CN104164441A - Three glufosinate-resistant rice cytoplasm type glutamine synthetase mutants - Google Patents
Three glufosinate-resistant rice cytoplasm type glutamine synthetase mutants Download PDFInfo
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Abstract
The invention relates to glutamine synthetases with substantially enhanced glufosinate resistance. The glutamine synthetases with substantially enhanced glufosinate resistance are mutants obtained through in vitro directed molecular evolution of wild rice cytoplasm type glutamine synthetase. The numbers, the amino acid sequences and corresponding amino acid mutation sites of the mutants are OsGS1m1, SEQIDNo2 and G245S/G318V; OsGS1m2, SEQIDNo4 and N54S/S115F/G245S; and OsGS1m3, SEQIDNo6 and V200A/T293A/R295K respectively. The glufosinate enzyme activity inhibition constants Ki of the mutants OsGS1m1, OsGS1m2 and OsGS1m3 are 43.7, 19 and 87.5 times higher than the resistance of the wild glutamine glutamine synthetase. Verification of the functions of the three mutation genes in Saccharomyces cerevisiae further proves that the expression in the three genes can improve the glufosinate tolerance of the Saccharomyces cerevisiae from 10mM to above 100mM. The expression mode of the above yeast is very similar to the expression mode of a plant, so the verification result of the functions in the yeast proves that the three mutants have a very good application potential in the cultivation of glufosinate-resistant plants.
Description
Technical field
The invention belongs to biological technical field, be specifically related to the mutator gene that one group of wild-type paddy rice glutamine synthetase by synthetic obtains through DNA reorganization (DNA shuffling) technology, the glutamine synthetase of this group genes encoding significantly strengthens the resistance of careless fourth phosphine.Gene described in this invention can be used for building the expression vector of conversion of plant.
Background technology
The anti-careless fourth phosphine crop of transgenosis is the second Resistant Herbicide Crops that is only second to glyphosate resistant crops, and business-like resistance to careless fourth phosphine crop has corn, rape, beet, cotton etc.In 4,000,000 hectares of rapes of calendar year 2001 Canada's plantation, the 80%th, antiweed kind, wherein resistance glyphosate rape accounts for 47%, and anti-careless fourth phosphine rape accounts for l3%.The herbicide principle of grass fourth phosphine is that it can suppress glutamine synthetase (Glutamine synthetase, GS), makes ammonia run-up in plant materials, and photosynthesis and photorespiration stop, and chloroplast(id) is impaired, thereby causes weeds death.Conventional crop does not have the ability of the careless fourth phosphine of degraded, if directly contact, careless fourth phosphine can kill conventional crop.Resistance to careless fourth phosphine crop (trade(brand)name is Liberty Link crop) contains and derives from microorganism
baror
patgene.The careless fourth phosphinothricin acetyl transferring enzyme (PAT) of these two genes encodings can be combined with careless fourth phosphine free amine group by catalysis acetyl-CoA, the careless fourth phosphine that degraded is absorbed by plants, thereby eliminate, careless fourth phosphine suppresses glutamine synthetase and the interference of plant ammonia metabolism, gives the patience of plant to careless fourth phosphine.
bargene come from moist streptomycete (
s. hygroscopicus), and
patgene come from green color-producing streptomycete (
s. Viridochromogenes).Although two gene source differences, they are very similar.Why resistance to careless fourth phosphine crop can resist careless fourth phosphine to be because this crop has the ability of decomposing careless fourth phosphine, that is to say, careless fourth phosphine was just divided and takes off by careless fourth phosphinothricin acetyl transferring enzyme before contact glutamine synthetase.In order to improve
bar(
pat) gene is in the expression of plant, great majority are produced the upper genes that utilize and have all been passed through the transformation of codon preference, but aminoacid sequence does not change.Although mouse feeds malicious test-results and shows that careless fourth phosphinothricin acetyl transferring enzyme can thoroughly decompose in stomach and enteron aisle, there will not be potential harm in edible process.But, because this gene source is in microorganism, instead of derive from farm crop itself, all can cause in varying degrees human consumer's conflict psychology.
Because glutamine synthetase is the target enzyme of careless fourth phosphine, although Bar energy fast degradation grass fourth phosphine, but careless fourth phosphine was difficult to thoroughly by its decomposition before contact glutamine synthetase, because a lot of glutamine synthetases are distributed on cytolemma, therefore careless fourth phosphine is turning while applying on Bar gene farm crop, can disturb to some extent the nitrogen metabolism of plant, affect the normal g and D of plant simultaneously.The cultivation of resistance glyphosate weedicide farm crop is at present used insensitive to weedicide mostly
ePSPSgene.But there is not yet research report or patent to the insensitive glutamine synthetase gene of careless fourth phosphine and commercial applications thereof, infer that possible reason is that nature lacks glyphosate lytic enzyme, bar has very strong decomposition effect to careless fourth phosphine.In plant, overexpression wild-type glutamine synthetase can only injure by partial rcsponse grass fourth phosphine, as the wild-type that pea is originated
gSgene transformation wheat, transfer-gen plant can be alleviated the injury of careless fourth phosphine, but patience degree is not enough to commercial applications.
Summary of the invention
The object of the present invention is to provide rice cytoplasmic type glutamine synthetase mutator gene of three careless fourth phosphine resistances raisings and preparation method thereof, the nucleotide sequence of described mutator gene is respectively as shown in SEQ ID No 1, SEQ ID No3, SEQ ID No 5; The aminoacid sequence of described mutator gene is respectively as shown in SEQ ID No 2, SEQ ID No4, SEQ ID No 6; Its corresponding amino acid mutation site is respectively: G245S/G318V, N54S/S115F/G245S, V200A/T293A/R295K.The glutamine synthetase of these three mutator gene codings significantly strengthens the resistance of careless fourth phosphine.The checking of the expression by described mutator gene in yeast saccharomyces cerevisiae and careless fourth phosphine tolerance, shows to reorganize gene and has structure plant expression vector, obtains the application potential of resistance to careless fourth phosphine plant.
