CN103757033A - Rice glutamine synthetase mutant gene capable of improving resistance of plant glufosinate, and preparation method and applications thereof - Google Patents
Rice glutamine synthetase mutant gene capable of improving resistance of plant glufosinate, and preparation method and applications thereof Download PDFInfo
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Abstract
The invention discloses a rice glutamine synthetase mutant gene capable of improving resistance of plant glufosinate, the nucleotide sequence of the rice glutamine synthetase mutant gene is shown as SEQ ID No3, and the coded amino acid sequence of the rice glutamine synthetase mutant gene is shown as SEQ ID No4. In addition, the invention also provides the applications of the mutant gene on culturing glufosinate-resistant transgenic crop. The resistance of the gene-coded glutamine synthetase on glufosinate can be obviously enhanced.
Description
Technical field
The invention belongs to biological technical field, relate to particularly a kind of paddy rice glutamine synthetase mutator gene that improves plant plait ammonium phosphine resistance and its preparation method and application.
Background technology
Grass ammonium phosphine (DL-phosphinotricinm) is second-biggest-in-the-world genetically modified crops herbicide-tolerant, and glutamine synthetase is the target enzyme of careless ammonium phosphine.
The advantages such as it is low that grass ammonium phosphine has toxicity, is easy to degraded in soil, is difficult for drift, active high, and consumption is few, and environmental stress is little, and herbicide is rapid.But careless ammonium phosphine is a kind of nonselective herbicide, can affect the growth of farm crop when killing weeds, limited careless ammonium phosphine as the use range of weedicide.From Streptomyce hygroscopicus clone's bar gene and the pat gene of cloning from viridochromogenes careless ammonium phosphinothricin acetyl transferring enzyme (the PPT acetyltransferase that all encodes; PAT); it can make the deactivation of careless ammonium phosphine by the acetylize of catalysis grass ammonium phosphine amino; applying gene recombinant technology is expressed this genoid, is a kind of method that obtains herbicide-resistant transgenic crop.
Overexpression glutamine synthetase in plant, can reduce equally transgenic plant to the sensitivity of Glufosinate ammonium (Pascual MB etc., Response of transgenic poplar overexpressing cytosolic glutamine synthetase to phosphinothricin.Phytochemistry?200869:382-9）。
But careless ammonium phosphine is very obvious to deriving from the restraining effect of wild-type glutamine synthetase of plant, transform this genoid to improve plant careless ammonium phosphine resistance action effect not clearly.
Summary of the invention
The object of the present invention is to provide a kind of paddy rice glutamine synthetase mutator gene that improves plant plait ammonium phosphine resistance and its preparation method and application, the glutamine synthetase of this mutator gene coding significantly strengthens the resistance of careless ammonium phosphine, and described mutator gene can be used for building the expression vector of conversion of plant.
For achieving the above object, the present invention mainly adopts following technical scheme:
A paddy rice glutamine synthetase mutator gene that improves plant plait ammonium phosphine resistance, its nucleotide sequence is as shown in SEQ ID No3.The aminoacid sequence of its coding is as shown in SEQ ID No4.
Adopt fallibility PCR method to set up random mutation library and obtain by screening, the nucleotide site of sudden change is C216T/T328C/A486G/T584C/A675G/T952C.Wherein, digitized representation mutational site, base before the representative sudden change above of each numeral, base after the representative sudden change below of each numeral.Amino acid mutation point position is His108Tyr/Val200Ala/Arg316Cys.
Meanwhile, the present invention also provides the expression vector pYM4807-OsGSM1 of described paddy rice glutamine synthetase mutator gene.
