CN105586324A - Glyphosate-resistant related protein originated from aspergillus flavus as well as coding gene and application thereof - Google Patents
Glyphosate-resistant related protein originated from aspergillus flavus as well as coding gene and application thereof Download PDFInfo
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- CN105586324A CN105586324A CN201610060452.8A CN201610060452A CN105586324A CN 105586324 A CN105586324 A CN 105586324A CN 201610060452 A CN201610060452 A CN 201610060452A CN 105586324 A CN105586324 A CN 105586324A
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- C12N9/1092—3-Phosphoshikimate 1-carboxyvinyltransferase (2.5.1.19), i.e. 5-enolpyruvylshikimate-3-phosphate synthase
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- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8274—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for herbicide resistance
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- C12Y205/01019—3-Phosphoshikimate 1-carboxyvinyltransferase (2.5.1.19), i.e. 5-enolpyruvylshikimate-3-phosphate synthase
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Abstract
The invention discloses a glyphosate-resistant related protein originated from aspergillus flavus as well as a coding gene and application thereof. The protein provided by the invention is named as EPSPS protein shown by the following (a) or (b), wherein (a) is a protein consisting of an amino acid sequence shown by a sequence 1 in a sequence table; and (b) is a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown by the sequence 1 in the sequence table, is related to glyphosate resistance and is derived from the sequence 1. The gene (EPSPS gene) for coding the EPSPS protein also belongs to the protection scope of the invention. The gene disclosed by the invention can be used for culturing a transgenic crop or transgenic microorganism with enhanced glyphosate resistance, and has a very high application value.
Description
Technical field
The present invention relates to a kind of resistance glyphosate GAP-associated protein GAP and encoding gene and application that is derived from aspergillus flavus.
Background technology
Glyphosate (glyphosate) is go out natural disposition, inner-adsorption conduction-type herbicide of a kind of wide spectrum, has physicochemical property steadyFixed, efficient, low toxicity, low-residual, easily by advantages such as microorganism decomposition. Glyphosate be widely used in corn, soybean,The production of the crops such as rape is one of herbicide of current use amount maximum. Glyphosate is PEPAnalog, is a kind of nonselective herbicide, can form with shikimic acid-3-phosphoric acid (S3P), epsp synthaseEPSP-S3P-glyphosate complex, and then stop PEP and epsp synthase combination, make the 5-alkene in shikimic acid pathwayAlcohol formula shikimic acid-3-phosphate synthase (EPSPS) is subject to competitive the inhibition, the biology of aromatic compound in organismSynthetic being interfered, thus biological death caused. Because herbicide does not have selection effect to weeds and crop, usuallyPhotosynthesis on crop and some metabolic processes produce certain impact and toxic action. This has just limited herbicideApplication, antiweed genetically modified crops research launches thereupon for this reason.
Antiglyphosate gene is the gene that is present in the antagonism glyphosate function in some specific species, sweet by blocking grassThe interference of phosphine to aromatic compound biosynthesis pathway, makes glyphosate lose the toxic action to plant. Adopt geneThe means such as engineering, cultivate glyphosate resistant crops new varieties, to controlling weeds in field and promoting that crop production has important meaningJustice.
Summary of the invention
The object of this invention is to provide a kind of resistance glyphosate GAP-associated protein GAP and encoding gene and application that is derived from aspergillus flavus.
Albumen provided by the invention, available from aspergillus flavus, called after EPSPS albumen is following (a) or (b):
(a) protein being formed by the amino acid sequence shown in sequence in sequence table 1;
(b) by amino acid sequence shown in sequence in sequence table 1 through the replacement of one or several amino acid residue and/Or disappearance and/or interpolation and the protein that by sequence 1 derived relevant to glyphosate resistance.
In order to make the protein in (a) be convenient to purifying, can be in by sequence table the amino acid order shown in sequence 1The amino terminal of the protein of row composition or carboxyl terminal connect upper label as shown in table 1.
The sequence of table 1 label
Protein in above-mentioned (b) can manually synthesize, and also can first synthesize its encoding gene, then carries out biological expression and obtain.The encoding gene of the protein in above-mentioned (b) can be by lacking one in the DNA sequence dna shown in sequence in sequence table 2Or the codon of several amino acid residues, and/or carry out the missense mutation of one or several base-pair, and/or its 5 'The coded sequence that end and/or 3 ' end connects the label shown in table 1 obtains.