For achieving the above object, the present invention mainly adopts following technical scheme:
According to known rice cytoplasmic type glutamine synthetase gene (
osGS1, Genbank accession number AB037595) be basis, synthetic this gene be building up to coli expression carrier pYM4807, further adopt DNA shuffling technology to set up random mutation library, and transform deficient strain JW3841-1, by the cultivation on different concns grass fourth phosphine (20-200 mM) M9 solid medium, screening obtains the mutant that careless fourth phosphine resistance is improved.Through sequencing, obtain Nucleotide and aminoacid sequence as shown in SEQ ID No 1 ~ 6.The numbering of mutant and corresponding amino acid mutation site are respectively: OsGS1m1, G245S/G318V; OsGS1m2, N54S/S115F/G245S; OsGS1m3, V200A/T293A/R295K.Contrasting careless fourth phosphine lives and suppresses constant enzyme
k i, mutant OsGS1m1, OsGS1m2, OsGS1m3 have improved 43.7,19,87.5 times than the resistance of wild-type glutamine synthetase respectively.By this functional verification of three mutator genes in yeast saccharomyces cerevisiae, further prove that the expression in these three genes can improve the tolerance (from 10 mMs bring up to 100 mMs more than) of yeast saccharomyces cerevisiae to careless fourth phosphine.Because yeast (
saccharomyces cerevisiae) not only there is prokaryotic system growth and soon, easily cultivate and be easy to the advantages such as genetic manipulation, and there is the characteristic of typical eukaryotic system, there is the processes such as translation post-treatment such as glycosylation, disulfide linkage formation and protein folding, its expression pattern and expression of plants pattern are very approaching, are having good application potential so the functional verification result in yeast shows three mutant aspect the anti-careless fourth phosphine plant of cultivation.
Compared with prior art, creativeness of the present invention is:
Utilize growth soon, easily to cultivate and be easy to the mutant that the prokaryotic system screening of genetic manipulation improves careless fourth phosphine resistance, having avoided the trouble brought because of plant-growth excessive cycle, having improved screening efficiency; The present invention simultaneously also adopts the yeast saccharomyces cerevisiae approaching with expression of plants pattern to verify the function of mutant.
Beneficial effect
The mutator gene that the resistance of careless fourth phosphine is significantly strengthened provided by the invention, has the expression vector for building conversion of plant, cultivates the application potential of anti-careless fourth phosphine genetically modified crops.
Brief description of the drawings
Fig. 1 transforms the intestinal bacteria of sudden change and OsGS1 in the comparison of the growing state of different concns grass fourth phosphine M9 solid medium.The concentration of grass fourth phosphine is respectively 0,10,25,75,100,200 mM.
Fig. 2 sudden change is compared with OsGS1 nucleotide sequence.
Fig. 3 sudden change is compared with OsGS1 aminoacid sequence.
The comparison of Fig. 4 mutant and OsGS1 sequence and enzyme characteristic alive.
Fig. 5 transforms the yeast saccharomyces cerevisiae of sudden change and OsGS1 in the comparison of the growing state of different concns grass fourth phosphine SD-Trp solid medium.The concentration of grass fourth phosphine is respectively 0,10,20,50,100 mM.
Embodiment
Further describe the present invention below in conjunction with embodiment, but described embodiment is only for illustrating the present invention instead of restriction the present invention.
Not marked experimental technique in the invention process, as the preparation of the substratum that connects, transforms, is correlated with etc. is carried out with reference to method in the molecular cloning experiment guide third edition (Huang Peitang etc. translate, China, Science Press, 2002).Intestinal bacteria glutamine synthetase deficient strain JW3841-1 (glnA732::Kan) preserves center (CGSC#10775) by Yale's coli strain and provides; Plasmid pBluescrip SK+ is purchased from Stratagene company of the U.S.; PYM4807 carrier builds preservation (Xu Hu etc., Characterization of a Glucose-, Xylose-, Sucrose-, and D-Galactose-Stimulated b-Glucosidase from the voluntarily by laboratory
alkalophilic Bacterium Bacillus, Curr Microbiol, 2011,62:833-839); The KOD FX taq enzyme that amplification is used is purchased from Japanese Toyobo company; Required primer is synthetic by Nanjing Genscript Biotechnology Co., Ltd., and pYF1274 carrier is purchased from Shanghai Yongye Agricultural Science Bioengineering Co., Ltd; Other not marked pharmaceutical chemicals is analytical pure level, purchased from Shanghai traditional Chinese medicines Group Co.,Ltd.
Embodiment
1, build the expression plasmid of the cytoplasm type paddy rice glutamine synthetase gene that contains restructuring
With cytoplasm type paddy rice glutamine synthetase gene
osGS1gene (GenBank Accession No. AB037595) is template, at the synthetic wild cytoplasm type paddy rice glutamine synthetase gene of Nanjing Genscript Biotechnology Co., Ltd., this gene warp
bamHi and
sacafter I double digestion, be inserted in coli expression carrier pYM4807 with correct reading frame, obtain the plasmid that contains wild cytoplasm type paddy rice glutamine synthetase gene, be defined as pYM4807-OsGS1.