And the present invention also provides a kind of preparation method of described paddy rice glutamine synthetase mutator gene, comprises the steps:
1) take the paddy rice glutamine synthetase gene sequence shown in sequence table SEQ ID No1 is template, synthesize 26 primers shown in SEQ ID No5-SEQ ID No30 as shown in sequence table, called after primer P1-P26 successively, adopt continuous extension PCR method amplification, synthetic water rice glutamine synthetase gene, and be building up to pBlueScriptII S+ carrier, and the plasmid of the paddy rice glutamine synthetase gene that obtains containing restructuring, concrete grammar is as follows:
A) take respectively primer P1, P10 as outside primer, P2-P9 is inner side primer synthetic fragment 1; Primer P11, P20 are outside primer, and P12-P19 is inner side primer synthetic fragment 2; Primer P21, P26 are outside primer, and P22-P25 is inner side primer synthetic fragment 3;
B) respectively get 1 μ L fragment 1, fragment 2, fragment 3 for template, with outside primer P1, P26, adopt continuous extension PCR method to increase to obtain the paddy rice glutamine synthetase gene that contains restructuring; Described reaction is in 50 μ L reaction systems, and the addition of inner side primer is 10ng, and outside primer addition is 100ng,
Concrete PCR reaction system is as follows:
Pcr amplification condition is: 94 ℃ of preheating 10min; 94 ℃, 30s, 54 ℃, 30s, 72 ℃, 80s, 35 circulations; Last 72 ℃ are extended 10min;
C) the paddy rice glutamine synthetase gene that contains restructuring amplification being obtained is cloned in pBlueScriptII S+ carrier, obtains the plasmid of the paddy rice glutamine synthetase gene that contains restructuring;
2) take the aforementioned plasmid obtaining is template, with sequence table SEQ ID No31 and SEQ ID No32, be depicted as primer, adopt fallibility PCR method to carry out random mutation to paddy rice glutamine synthetase gene, amplified production is after BamHI and SacI double digestion, with correct reading frame, be inserted in coli expression carrier pYM4807, by electric shock, transform bacillus coli DH 5 alpha competence, obtain the sudden change library of paddy rice glutamine synthetase gene;
Concrete PCR reaction system is as follows:
10mM Tris-HCl, pH8.3; 50mM KCl; 7mM MgCl
2; 0.3mM MnCl
2, 0.2mM dATPs; 0.2mM dGTPs; 1mM dGTPs; 1mM dTTPs; 5r U Taq0.3 μ L; Each 50p mol of primer, template 10ng; Pcr amplification condition is: 94 ℃ of preheating 10min; 94 ℃, 30s, 54 ℃, 30s, 72 ℃, 80s, 35 circulations; Last 72 ℃ are extended 10min;
3) the aforementioned sudden change library obtaining is transformed into deficient strain JW3841-1, be coated on the substratum that contains 20mM grass ammonium phosphine and cultivate, the normal single bacterium colony of picking growth, be inoculated into the LB(100mg/L containing Ampicillin Trihydrate) cultivate in liquid nutrient medium, tentatively obtain containing the bacterium sample of the paddy rice glutamine synthetase mutator gene that careless ammonium phosphine resistance improves;
4) the bacterium sample tentatively obtaining and the bacterium sample that contains wild-type glutamine synthetase are diluted to same concentrations, be added to concentration and be respectively 0, 10mM, 20mM, on the substratum of 100mM grass ammonium phosphine, cultivate, the growing state of observation on different concns Glufosinate substratum, raising along with careless ammonium phosphine concentration, the growing state of the bacterium sample of the paddy rice glutamine synthetase mutator gene that contains careless ammonium phosphine resistance raising is normal, the growth of the bacterium sample that contains wild-type glutamine synthetase is obviously suppressed, the bacterium sample of the plasmid of the plasmid vector pYM4807-OsGSM1 that extraction contains described mutator gene checks order, obtain described paddy rice glutamine synthetase mutator gene.
Meanwhile, the present invention also provides the application of described paddy rice glutamine synthetase mutator gene in cultivating anti-careless ammonium phosphine genetically modified crops.
Compared with prior art, beneficial effect of the present invention is:
The paddy rice glutamine synthetase mutator gene that the resistance of careless ammonium phosphine is significantly strengthened provided by the invention, has for building the expression vector of conversion of plant, and cultivates the application potential of anti-careless ammonium phosphine genetically modified crops.
