CN116445521A - Herbicide-resistant gene and application thereof - Google Patents
Herbicide-resistant gene and application thereof Download PDFInfo
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- CN116445521A CN116445521A CN202310433360.XA CN202310433360A CN116445521A CN 116445521 A CN116445521 A CN 116445521A CN 202310433360 A CN202310433360 A CN 202310433360A CN 116445521 A CN116445521 A CN 116445521A
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- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- 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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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
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Abstract
The invention belongs to the technical field of plant genetic engineering, and discloses a herbicide-resistant gene and application thereof. In particular, the invention relates to herbicide resistant genes and proteins encoded thereby, constructed vectors and their use. The gene can be used for expression in crops to enhance the resistance of the crops to herbicides. The invention can be applied to the field of herbicide-resistant transgenic crop development and has good application prospect.
Description
Technical Field
The invention belongs to the technical field of plant genetic engineering, and in particular relates to herbicide-resistant genes, coded proteins, constructed vectors and application thereof.
Background
With the large-area application of the plant direct seeding cultivation technology in agricultural production, the field weed problem in the plant direct seeding production becomes an important factor affecting the crop yield. In particular, the physiological and metabolic processes of the grassy weeds in paddy fields are highly similar to those of paddy rice, and proper herbicide is lacked for prevention and control. Since the traditional breeding and domestication process does not involve herbicide tolerance traits, resistant resources in natural genetic resources are extremely rare. Screening and identifying endogenous resistance mutants by using herbicide inhibition target genes is a feasible way for developing herbicide-resistant germplasm resources of main grain crops at present. For example, resistance resources can be obtained by EMS mutagenesis of the imidazole herbicide target gene ALS. In recent years, the mutation is widely applied to varieties such as Jin Jing 818, and the like, and has obvious cultivation advantages in chemical weeding. However, it is known that the endogenous resistance sites of major herbicide target genes such as EPSPS, ACCase, ALS, HPPD and the like are mostly protected by foreign commercial company patents. At the same time, field weed acquired resistance increases rapidly with repeated application of a single type of herbicide. Therefore, development of novel herbicide resistance genes of plants and expression of heterologous metabolic genes by transgenic means to obtain herbicide resistance traits are necessary for cultivation of herbicide resistant plants.
Aryloxy-phenoxy-propionate (Aryloxyphenoxy propinoate, AOPP) herbicides are a class of highly active herbicides developed by the company helst in germany in the 70 th century. Has the advantages of high efficiency, broad spectrum, low toxicity, low residue and the like, and has been widely applied to agricultural production. Currently, there are about 20 commercial AOPP herbicides. By 2014, global AOPP herbicides were sold in excess of dollars 121.7 billion, one of the most widely used classes of herbicides in the world following glyphosate. AOPP herbicides generally belong to systemic selective herbicides and are mainly used in broadleaf crop fields (such as rape, peanut, soybean, tomatoes and the like) for preventing and killing annual and perennial grassy weeds, and the weeds can be killed after application for 2 weeks. However, certain herbicides of this type (e.g., quizalofop-p-ethyl, haloxyfop-methyl, etc.) are equally biocidal to gramineous crops, and their residues in the ecological environment can harm succeeding crops and non-target organisms. This limits the scope and duration of use of these herbicides to some extent. Therefore, the cultivation of gramineous crops with the herbicide resistance has great significance, not only can expand the application range of the herbicide, but also can reduce the agricultural production cost.
The mechanism of action of AOPP herbicides is mainly to kill plants by inhibiting acetyl-CoA carboxylase (ACCase) activity. This enzyme catalyzes the ATP-dependent carboxylation of acetyl-coa to induce malonyl-coa, a key metabolite in fatty acid and flavonoid biosynthesis, which is the first key step in fatty acid biosynthesis in plants, and then forms fatty acids under the action of fatty acid synthase. If synthesis of malonyl-coa is stopped, inhibition of ACCase will cause the plant to lose this intermediate, resulting in disruption of membrane structure, enhanced permeability, and cell destruction. The ACCase site mutation of part of gramineous crops (such as wheat and rice) can reduce the sensitivity of AOPP herbicide, and plant expression of the mutant protein can resist AOPP herbicide. The expression of ACCase mutated at the relevant sites of wheat and rice in plants to obtain resistance has been patented (chinese patent: 112410308a,109355264 b). However, in order to increase the level of resistance of transgenic crops and to increase the diversity of resistance genes, there is still a need for new herbicide-resistant genes and transgenic herbicide-resistant plants based thereon in production applications.
glutathione-S-transferases (GSTs) catalyze the coupling of tripeptide glutathione with several electrophilic, lipophilic compounds to form water-soluble products. This enzyme is present in a wide range of species from mammals to insects and plants and plays an important role in detoxification of many toxic substances including carcinogens, pesticides and herbicides, which has attracted considerable attention from pesticide researchers. In plants, GSTs are largely divided into six classes, phi, zeta, tau, theta, lambda and dhar. Although researchers have identified GST genes from rice, maize, wheat and other plant species that have herbicide resistance. However, a gene of GST capable of catalyzing coupling of quizalofop-p-ethyl with glutathione and causing resistance has not been found yet.