2 DNA Shuffling Methods build wild cytoplasm type paddy rice glutamine synthetase gene
osGS1gene mutation library
The structure in 2.1 sudden change libraries
1) amplification of gene: according to known pYM4807 carrier sequence, design P1(GAGACGGTCACAGCTTGTCTG with Primer 5.0) and P2(ATGCCTGGCAGTTCCCTACTC) two primers for PCR reaction.Reaction system comprises: 1 μ L plasmid pYM4807-OsGS1; 4 μ L 2.5 mmol/L dNTPs; 5 μ L 10XPCR Buffer; 0.3 μ L rTaq; The each 1 μ L of primer; Supplement ddH
2o to 50 μ L.PCR reaction conditions is, 94 denaturation 10min; 94 DEG C of sex change 30s, 72 DEG C of anneal 30s and extension 1.5 min, totally 35 circulations, 1.5% agarose gel reclaims the gene fragment of 1.5 Kb, reclaims product with entering 100 μ L DNase I damping fluid (50mmol/L Tris-Cl pH7.4+l mmol/L MgCl
2) dissolve.
2) DNase I enzymolysis: add 0.I U DNase I in the gene fragment reclaiming, 30 DEG C of enzymolysis 3 min, process after 10min inactivation DNase I through 80 DEG C, enzymolysis product is separated with 12% acrylamide gel electrophoresis, finally reclaim the small segment of 50-100bp by saturating suction bag method.
3) without primer PCR (Primerless PCR): reaction system comprises 25 μ L small pieces segment DNAs; 4 μ L 2.5 mmol/L dNTPs; 5 μ L 10XPCR Buffer; 0.3 μ L rTaq; 15.7 μ L ddH
2o; Response procedures is, 94 denaturation 2 min; 94 DEG C of sex change 30s, 42 DEG C of annealing 30s; 72 DEG C are extended 2 min, totally 45 circulations, 1% Agrose electrophoresis detection pcr amplification result.
4) have primer PCR (Primer PCR): with 1 μ L be template without primer PCR amplified production, other carries out pcr amplification with reference to step 1), reclaim 1.5 kp amplified productions.
5) amplified production warp
bamHi and
sacafter I double digestion, be inserted in coli expression carrier pYM4807 with correct reading frame, transform bacillus coli DH 5 alpha competence by electric shock, obtain paddy rice glutamine synthetase gene
osGSfirst run sudden change library.
Many wheels in 2.2 sudden change libraries build and screening
Sudden change library described in embodiment 2.1 is transformed into after deficient strain JW3841-1, is coated on the M9 flat board that contains 20 mM grass fourth phosphorus, 28 ° of C cultivate 48 h.With single bacterium colony of 10 normal growths of toothpick picking, extraction plasmid continues to build second as template according to embodiment 2.1 and takes turns sudden change library; Take turns sudden change library by second and be coated on the M9 flat board that contains 50 mM grass fourth phosphorus and screen, single bacterium colony of same 10 normal growths of picking, extracts plasmid and builds the sudden change library of third round as template; On this basis, set up the sudden change library of fourth round, be coated on the M9 flat board of 200 mM grass fourth phosphorus and screen, through revolution checking, finally obtain resisting 3 mutant (Fig. 1) of this concentration, numbering is respectively: OsGS1m1, OsGS1m2, OsGS1m3.Through sequencing, obtain the nucleotide sequence of described mutant, as shown in SEQ ID No 1, SEQ ID No3, SEQ ID No 5, find that by comparison change has as shown in Figure 2 occurred Nucleotide respectively; Obtain the aminoacid sequence of its coding through translation, respectively as shown in SEQ ID No 2, SEQ ID No4, SEQ ID No 6, compare and find that its corresponding amino acid mutation site is respectively by aminoacid sequence: G245S/G318V, N54S/S115F/G245S, V200A/T293A/R295K(Fig. 3, Fig. 4).
3 mutant enzymes characteristic research alive
3.1 protein expressions and purifying
The plasmid that contains glutamine synthetase mutator gene that wild-type and embodiment 2.3 are obtained proceeds to respectively in escherichia coli expression Host Strains BL21 (DE3), coat and contain Ap(penbritin, 100 μ g/ml) 2YT solid medium in, 37 DEG C of overnight incubation are to growing bacterium colony.The fresh conversion of picking 3-4 clone, access 100 ml contain Ap(100 μ g/ml) in antibiotic LB liquid nutrient medium, use 250ml shaking flask, 37 DEG C jolt and are cultured to OD600 value and reach 0.6-1.0.Shaking flask is placed in 5 minutes to 4 DEG C of centrifugal 5 minutes collection thalline of 5000g on ice.Re-suspended cell is in the broken damping fluid (100 mM This-hydrochloride buffers, 0.5M NaCl, 0.5mg/ml N,O-Diacetylmuramidase, 1mM PMSF, 1mM MgCl2) of 5ml precooling.In frozen water, use Ultrasonic Cell Disruptor fragmentation mixing thalline.Broken good bacterium liquid is through 12000 rpm, 4 DEG C after centrifugal 30 minutes, collects supernatant.After membrane filtration with 0.45 μ m, filtrate is carried out affinitive layer purification with Ni-NTA post.
Concrete purge process is: with 1.5 mL level pads (50 mM Tris-HCl, pH 7.5,300mM NaCl, and 10 mM imidazole) balance Ni-NTA post 5 times; Draw 1.5 mL filtrate loadings, after flowing out Ni-NTA post completely, 1.5 mL cleaning buffer solutions (50 mM Tris-HCl, pH 7.5,300mM NaCl, and 20 mM imidazole) clean five times; Finally use 1 mL elution buffer (50 mM Tris-HCl, pH 7.5,300mM NaCl, and 250 mM imidazole) wash-out 2 times.The elutriant that contains target protein is placed in after dialyzate (50 mM Tris-HCl, pH 7.5,0.1 mM EDTA, and 10% glycerol) dialysis 12 h, lives for SDS-PAGE electrophoresis and mensuration enzyme.