By contrasting careless ammonium phosphine, enzyme is lived and suppressed constant K
i, show that the paddy rice glutamine synthetase mutant that contains mutator gene provided by the invention has improved 28 times than wild-type glutamine synthetase to the resistance of careless ammonium phosphine.
Accompanying drawing explanation
Fig. 1 is that in paddy rice glutamine synthetase mutator gene provided by the invention, nucleotide sequence and wild-type paddy rice glutamine synthetase nucleotide sequence are compared.
Fig. 2 is that in paddy rice glutamine synthetase mutator gene provided by the invention, aminoacid sequence and wild-type paddy rice glutamine synthetase aminoacid sequence are compared.
Fig. 3 is the paddy rice glutamine synthetase mutant bacterium sample that contains mutator gene provided by the invention and contains wild-type glutamine synthetase bacterium sample in the comparison of the growing state of different concns grass ammonium phosphine M9 solid medium; Wherein, wild-type: contain wild-type glutamine synthetase bacterium sample; OsGS-M1: the paddy rice glutamine synthetase mutant bacterium sample that contains mutator gene provided by the invention.
Embodiment
Below in conjunction with embodiment, further describe the present invention, but described embodiment is only for illustrating the present invention rather than restriction the present invention.
Not marked experimental technique in the invention process, as connected, transform, the preparation of relevant substratum etc. carries 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 U.S. Stratagene company; PYM4807 carrier builds preservation (Xu Hu etc. voluntarily by laboratory, Characterization of a Glucose-, Xylose-, Sucrose-, and D-Galactose-Stimulated b-Glucosidase from the 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 other not marked pharmaceutical chemicals is analytical pure level, purchased from Shanghai traditional Chinese medicines Group Co.,Ltd.
Embodiment
1, the plasmid of the synthetic paddy rice glutamine synthetase gene that contains restructuring
The paddy rice glutamine synthetase gene OsGS sequence (GenBank Accession No.X14245) shown in sequence table SEQ ID No1 of take is template, synthetic 26 primers as shown in SEQ ID No5-SEQ ID No30 in sequence table, called after primer P1-P26 successively, adopt continuous extension PCR method amplification, the plasmid of the synthetic paddy rice glutamine synthetase gene that contains restructuring.
Concrete synthetic method is as follows:
The first step: take respectively primer P1, P10 as outside primer, P2-P9 is inner side primer synthetic fragment 1; Primer P11, P20 are outside primer, and P12-P19 is inner side primer synthetic fragment 2; Primer P21, P26 are outside primer, and P22-P25 is inner side primer synthetic fragment 3.
Second step, respectively gets 1 μ L fragment 1, fragment 2, fragment 3 for template, with outside primer P1, P26, increases and obtains the paddy rice glutamine synthetase gene that contains restructuring.
Above-mentioned reaction is all in 50 μ L reaction systems, and the addition of inner side primer is 10ng, and outside primer addition is 100ng.
Concrete PCR reaction system is as follows:
Pcr amplification condition is: 94 ℃ of preheating 10min; 94 ℃, 30s, 54 ℃, 30s, 72 ℃, 80s, 35 circulations; Last 72 ℃ are extended 10min.
The 3rd step, the paddy rice glutamine synthetase gene that contains restructuring that amplification is obtained is cloned in pBluescriptII SK+ carrier, obtains the plasmid of the paddy rice glutamine synthetase gene OsGS that contains restructuring.
2, structure and the screening in paddy rice glutamine synthetase gene OsGS sudden change library
The structure in 2.1 sudden change libraries
The aforementioned plasmid obtaining of take is template, with sequence table SEQ ID No31 and SEQ ID No32, be depicted as primer, adopt fallibility PCR method amplifying rice glutamine synthetase gene OsGS, described reaction is carried out in 100 μ LPCR reaction systems, described PCR reaction system is: 10mM Tris-HCl, pH8.3; 50mM KCl; 7mM MgCl
2; 0.3mM MnCl
2, 0.2mM dATPs; 0.2mM dGTPs; 1mM dGTPs; 1mM dTTPs; 5U rTaq0.3 μ L; Each 50p mol of primer, template 10ng; PCR response procedures: 94 ℃ of denaturation 10min; 94 ℃ of sex change 30s, 54 ℃ of annealing 30s and 72 ℃ of extensions 2min, totally 35 circulations.
