA85002.1》;20111129;第1页 |
| Aspergillus niger CBS 513.88 acetolactate synthase catalytic subunit, mRNA;匿名;《NCBI Reference Sequence： XM_001397691.2》;20110303;第1页 |
| 乙酰乳酸合成酶及ALS 基因研究概述;任洪雷;《中国农学通报》;20160915;第32卷(第26期);37-42 |
| 抗氯嘧磺隆黑曲霉乙酰乳酸合成酶(ALS)酶学特性研究;张洪岩 等;《中国油料作物学报》;20101231;第32卷(第4期);563-566 |
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