32 enzyme activity determinations
Glutamine synthetase enzyme activity determination reaction solution component is: 100 mM Tris-HCl (pH 7.5), 20 mM ATP, 10 mM L-glutamate, 30 mM hydroxylamine, 20 mM MgCl
2.After 35 ° of C preheatings of 190 μ l reaction solutions minute, add 10 μ l enzyme liquid to start reaction, after reaction 30 min, add 200 μ l nitrite ion (55 g/L FeCl
36H
2o, 20 g/L trichoroacetic acid(TCA)s, 2.1% concentrated hydrochloric acid) termination reaction.Be determined at the absorbance value of 540nM.Enzyme work is defined as per minute and discharges the needed enzyme amount of 1 μ mol gamma-glutamyl hydroximic acid.
3.3 suppress constant
k imeasure
In the survey live body system of the careless fourth phosphine (0,20,50,100 μ M) containing different concns, change the concentration (2,4,8,12,16,32,50 mM) of substrate L-glutamate, measure the enzymatic reaction speed under different substrates, by Lineweaver – Burk double-reciprocal plot, calculate respectively the inhibition constant of careless fourth phosphine to paddy rice wild-type glutamine synthetase and mutant, result is auspicious sees Fig. 4.
4. the functional verification of mutant in yeast saccharomyces cerevisiae
The structure of 4.1 saccharomyces cerevisiae expressions and conversion
Said mutation body is connected into Yeast expression carrier pYF1274, and (shuttle vectors that this carrier is intestinal bacteria-yeast saccharomyces cerevisiae, with colibacillary colE1 replication orgin and ammonia benzyl (Ap) antibiotic-screening mark; While is with yeast replication orgin (centromere, CEN), and the marker gene of coding TRP synthetic enzyme, is not therefore screening yeast transformant containing on the substratum of tryptophane.PYF1274 Yeast expression carrier is with yeast PGK strong promoter and ADC1 gene terminator, inserted the Cm resistant gene of 630 bp in promotor downstream, can replace to other foreign gene, realize the high efficient expression of foreign gene in yeast saccharomyces cerevisiae), the plasmid obtaining after connection is respectively used to yeast saccharomyces cerevisiae EGY48 (MAT α, his3, trp1, ura3-52, leu::pLeu2-LexAop6) conversion.
Yeast saccharomyces cerevisiae transforms the Lithium Acetate method of selecting.5 ml yeast saccharomyces cerevisiaes are 0.5 left and right in 30 DEG C of incubated overnight to OD600, and enlarged culturing is to 50ml, and regrowth is after 4 hours, and 5000rpm/ divides centrifugal 8 minutes collecting cells.With the aseptic washing of 20ml, but the TE/LiAc that the yeast of centrifugal collection is now joined with 10ml (100 mM LiAc in TE) washes once.7000 revs/min after centrifugal 8 minutes, yeast is suspended from the TE/LiAc of 0.5ml the most at last.In the sterilised yeast suspension of 50 μ l, add the salmon sperm dna of plasmid DNA, 50 μ g of 1 μ g and the 300 μ l TE/LiAc solution containing 40%PEG3350, after fully mixing in 30 DEG C of insulations 30 minutes.Then in 42 DEG C of heat shocks 15 minutes.The yeast of centrifugal collection is resuspended in TE solution, and is applied to the not yeast selection substratum containing tryptophane.
4.2 functional verification
Picking is selected the normal clone of substratum growth at the yeast that does not contain tryptophane, be inoculated in SD liquid nutrient medium, 28 ° of C overnight incubation, collect bacterium sample, be diluted to respectively identical concentration, get 50 μ L and be transferred in the 1 ml SD-Trp liquid nutrient medium that contains different concns grass fourth phosphine.After 28 ° of C shaking culture 36 h, observe growing state.Result shows that the expression in these three genes can improve the tolerance (from 10 mMs bring up to 100 mMs more than) of yeast saccharomyces cerevisiae to careless fourth phosphine (Fig. 5), similar because of the expression pattern of yeast saccharomyces cerevisiae and plant, show that three mutant are having good application potential aspect the anti-careless fourth phosphine plant of cultivation.
Ultimate principle of the present invention, principal character and advantage of the present invention have more than been described.The technician of the industry should understand; the present invention is not restricted to the described embodiments; that in above-described embodiment and specification sheets, describes just illustrates principle of the present invention; the present invention also has various changes and modifications without departing from the spirit and scope of the present invention, and these changes and improvements all fall in the claimed scope of the invention.The claimed scope of the present invention is defined by appending claims and equivalent thereof.