Amplified production is after BamHI and SacI double digestion, with correct reading frame, be inserted in coli expression carrier pYM4807, the plasmid vector that acquisition contains described mutator gene, be defined as pYM4807-OsGSM1, by electric shock, transform bacillus coli DH 5 alpha competence, obtain the sudden change library of paddy rice glutamine synthetase gene OsGS.
The preliminary screening in 2.2 sudden change libraries
Described sudden change library is transformed into after deficient strain JW3841-1, is coated on the M9 flat board that contains 20mM grass ammonium phosphine, cultivate 48h for 28 ℃.Single bacterium colony with toothpick picking normal growth, be inoculated into fresh the containing in the LB liquid nutrient medium of 100mg/L Ampicillin Trihydrate of 2ml, in temperature, it is 37 ℃, rotating speed is to cultivate 12h under 180rpm condition, tentatively obtains containing the mutant bacterium sample of the paddy rice glutamine synthetase mutator gene that careless ammonium phosphine resistance improves.
2.3 mutant sieve again
The mutant bacterium sample of the paddy rice glutamine synthetase mutator gene that contains careless ammonium phosphine resistance raising that preliminary screening is obtained, the wild-type bacteria sample that contains wild-type glutamine synthetase gene are diluted to after same concentrations, drawing 2 μ L is added drop-wise to concentration and is respectively 0,10mM, 20mM, on the M9 culture medium flat plate of 100mM grass ammonium phosphine, cultivate 2 days for 28 ℃, observe growing state.
Result is as shown in Figure 3: on the substratum that does not add careless ammonium phosphine (being that careless ammonium phosphine concentration is 0), wild-type glutamine synthetase bacterium sample and the paddy rice glutamine synthetase mutant bacterium sample growth that contains mutator gene provided by the invention are basically identical; But along with the raising of careless ammonium phosphine concentration (as is promoted to 10mM, be promoted to 20mM, be promoted to 100mM), the growth of wild-type glutamine synthetase bacterium sample is obviously suppressed, and the paddy rice glutamine synthetase mutant bacterium sample that contains mutator gene provided by the invention careless ammonium phosphine concentration lower than the culture medium flat plate of 100mM on growing state substantially there is no difference.
Result verification the raising of paddy rice glutamine synthetase mutant to careless ammonium phosphine resistance that contains mutator gene provided by the invention.Extract and obtain the plasmid vector that contains described mutator gene, be defined as pYM4807-OsGSM1, this plasmid is after order-checking, obtain the nucleotide sequence of mutator gene as shown in SEQ ID No3, after translation, obtain the aminoacid sequence of mutator gene coding as shown in SEQ ID No4, the nucleotide site of sudden change be C216T/T328C/A486G/T584C/A675G/T952C(as shown in Figure 1).Wherein, digitized representation mutational site, base before the representative sudden change above of each numeral, base after the representative sudden change below of each numeral.Amino acid mutation point position be His108Tyr/Val200Ala/Arg316Cys(as shown in Figure 2).