The gene (EPSPS gene) of described EPSPS albumen of encoding also belongs to protection scope of the present invention.
Described EPSPS gene is the DNA molecular of following (1) or (2) or (3):
(1) DNA molecular of code area as shown in sequence in sequence table 2;
(2) DNA of the DNA sequence dna hybridization limiting with (1) under stringent condition and coding glyphosate resistance GAP-associated protein GAPMolecule;
(3) DNA sequence dna limiting with (1) at least has 70%, at least have 75%, at least have 80%, at leastHave 85%, at least have 90%, at least have 95%, at least have 96%, at least have 97%, at least have 98%Or at least there is the DNA molecular of 99% homology and the coding albumen relevant to glyphosate resistance.
Described stringent condition can be as follows: at 6 × SSC, in the solution of 0.5%SDS, 65℃Under C, hybridize, thenWith 2 × SSC, 0.1%SDS and 1 × SSC, 0.1%SDS respectively washes film once.
The recombinant vector, expression cassette, transgenic cell line or the recombinant bacterium that contain described EPSPS gene all belong to the present inventionProtection domain.
Described recombinant vector specifically can be described EPSPS gene insertion pGEX-4T-1 carrier or pET-30a (+) is carriedThe recombinant plasmid that body obtains. Described recombinant vector specifically can be in the EcoR of pGEX-4T-1 carrier I and Sal I enzyme and cutsThe recombinant plasmid that between site, the double chain DNA molecule shown in the sequence 2 of insetion sequence table obtains. Described recombinant vector toolBody can be shown in the sequence 2 of insetion sequence table between the EcoR I of pET-30a (+) carrier and Sal I restriction enzyme siteThe recombinant plasmid that obtains of double chain DNA molecule.
Described recombinant bacterium is that above arbitrary described recombinant vector is imported to the recombinant bacterium that Host Strains obtains. Described Host Strains canFor Escherichia coli, specifically can be e. coli bl21 (DE3).
The present invention also protects a kind of method of cultivating recombinant microorganism, comprises the steps: described EPSPS gene to leadThe microorganism that enters to set out, obtains the recombinant microorganism higher than the described microorganism that sets out to glyphosate resistance. Described EPSPS baseMicroorganism sets out described in cause specifically can import by above arbitrary described recombinant vector. The described microorganism that sets out can be bacterium,Specifically can be Escherichia coli, more specifically can be e. coli bl21 (DE3).
The present invention also protects the application of described EPSPS albumen or described EPSPS gene, for following (c1), (c2) or(c3) at least one in:
(c1) regulating plant or the microorganism resistance to glyphosate;
(c2) increase plant or the resistance of microorganism to glyphosate;
(c3) cultivate plant or microorganism that glyphosate resistance is increased.
Described microorganism can be bacterium, specifically can be Escherichia coli, more specifically can be e. coli bl21 (DE3).
The present invention has found a new albumen, and its encoding gene is proceeded to expression in escherichia coli, can improve transgenosisThe resistance of bacterial strain to glyphosate. Therefore, gene of the present invention can be used for cultivating the transgenosis work that glyphosate resistance is strengthenedArticle kind or transgenic microorganism, have very high using value.
Brief description of the drawings
Fig. 1 is the result of the step 3 of embodiment 1.
Fig. 2 is the result of the step 4 of embodiment 1.
Detailed description of the invention
Following embodiment is convenient to understand better the present invention, but does not limit the present invention. Experiment in following embodimentMethod, if no special instructions, is conventional method. Test material used in following embodiment, if no special instructions,Be and purchase available from routine biochemistry reagent shop. Quantitative test in following examples, all arranges three times and repeats in factTest results averaged. PGEX-4T-1 carrier: GEHealthcare company. PET-30a (+) carrier: NovagenCompany. E. coli bl21 (DE3): Tian Gen biochemical technology Co., Ltd, catalog number is CB105-02. Grass is sweetPhosphine: About Monsanto Chemicals.
Aspergillus flavus TZ1985 is the strain resistance glyphosate bacterial strain that inventor separates preservation. From aspergillus flavus TZ1985, find oneIndividual new albumen, as shown in the sequence 1 of sequence table, by its called after EPSPS albumen. By the gene life of coding EPSPS albumenEPSPS gene by name, its ORFs is as shown in the sequence 2 of sequence table.
The structural formula of glyphosate is as follows:
The functional verification of embodiment 1, EPSPS albumen
One, construction recombination plasmid
1, the double chain DNA molecule shown in the sequence 2 of composition sequence table.