<110> Academy of Agricultural Sciences, Shanghai City
<120> utilizes DNA reorganization rice cytoplasmic type glutamine synthetase to obtain anti-careless fourth phosphorus mutant
<160> 7
<170> PatentIn version 3.3
<210> 1
<211> 356
<212> PRT
Rice cytoplasmic type glutamine synthetase (OsGS1) aminoacid sequence that <213> is original
<400> 1
Met Ala Ser Leu Thr Asp Leu Val Asn Leu Asn Leu Ser Asp Thr Thr
1 5 10 15
Glu Lys Ile Ile Ala Glu Tyr Ile Trp Ile Gly Gly Ser Gly Met Asp
20 25 30
Leu Arg Ser Lys Ala Arg Thr Leu Ser Gly Pro Val Thr Asp Pro Ser
35 40 45
Lys Leu Pro Lys Trp Asn Tyr Asp Gly Ser Ser Thr Gly Gln Ala Pro
50 55 60
Gly Glu Asp Ser Glu Val Ile Leu Tyr Pro Gln Ala Ile Phe Lys Asp
65 70 75 80
Pro Phe Arg Lys Gly Asn Asn Ile Leu Val Met Cys Asp Cys Tyr Thr
85 90 95
Pro Ala Gly Glu Pro Ile Pro Thr Asn Lys Arg His Asn Ala Ala Lys
100 105 110
Ile Phe Ser Ser Pro Glu Val Ala Ser Glu Glu Pro Trp Tyr Gly Ile
115 120 125
Glu Gln Glu Tyr Thr Leu Leu Gln Lys Asp Ile Asn Trp Pro Leu Gly
130 135 140
Trp Pro Val Gly Gly Phe Pro Gly Pro Gln Gly Pro Tyr Tyr Cys Gly
145 150 155 160
Ile Gly Ala Asp Lys Ser Phe Gly Arg Asp Ile Val Asp Ser His Tyr
165 170 175
Lys Ala Cys Leu Tyr Ala Gly Ile Asn Ile Ser Gly Ile Asn Gly Glu
180 185 190
Val Met Pro Gly Gln Trp Glu Phe Gln Val Gly Pro Ser Val Gly Ile
195 200 205
Ser Ala Gly Asp Gln Val Trp Val Ala Arg Tyr Ile Leu Glu Arg Ile
210 215 220
Thr Glu Ile Ala Gly Val Val Val Ser Phe Asp Pro Lys Pro Ile Pro
225 230 235 240
Gly Asp Trp Asn Gly Ala Gly Ala His Thr Asn Tyr Ser Thr Lys Ser
245 250 255
Met Arg Asn Asp Gly Gly Tyr Glu Ile Ile Lys Ser Ala Ile Glu Lys
260 265 270
Leu Lys Leu Arg His Lys Glu His Ile Ser Ala Tyr Gly Glu Gly Asn
275 280 285
Glu Arg Arg Leu Thr Gly Arg His Glu Thr Ala Asp Ile Asn Thr Phe
290 295 300
Ser Trp Gly Val Ala Asn Arg Gly Ala Ser Val Arg Val Gly Arg Glu
305 310 315 320
Thr Glu Gln Asn Gly Lys Gly Tyr Phe Glu Asp Arg Arg Pro Ala Ser
325 330 335
Asn Met Asp Pro Tyr Ile Val Thr Ser Met Ile Ala Glu Thr Thr Ile
340 345 350
Ile Trp Lys Pro
355
<210> 2
<211> 1071
<212> DNA
The nucleotide acid sequence of the coding OsGS1 of <213> synthetic
<400> 2
atggcttctc tcaccgacct tgtcaacctc aacctctctg acactactga gaagatcatc 60
gctgagtaca tctggatcgg tggatctggc atggatctca ggtctaaggc taggactctc 120
tctggtccag tcactgatcc atctaagctg ccaaagtgga actacgatgg ttcttctact 180
ggtcaagcac caggagagga ctctgaggtc atcctgtatc cacaggctat cttcaaggac 240
ccattcagga agggaaacaa catccttgtc atgtgtgact gttacactcc agcaggagaa 300
ccaatcccaa ccaacaagag gcacaatgct gccaaaatct tctcttctcc agaggttgct 360
tccgaagaac cttggtatgg tatcgaacaa gagtacactc tcctccagaa ggacatcaac 420
tggccacttg gttggccagt tggaggattc cctggaccac agggtccata ctactgtgga 480
atcggtgctg acaagtcctt tggtcgtgac atcgtcgatt ctcactacaa agcctgtctg 540
tatgctggca tcaacatctc tggcatcaat ggtgaagtca tgccaggaca gtgggagttc 600
caagttggtc catctgttgg catctctgct ggagaccaag tctgggtcgc acgttacatc 660
ctggaaagaa tcactgaaat cgcaggtgtt gtcgtctctt tcgacccaaa gccaatccca 720
ggtgactgga atggtgctgg tgctcatacc aactactcta ctaagtccat gaggaacgat 780
ggtggctacg agatcatcaa gtctgctatc gagaaactca agctcaggca caaggagcac 840
atctctgcct atggtgaagg caacgaacgt cgtctcactg gtaggcacga gactgctgac 900
atcaacacct tctcttgggg agttgccaac cgtggtgcat ctgttcgtgt tggtcgtgag 960
actgaacaga atggtaaggg ttacttcgaa gacagaagac cagcctctaa catggaccca 1020
tacatcgtta cttctatgat tgctgagact actatcatct ggaaaccata a 1071
<210> 3
<211> 1071
<212> DNA
The nucleotide acid sequence of <213> OsGSm1 mutant
<400> 3
atggcttctc tcaccgacct tgtcaacctc aacctctctg acactactga gaagatcatc 60
gctgagtaca tctggatcgg tggatctggc atggatctca ggtctaaggc taggactctc 120
tctggtccag tcactgatcc atctaagctg ccaaagtgga actacgatgg ttcttctact 180
ggtcaagcac caggagagga ctctgaggtc atcctgtatc cacaggctat cttcaaggac 240