3, mutant enzyme characteristic research alive
3.1 protein expressions and purifying
The mutant bacterium sample that contains paddy rice glutamine synthetase mutator gene that the screening of wild-type glutamine synthetase bacterium sample and step 2.3 is obtained proceeds to respectively in escherichia coli expression Host Strains BL21 (DE3), coat and contain Ap(penbritin, 100 μ g/ml), in 2YT solid medium, 37 ℃ of overnight incubation are to growing bacterium colony.The 3-4 of the fresh conversion of picking clone, access 100ml contains Ap(100 μ g/ml) in antibiotic LB liquid nutrient medium, use 250ml shaking flask, 37 ℃ jolt and are cultured to OD600 value and reach 0.6-1.0.Shaking flask is placed in 5 minutes on ice, within centrifugal 5 minutes, collects thalline for 5000g4 ℃.Re-suspended cell is in broken damping fluid (100mM This-hydrochloride buffer, 0.5M NaCl, 0.5mg/ml N,O-Diacetylmuramidase, 1mM PMSF, the 1mM MgCl of 5ml precooling
2) in.Broken with Ultrasonic Cell Disruptor in frozen water mixing thalline.Broken good bacterium liquid is through 12000rpm, 4 ℃ 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: use 1.5mL level pad (50mM Tris-HCl, pH7.5,300mM NaCl, and10mM imidazole) balance Ni-NTA post 5 times; Draw 1.5mL filtrate loading, after flowing out Ni-NTA post completely, 1.5mL cleaning buffer solution (50mM Tris-HCl, pH7.5,300mM NaCl, and 20mM imidazole) cleans five times; Finally use 1mL elution buffer (50mM Tris-HCl, pH7.5,300mM NaCl, and 250mM imidazole) wash-out 2 times.The elutriant that contains target protein is placed in after dialyzate (50mM Tris-HCl, pH7.5,0.1mM EDTA, and 10% glycerol) dialysis 12h, for SDS-PAGE electrophoresis and mensuration enzyme, lives.
3.2 enzyme activity determination
Glutamine synthetase enzyme activity determination reaction solution component is: 100mM Tris-HCl (pH7.5), 20mM ATP, 10mM L-glutamate, 30mM hydroxylamine, 20mM MgCl
2.After 35 ℃ of preheatings of 190 μ l reaction solutions minute, add 10 μ l enzyme liquid to start reaction, after reaction 30min, add 200 μ l nitrite ion (55g/L FeCl
36H
2o, 20g/L trichoroacetic acid(TCA), 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 ammonium phosphine (0,20,50 μ M) containing different concns, change the concentration (1,2,4,8,12,16,32mM) 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 ammonium phosphine to paddy rice wild-type glutamine synthetase and the paddy rice glutamine synthetase mutant that contains mutator gene provided by the invention, the results are shown in following table:
By contrast Glufosinate, enzyme is lived and suppressed constant K
i, show that the paddy rice glutamine synthetase mutant that contains mutator gene provided by the invention has improved 28 times than wild-type glutamine synthetase to the resistance of careless ammonium phosphine.
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.
Claims (5)
1. a paddy rice glutamine synthetase mutator gene that improves plant plait ammonium phosphine resistance, is characterized in that, its nucleotide sequence is as shown in SEQ ID No3.
2. paddy rice glutamine synthetase mutator gene according to claim 1, is characterized in that, the aminoacid sequence of its coding is as shown in SEQ ID No4.
3. the expression vector pYM4807-OsGSM1 of paddy rice glutamine synthetase mutator gene as claimed in claim 1.