2, the double chain DNA molecule obtaining taking step 1 is template, adopts drawing of EcoRTZEPF and SalTZEPR compositionThing, to carrying out pcr amplification, obtains pcr amplification product.
EcoRTZEPF：5'-CGGAATTCATGGTACATCCTGGCGTAGAGC-3'；
SalTZEPR：5'-GCGTCGACCTACTCCAACTTGGCCGAGAAC-3'。
3, the pcr amplification product obtaining by restriction enzyme EcoR I and Sal I double digestion step 2, reclaims enzyme and cutsProduct.
4,, with restriction enzyme EcoR I and Sal I double digestion pGEX-4T-1 carrier, reclaim the carrier of about 4.9kbSkeleton.
5, the carrier framework of the enzyme of step 3 being cut to product and step 4 is connected, and obtains recombinant plasmid pGEXTZ. According toSequencing result, carries out structure to recombinant plasmid pGEXTZ and is described below: at the EcoR of pGEX-4T-1 carrier I and SalBetween I restriction enzyme site, insert the double chain DNA molecule shown in the sequence 2 of sequence table.
6, with restriction enzyme EcoR I and Sal I double digestion pET-30a (+) carrier, reclaim about 5.4kb'sCarrier framework.
7, the carrier framework of the enzyme of step 3 being cut to product and step 6 is connected, and obtains recombinant plasmid pETTZ. According to surveyOrder result, carries out structure to recombinant plasmid pETTZ and is described below: at EcoR I and the Sal of pET-30a (+) carrierBetween I restriction enzyme site, insert the double chain DNA molecule shown in the sequence 2 of sequence table.
Two, prepare recombinant bacterium
Recombinant plasmid pGEXTZ is imported to e. coli bl21 (DE3), obtain recombinant bacterium pGEXZ.
Recombinant plasmid pETTZ is imported to e. coli bl21 (DE3), obtain recombinant bacterium pETZ.
PGEX-4T-1 carrier is imported to e. coli bl21 (DE3), obtain recombinant bacterium pGEX.
PET-30a (+) carrier is imported to e. coli bl21 (DE3), obtain recombinant bacterium pET.
Three, the resistance of recombinant bacterium to glyphosate
Respectively recombinant bacterium pETZ, recombinant bacterium pET and e. coli bl21 (DE3) are put down as follows as bacterium to be measuredRow test: bacterium monoclonal to be measured is inoculated in to the LB fluid nutrient medium containing variable concentrations glyphosate, and glyphosate concentration respectivelyFor 25mmol/L, 50mmol/L, 75mmol/L or 100mmol/L, arrange do not add glyphosate control treatment (Glyphosate concentration is 0mmol/L), 37 DEG C, 200rpm shaken cultivation 7d, then detect OD600nmLight absorption value.
Each glyphosate concentration arranges five reprocessings, results averaged.
The results are shown in Figure 1. In the time that glyphosate concentration is 0-50mmol/L, e. coli bl21 (DE3), recombinant bacterium pETZCan grow with recombinant bacterium pET. In the time that glyphosate concentration is 75mmol/L, only have recombinant bacterium pETZ normally to give birth toLong. In the time that glyphosate concentration is 100mmol/L, each bacterium to be measured all can not grow. Result shows, proceeds to EPSPSThe Escherichia coli of gene have been improved the resistance to glyphosate.
Four, abduction delivering EPSPS albumen in Escherichia coli
Carry out recombinant bacterium pGEXZ, recombinant bacterium pETZ, recombinant bacterium pGEX and recombinant bacterium pET as bacterium to be measured respectively asLower parallel test:
1, bacterium to be measured is seeded to LB fluid nutrient medium, 37 DEG C, 200rpm shaken cultivation are to OD600nm＝0.6。
2,, after completing steps 1, adding IPTG and making its concentration is 0.4mmol/L, 30 DEG C, 170rpm shaken cultivation,Every sampling in 2 hours, carry out SDS-PAGE.
Fig. 2 is shown in by SDS-PAGE collection of illustrative plates. In Fig. 2: A1 is recombinant bacterium pGEX, A2 is recombinant bacterium pET, and B1 is restructuringBacterium pGEXZ, B2 is recombinant bacterium pETZ; 1-6: be followed successively by when IPTG induces 0,2,4,6,8 and 10 hour and getThe sample of sample; M: albumen Marker.