ccattcagga agggaaacaa catccttgtc atgtgtgact gttacactcc agcaggagaa 300
ccaatcccaa ccaacaagag gcacaatgct gccaaaatct tctcttctcc agaggttgct 360
tccgaagaac cttggtatgg tatcgaacaa gagtacactc tcctccagaa ggacatcaac 420
tggccacttg gttggccagt tggaggattc cctggaccac agggtccgta ctactgtgga 480
atcggtgctg acaagtcctt tggtcgtgac atcgtcgatt ctcactacaa agcctgtctg 540
tatgctggca tcaacatctc tggcatcaat ggtgaagtca tgccaggaca gtgggagttc 600
caagttggtc catctgttgg catctctgct ggagaccaag tctgggtcgc acgttacatc 660
ctggaaagaa tcactgaaat cgcaggtgtt gtcgtctctt tcgacccaaa gccaatccca 720
ggtgactgga atagtgctgg tgctcatacc aactactcta ctaagtccat gaggaacgat 780
ggtggctacg agatcatcaa gtctgctatc gagaaactca agctcaggca caaggagcac 840
atctctgcct atggtgaagg caacgaacgt cgtctcactg gtaggcacga gactgctgac 900
atcaacacct tctcttgggg agttgccaac cgtggtgcat ctgttcgtgt tgttcgtgag 960
actgaacaga atggtaaggg ttacttcgaa gacagaagac cagcctctaa catggaccca 1020
tacatcgtta cttctatgat tgctgagact actatcatct ggaaaccata a 1071
<210> 4
<211> 356
<212> PRT
The aminoacid sequence of <213> OsGSm1 mutant
<400> 4
Met Ala Ser Leu Thr Asp Leu Val Asn Leu Asn Leu Ser Asp Thr Thr
1 5 10 15
Glu Lys Ile Ile Ala Glu Tyr Ile Trp Ile Gly Gly Ser Gly Met Asp
20 25 30
Leu Arg Ser Lys Ala Arg Thr Leu Ser Gly Pro Val Thr Asp Pro Ser
35 40 45
Lys Leu Pro Lys Trp Asn Tyr Asp Gly Ser Ser Thr Gly Gln Ala Pro
50 55 60
Gly Glu Asp Ser Glu Val Ile Leu Tyr Pro Gln Ala Ile Phe Lys Asp
65 70 75 80
Pro Phe Arg Lys Gly Asn Asn Ile Leu Val Met Cys Asp Cys Tyr Thr
85 90 95
Pro Ala Gly Glu Pro Ile Pro Thr Asn Lys Arg His Asn Ala Ala Lys
100 105 110
Ile Phe Ser Ser Pro Glu Val Ala Ser Glu Glu Pro Trp Tyr Gly Ile
115 120 125
Glu Gln Glu Tyr Thr Leu Leu Gln Lys Asp Ile Asn Trp Pro Leu Gly
130 135 140
Trp Pro Val Gly Gly Phe Pro Gly Pro Gln Gly Pro Tyr Tyr Cys Gly
145 150 155 160
Ile Gly Ala Asp Lys Ser Phe Gly Arg Asp Ile Val Asp Ser His Tyr
165 170 175
Lys Ala Cys Leu Tyr Ala Gly Ile Asn Ile Ser Gly Ile Asn Gly Glu
180 185 190
Val Met Pro Gly Gln Trp Glu Phe Gln Val Gly Pro Ser Val Gly Ile
195 200 205
Ser Ala Gly Asp Gln Val Trp Val Ala Arg Tyr Ile Leu Glu Arg Ile
210 215 220
Thr Glu Ile Ala Gly Val Val Val Ser Phe Asp Pro Lys Pro Ile Pro
225 230 235 240
Gly Asp Trp Asn Ser Ala Gly Ala His Thr Asn Tyr Ser Thr Lys Ser
245 250 255
Met Arg Asn Asp Gly Gly Tyr Glu Ile Ile Lys Ser Ala Ile Glu Lys
260 265 270
Leu Lys Leu Arg His Lys Glu His Ile Ser Ala Tyr Gly Glu Gly Asn
275 280 285
Glu Arg Arg Leu Thr Gly Arg His Glu Thr Ala Asp Ile Asn Thr Phe
290 295 300
Ser Trp Gly Val Ala Asn Arg Gly Ala Ser Val Arg Val Val Arg Glu
305 310 315 320
Thr Glu Gln Asn Gly Lys Gly Tyr Phe Glu Asp Arg Arg Pro Ala Ser
325 330 335
Asn Met Asp Pro Tyr Ile Val Thr Ser Met Ile Ala Glu Thr Thr Ile
340 345 350
Ile Trp Lys Pro
355
<210> 5
<211> 1071
<212> DNA
The nucleotide acid sequence of <213> OsGSm2 mutant
<400> 5
atggcttctc tcaccgacct tgtcaacctc aacctctctg acactactga gaagatcatc 60
gctgagtaca tctggatcgg tggatctggc atggatctca ggtctaaggc taggactctc 120
tctggtccag tcactgatcc atctaagctg ccaaagtgga gctacgatgg ttcttctact 180
ggtcaagcac caggagagga ctctgaggtc atcctgtatc cacaggctat cttcaaggac 240
ccattcagga agggaaacaa catccttgtc atgtgtgact gttacactcc agcaggagaa 300
ccaatcccaa ccaacaagag gcacaatgct gccaaaatct tcttctctcc agaggttgct 360
tccgaagaac cttggtatgg tatcgaacaa gagtacactc tcctccagaa ggacatcaac 420
tggccacttg gttggccagt tggaggattc cctggaccac agggtccata ctactgtgga 480
atcggtgctg acaagtcctt tggtcgtgac atcgtcgatt ctcactacaa agcctgtctg 540
tatgctggca tcaacatctc tggcatcaat ggtgaagtca tgccaggaca gtgggagttc 600
caagttggtc catctgttgg catctctgct ggagaccaag tctgggtcgc acgttacatc 660
ctggaaagaa tcactgaaat cgcaggtgtt gtcgtctctt tcgacccaaa gccaatccca 720
ggtgactgga atagtgctgg tgctcatacc aactactcta ctaagtccat gaggaacgat 780