4. a preparation method for the paddy rice glutamine synthetase mutator gene as described in claim 1-3 any one, is characterized in that, comprises the steps:
1) the paddy rice glutamine synthetase gene sequence of take as shown in SEQ ID No1 is template, synthetic 26 primers shown in SEQ ID No5～SEQ ID No30 as shown, called after primer P1-P26 successively, adopt continuous extension PCR method amplification, synthetic water rice glutamine synthetase gene, and be building up to pBlueScriptII S+ carrier, and the plasmid of the paddy rice glutamine synthetase gene that obtains containing restructuring, concrete grammar is as follows:
A) take respectively primer P1, P10 as outside primer, P2-P9 is inner side primer synthetic fragment 1; Primer P11, P20 are outside primer, and P12-P19 is inner side primer synthetic fragment 2; Primer P21, P26 are outside primer, and P22-P25 is inner side primer synthetic fragment 3;
B) respectively get 1 μ L fragment 1, fragment 2, fragment 3 for template, with outside primer P1, P26, adopt continuous extension PCR method to increase to obtain the paddy rice glutamine synthetase gene that contains restructuring; Described reaction is in 50 μ L reaction systems, and the addition of inner side primer is respectively 10ng, and outside primer addition is respectively 100ng;
C) the paddy rice glutamine synthetase gene that contains restructuring amplification being obtained is cloned in pBluescriptII SK+ carrier, obtains the plasmid of the paddy rice glutamine synthetase gene that contains restructuring;
2) take the aforementioned plasmid obtaining is template, take is primer as shown in SEQ ID No31 and SEQ ID No32, adopt fallibility PCR method to carry out random mutation to paddy rice glutamine synthetase gene, amplified production is after BamHI and SacI double digestion, with correct reading frame, be inserted in coli expression carrier pYM4807, by electric shock, transform bacillus coli DH 5 alpha competent cell, obtain the sudden change library of paddy rice glutamine synthetase gene;
3) the aforementioned sudden change library obtaining is transformed into deficient strain JW3841-1, be coated on the substratum that contains 20-100mM grass ammonium phosphine and cultivate, the normal single bacterium colony of picking growth, be inoculated into containing cultivating in the LB liquid nutrient medium of Ampicillin Trihydrate, tentatively obtain containing the bacterium sample of the paddy rice glutamine synthetase mutator gene that careless ammonium phosphine resistance improves;
4) the bacterium sample tentatively obtaining and the bacterium sample that contains wild-type glutamine synthetase are diluted to same concentrations, being added drop-wise to respectively concentration is 0,10mM, 20mM, on the substratum of 100mM grass ammonium phosphine, cultivate, observe the growing state on different concns Glufosinate substratum, extraction obtains the plasmid vector that contains described mutator gene, be defined as pYM4807-OsGSM1, this plasmid obtains described paddy rice glutamine synthetase mutator gene through order-checking.
5. paddy rice glutamine synthetase mutator gene claimed in claim 1 is in the application of cultivating on anti-careless ammonium phosphine genetically modified crops.
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| WO2022142936A1 (en) * | 2020-12-31 | 2022-07-07 | 四川天豫兴禾生物科技有限公司 | Plant-derived glufosinate-ammonium-resistant glutamine synthase mutant, nucleic acid molecule, and applications |
| WO2023040564A1 (en) * | 2021-09-15 | 2023-03-23 | 四川天豫兴禾生物科技有限公司 | Glutamine synthetase mutant and application thereof in breeding of glufosinate-ammonium-resistant plant varieties |
| CN113736757A (en) * | 2021-09-15 | 2021-12-03 | 四川天豫兴禾生物科技有限公司 | Glutamine synthetase mutant with glufosinate-ammonium resistance, nucleic acid molecule and application |
| CN113736757B (en) * | 2021-09-15 | 2023-12-15 | 四川天豫兴禾生物科技有限公司 | Glutamine synthetase mutant with glufosinate resistance, nucleic acid molecule and application |
| CN113957060A (en) * | 2021-10-26 | 2022-01-21 | 四川天豫兴禾生物科技有限公司 | Glutamine synthetase mutant and application thereof |
| CN113957060B (en) * | 2021-10-26 | 2024-04-23 | 四川天豫兴禾生物科技有限公司 | Glutamine synthetase mutant and application thereof |
| CN114058600A (en) * | 2021-11-16 | 2022-02-18 | 四川天豫兴禾生物科技有限公司 | Glutamine synthetase mutant with glufosinate-ammonium resistance and application thereof |
| CN114058600B (en) * | 2021-11-16 | 2023-12-08 | 四川天豫兴禾生物科技有限公司 | Glutamine synthetase mutant with glufosinate resistance and application thereof |
| WO2023207669A1 (en) * | 2022-04-25 | 2023-11-02 | 四川天豫兴禾生物科技有限公司 | Method for acquiring protein with glufosinate resistance and glutamine synthetase mutant |
| CN115976042A (en) * | 2022-09-28 | 2023-04-18 | 上海市农业科学院 | Phosphorus efficient gene applied to rice and germplasm cultivation of rice |
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