Result shows, after IPTG induction 2h, the destination protein in recombinant bacterium pGEXZ and recombinant bacterium pETZ starts to express,Along with the growth of induction time, expressing quantity increases to some extent, in the time that induction reaches 8h, expressing quantity substantially inStable state, protein induced expression success.
Claims (10)
1. an albumen is following (a) or (b):
(a) protein being formed by the amino acid sequence shown in sequence in sequence table 1;
(b) by amino acid sequence shown in sequence in sequence table 1 through the replacement of one or several amino acid residue and/Or disappearance and/or interpolation and the protein that by sequence 1 derived relevant to glyphosate resistance.
2. the gene of albumen described in coding claim 1.
3. gene as claimed in claim 2, is characterized in that: described gene is following (1) or (2) or (3)DNA molecular:
(1) DNA molecular of code area as shown in sequence in sequence table 2;
(2) DNA of the DNA sequence dna hybridization limiting with (1) under stringent condition and coding glyphosate resistance GAP-associated protein GAPMolecule;
(3) DNA sequence dna limiting with (1) at least has 70%, at least have 75%, at least have 80%, at leastHave 85%, at least have 90%, at least have 95%, at least have 96%, at least have 97%, at least have 98%Or at least there is the DNA molecular of 99% homology and the coding albumen relevant to glyphosate resistance.
4. contain recombinant vector, expression cassette, transgenic cell line or the recombinant bacterium of gene described in claim 2 or 3.
5. recombinant vector as claimed in claim 4, is characterized in that: described recombinant vector is by claim 2Or described in 3, gene inserts the recombinant plasmid that pGEX-4T-1 carrier or pET-30a (+) carrier obtain.
6. recombinant bacterium as claimed in claim 4, is characterized in that: described recombinant bacterium is for weighing described in claim 5Group carrier imports the recombinant bacterium that Host Strains obtains.
7. cultivate a method for recombinant microorganism, comprise the steps: gene described in claim 2 or 3 to importThe microorganism that sets out, obtains the recombinant microorganism higher than the described microorganism that sets out to the resistance of glyphosate.
8. method as claimed in claim 7, is characterized in that: described microorganism is Escherichia coli.
9. encoding gene described in encoding gene or claim 3 described in albumen or claim 2 described in claim 1Application, for following (c1), (c2) or (c3):
(c1) regulating plant or the microorganism resistance to glyphosate;
(c2) increase plant or the resistance of microorganism to glyphosate;
(c3) cultivate plant or microorganism that glyphosate resistance is increased.
10. application as claimed in claim 9, is characterized in that: described microorganism is Escherichia coli.
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| CN201610060452.8A CN105586324A (en) | 2016-01-28 | 2016-01-28 | Glyphosate-resistant related protein originated from aspergillus flavus as well as coding gene and application thereof |
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| CN201610060452.8A CN105586324A (en) | 2016-01-28 | 2016-01-28 | Glyphosate-resistant related protein originated from aspergillus flavus as well as coding gene and application thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106699854A (en) * | 2017-01-09 | 2017-05-24 | 广西大学 | Functional protein POX04420 and encoding gene and application thereof |
| CN113185589A (en) * | 2021-04-23 | 2021-07-30 | 东北农业大学 | Glyphosate-resistant related protein and coding gene and application thereof |
-
2016
- 2016-01-28 CN CN201610060452.8A patent/CN105586324A/en active Pending
Non-Patent Citations (3)
| Title |
|---|
| MARTIN M. VILA‑AIUB ET AL.: "No fitness cost of glyphosate resistance endowed by massive EPSPS gene amplification in Amaranthus palmeri", 《PLANTA》 * |
| 于海涛: "抗草甘膦真菌的分离及EPSPS基因克隆与表达研究", 《中国优秀硕士学位论文全文数据库农业科技辑》 * |
| 吴丹丹等: "草甘膦抗性真菌的分离与鉴定", 《环境昆虫学报》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106699854A (en) * | 2017-01-09 | 2017-05-24 | 广西大学 | Functional protein POX04420 and encoding gene and application thereof |
| CN113185589A (en) * | 2021-04-23 | 2021-07-30 | 东北农业大学 | Glyphosate-resistant related protein and coding gene and application thereof |
| CN113185589B (en) * | 2021-04-23 | 2023-05-30 | 东北农业大学 | Glyphosate-resistant related protein, and coding gene and application thereof |
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