ggtggctacg agatcatcaa gtctgctatc gagaaactca agctcaggca caaggagcac 840
atctctgcct atggtgaagg caacgaacgt cgtctcactg gtaggcacga gactgctgac 900
atcaacacct tctcttgggg agttgccaac cgtggtgcat ctgttcgtgt tggtcgtgag 960
actgaacaga atggtaaggg ttacttcgaa gacagaagac cagcctctaa catggaccca 1020
tacatcgtta cttctatgat tgctgagact actatcatct ggaaaccata a 1071
<210> 6
<211> 356
<212> PRT
The aminoacid sequence of <213> OsGSm2 mutant
<400> 6
Met Ala Ser Leu Thr Asp Leu Val Asn Leu Asn Leu Ser Asp Thr Thr
1 5 10 15
Glu Lys Ile Ile Ala Glu Tyr Ile Trp Ile Gly Gly Ser Gly Met Asp
20 25 30
Leu Arg Ser Lys Ala Arg Thr Leu Ser Gly Pro Val Thr Asp Pro Ser
35 40 45
Lys Leu Pro Lys Trp Ser Tyr Asp Gly Ser Ser Thr Gly Gln Ala Pro
50 55 60
Gly Glu Asp Ser Glu Val Ile Leu Tyr Pro Gln Ala Ile Phe Lys Asp
65 70 75 80
Pro Phe Arg Lys Gly Asn Asn Ile Leu Val Met Cys Asp Cys Tyr Thr
85 90 95
Pro Ala Gly Glu Pro Ile Pro Thr Asn Lys Arg His Asn Ala Ala Lys
100 105 110
Ile Phe Phe Ser Pro Glu Val Ala Ser Glu Glu Pro Trp Tyr Gly Ile
115 120 125
Glu Gln Glu Tyr Thr Leu Leu Gln Lys Asp Ile Asn Trp Pro Leu Gly
130 135 140
Trp Pro Val Gly Gly Phe Pro Gly Pro Gln Gly Pro Tyr Tyr Cys Gly
145 150 155 160
Ile Gly Ala Asp Lys Ser Phe Gly Arg Asp Ile Val Asp Ser His Tyr
165 170 175
Lys Ala Cys Leu Tyr Ala Gly Ile Asn Ile Ser Gly Ile Asn Gly Glu
180 185 190
Val Met Pro Gly Gln Trp Glu Phe Gln Val Gly Pro Ser Val Gly Ile
195 200 205
Ser Ala Gly Asp Gln Val Trp Val Ala Arg Tyr Ile Leu Glu Arg Ile
210 215 220
Thr Glu Ile Ala Gly Val Val Val Ser Phe Asp Pro Lys Pro Ile Pro
225 230 235 240
Gly Asp Trp Asn Ser Ala Gly Ala His Thr Asn Tyr Ser Thr Lys Ser
245 250 255
Met Arg Asn Asp Gly Gly Tyr Glu Ile Ile Lys Ser Ala Ile Glu Lys
260 265 270
Leu Lys Leu Arg His Lys Glu His Ile Ser Ala Tyr Gly Glu Gly Asn
275 280 285
Glu Arg Arg Leu Thr Gly Arg His Glu Thr Ala Asp Ile Asn Thr Phe
290 295 300
Ser Trp Gly Val Ala Asn Arg Gly Ala Ser Val Arg Val Gly Arg Glu
305 310 315 320
Thr Glu Gln Asn Gly Lys Gly Tyr Phe Glu Asp Arg Arg Pro Ala Ser
325 330 335
Asn Met Asp Pro Tyr Ile Val Thr Ser Met Ile Ala Glu Thr Thr Ile
340 345 350
Ile Trp Lys Pro
355
<210> 7
<211> 1071
<212> DNA
The nucleotide acid sequence of <213> OsGSm3 mutant
<400> 7
atggcttctc tcaccgacct tgtcaacctc aacctctctg acactactga gaagatcatc 60
gctgagtaca tctggatcgg tggatctggc atggatctca ggtctaaggc taggactctc 120
tctggtccag tcactgatcc atctaagctg ccaaagtgga actacgatgg ttcttctact 180
ggtcaagaac caggagagga ctctgaggtc atcctgtatc cacaggctat cttcaaggac 240
ccattcagga agggaaacaa catccttgtc atgtgtgact gttacactcc agcaggagaa 300
ccaatcccaa ccaacaagag gcacaatgct gccaaaatct tctcttctcc agaggttgct 360
tccgaagaac cttggtatgg tatcgaacaa gagtacactc tcctccagaa ggacatcaac 420
tggccacttg gttggccagt tggaggattc cctggaccac agggtccata ctactgtgga 480
atcggtgctg acaagtcctt tggtcgtgac atcgtcgatt ctcactacaa agcctgtctg 540
tatgctggca tcaacatctc tggcatcaat ggtgaagcca tgccaggaca gtgggagttc 600
caagttggtc catctgttgg catctctgct ggagaccaag tctgggtcgc acgttacatc 660
ctggaaagga tcactgaaat cgcaggtgtt gtcgtctctt tcgacccaaa gccaatccca 720
ggtgactgga atggtgctgg tgctcatacc aactactcta ctaagtccat gaggaacgat 780
ggtggctacg agatcatcaa gtctgctatc gagaaactca agctcaggca caaggagcac 840
atctctgcct atggtgaagg caacgaacgt cgtctcgctg gtaagcacga gactgctgac 900
atcaacacct tctcttgggg agttgccaac cgtggtgcat ctgttcgtgt tggtcgtgag 960
actgaacaga atggtaaggg ttacttcgaa gacagaagac cagcctctaa catggaccca 1020
tacatcgtta cttctatgat tgctgagact actatcatct ggaaaccata a 1071
<210> 8
<211> 356
<212> PRT
The aminoacid sequence of <213> OsGSm3 mutant
<400> 8
Met Ala Ser Leu Thr Asp Leu Val Asn Leu Asn Leu Ser Asp Thr Thr
1 5 10 15
Glu Lys Ile Ile Ala Glu Tyr Ile Trp Ile Gly Gly Ser Gly Met Asp
20 25 30
Leu Arg Ser Lys Ala Arg Thr Leu Ser Gly Pro Val Thr Asp Pro Ser
35 40 45
Lys Leu Pro Lys Trp Asn Tyr Asp Gly Ser Ser Thr Gly Gln Glu Pro
50 55 60
Gly Glu Asp Ser Glu Val Ile Leu Tyr Pro Gln Ala Ile Phe Lys Asp
65 70 75 80
Pro Phe Arg Lys Gly Asn Asn Ile Leu Val Met Cys Asp Cys Tyr Thr
85 90 95
Pro Ala Gly Glu Pro Ile Pro Thr Asn Lys Arg His Asn Ala Ala Lys
100 105 110
Ile Phe Ser Ser Pro Glu Val Ala Ser Glu Glu Pro Trp Tyr Gly Ile
115 120 125
Glu Gln Glu Tyr Thr Leu Leu Gln Lys Asp Ile Asn Trp Pro Leu Gly
130 135 140
Trp Pro Val Gly Gly Phe Pro Gly Pro Gln Gly Pro Tyr Tyr Cys Gly
145 150 155 160
Ile Gly Ala Asp Lys Ser Phe Gly Arg Asp Ile Val Asp Ser His Tyr
165 170 175
Lys Ala Cys Leu Tyr Ala Gly Ile Asn Ile Ser Gly Ile Asn Gly Glu
180 185 190
Ala Met Pro Gly Gln Trp Glu Phe Gln Val Gly Pro Ser Val Gly Ile
195 200 205
Ser Ala Gly Asp Gln Val Trp Val Ala Arg Tyr Ile Leu Glu Arg Ile
210 215 220
Thr Glu Ile Ala Gly Val Val Val Ser Phe Asp Pro Lys Pro Ile Pro
225 230 235 240
Gly Asp Trp Asn Gly Ala Gly Ala His Thr Asn Tyr Ser Thr Lys Ser
245 250 255
Met Arg Asn Asp Gly Gly Tyr Glu Ile Ile Lys Ser Ala Ile Glu Lys
260 265 270
Leu Lys Leu Arg His Lys Glu His Ile Ser Ala Tyr Gly Glu Gly Asn
275 280 285
Glu Arg Arg Leu Ala Gly Lys His Glu Thr Ala Asp Ile Asn Thr Phe
290 295 300
Ser Trp Gly Val Ala Asn Arg Gly Ala Ser Val Arg Val Gly Arg Glu
305 310 315 320
Thr Glu Gln Asn Gly Lys Gly Tyr Phe Glu Asp Arg Arg Pro Ala Ser
325 330 335
Asn Met Asp Pro Tyr Ile Val Thr Ser Met Ile Ala Glu Thr Thr Ile
340 345 350
Ile Trp Lys Pro
355
Claims (3)
1. three paddy rice glutamine synthetase mutant that careless fourth phosphine resistance is significantly improved, is characterized in that, it is encoded by the nucleotide acid sequence as shown in SEQ ID No 1, SEQ ID No3, SEQ ID No 5.
2. mutant described in claim 1, is characterized in that, its aminoacid sequence is respectively as shown in SEQ ID No 2, SEQ ID No 4, SEQ ID No 6.
3. contain yeast and plant expression vector and the application thereof of mutator gene described in claim 1.
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|---|---|---|---|
| CN201410207844.3A CN104164441A (en) | 2014-05-17 | 2014-05-17 | Three glufosinate-resistant rice cytoplasm type glutamine synthetase mutants |
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|---|---|---|---|
| CN201410207844.3A CN104164441A (en) | 2014-05-17 | 2014-05-17 | Three glufosinate-resistant rice cytoplasm type glutamine synthetase mutants |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021000870A1 (en) * | 2019-07-01 | 2021-01-07 | 四川天豫兴禾生物科技有限公司 | Glutamine synthetase mutant having glufosinate ammonium resistance and application thereof and cultivation method therefor |
| CN114807064A (en) * | 2022-06-06 | 2022-07-29 | 四川天豫兴禾生物科技有限公司 | Method for obtaining protein with glufosinate-ammonium resistance and mutant thereof |
| WO2023071438A1 (en) * | 2021-10-26 | 2023-05-04 | 四川天豫兴禾生物科技有限公司 | Glutamine synthetase mutant and application |
-
2014
- 2014-05-17 CN CN201410207844.3A patent/CN104164441A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021000870A1 (en) * | 2019-07-01 | 2021-01-07 | 四川天豫兴禾生物科技有限公司 | Glutamine synthetase mutant having glufosinate ammonium resistance and application thereof and cultivation method therefor |
| WO2023071438A1 (en) * | 2021-10-26 | 2023-05-04 | 四川天豫兴禾生物科技有限公司 | Glutamine synthetase mutant and application |
| CN114807064A (en) * | 2022-06-06 | 2022-07-29 | 四川天豫兴禾生物科技有限公司 | Method for obtaining protein with glufosinate-ammonium resistance and mutant thereof |
| CN114807064B (en) * | 2022-06-06 | 2024-04-19 | 四川天豫兴禾生物科技有限公司 | Method for obtaining protein with glufosinate resistance and mutant thereof |
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