The invention firstly discovers the grass variety with enhanced herbicide metabolism, then clones a GSTs gene GST1 by utilizing the information of transcriptome sequences, and further proves the herbicide resistance of the GST1 gene in rice callus and the application thereof in developing transgenic herbicide resistant crops. The invention relates to a glutathione-S-transferase gene GST1, and the transgene at least can lead rice to generate resistance to at least one of AOPP herbicides such as quizalofop-p-ethyl, high-efficiency haloxyfop-methyl and the like.
Disclosure of Invention
The primary object of the present invention is to provide a herbicide-resistant gene. The introduction of the herbicide resistance gene into quizalofop-p-ethyl sensitive plants can significantly increase the tolerance of these sensitive plants to herbicides, so that herbicides which would not have been used on these plants to control weeds can be used on plants into which the herbicide resistance gene has been introduced.
In order to solve the above-mentioned technical problems,
the invention provides a herbicide-resistant gene, the nucleotide sequence of which is any one of the following: SEQ ID NO. 1, SEQ ID NO. 2, and functionally identical homologous gene sequences.
The herbicide is aryloxy-phenoxy propionate, and further comprises at least one of quizalofop-p-ethyl, haloxyfop-methyl, fenoxaprop-p-ethyl, clodinafop-propargyl, metamifop and cyhalofop-butyl.
The object of the second aspect of the present invention is to provide a protein polypeptide encoded by the herbicide resistance gene, the amino acid sequence comprising: SEQ ID NO. 3, SEQ ID NO. 4.
It is an object of the third aspect of the present invention to provide a plasmid for expressing a herbicide resistance gene, comprising the nucleotide sequence.
The plasmid comprises an expression frame formed by connecting a nucleotide sequence molecule and a nucleotide sequence for controlling expression, wherein the nucleotide sequence codes for the protein polypeptide.
As a preferred mode of the present invention, a glutathione-S-transferase gene GST1 gene fragment was ligated with double digested pET-28a (+) IN FUSION (Takara, inc.) kit to obtain a recombinant plasmid pET-28a (+) containing a glutathione-S-transferase gene.
The object of the fourth aspect of the present invention is to provide a recombinant strain expressing herbicide-resistant genes, which is transformed into E.coli BL21 (DE 3) to obtain recombinant strain E.coli BL21 (DE 3).
As a preferred mode of the invention, the constructed pET-28a (+) recombinant plasmid containing the glutathione-S-transferase gene is transformed into escherichia coli BL21 (DE 3) to obtain a recombinant strain E.coli BL21 (DE 3).
The object of the fifth aspect of the present invention is to provide the use of the herbicide-resistant gene, and to transfer the gene into a plant to obtain a resistant plant.
Further, constructing a plasmid for expressing the herbicide-resistant gene, transferring the plasmid into plant callus to obtain herbicide-resistant plant callus, and differentiating a resistant plant.
Further, the plants include dicots and monocots.
Still further, the plant comprises a gramineous crop.
Still further, the plant is rice, maize, cotton, wheat, soybean, turf grass or pasture grass.
The object of the sixth aspect of the present invention is to provide a system for rapidly detecting the detoxification metabolism of a herbicide by recombinant expression of a protein in vitro, comprising the steps of:
(1) Recombining sequences shown in SEQ ID NO. 1 and SEQ ID NO. 2 in a protein expression vector;
(2) Transferring the recombinant expression vector of step (1) into a host cell;
(3) Inducing the host cell to express a plurality of proteins of interest;
(4) Purifying the target protein to detect the metabolism of herbicide.
The seventh aspect of the present invention is to provide a system for rapidly detecting the antioxidant enzyme activity of the recombinant expressed glutathione-S-transferase in vitro, comprising the steps of:
(1) Recombining sequences shown in SEQ ID NO. 1 and SEQ ID NO. 2 in a protein expression vector;
(2) Transferring the recombinant expression vector of step (1) into a host cell;
(3) Detecting sensitivity of host cells into which glutathione-S-transferase is transferred to CHP on an induction plate medium;
(4) The size of the inhibition zone is recorded.
The object of the eighth aspect of the present invention is to provide the use of said herbicide resistant gene as a screening marker in transgenic plant cultures.
Improvement of herbicide resistance genes as the present invention: 1) An amino acid sequence having an identity of not less than 80% compared to SEQ ID NO. 3, SEQ ID NO. 4. 2) And a plant which, after introduction of the gene, is at least capable of increasing resistance to one or more of the following herbicides: AOPP herbicides, including but not limited to quizalofop-p-ethyl, haloxyfop-methyl, fenoxaprop-p-ethyl, sethoxydim, clodinafop-propargyl, oxazoxamide, cyhalofop-butyl, and the like.
The present invention provides a GST gene or a variant of the gene, the expression in a plant of a protein encoded by its nucleotide sequence being capable of causing resistance of a transgenic plant to at least one or more of the following herbicides: including but not limited to quizalofop-p-ethyl, haloxyfop-methyl, fenoxaprop-p-ethyl, clodinafop-propargyl, metamifop, cyhalofop-butyl, and the like. The genes of the invention can also be introduced into plants together with other herbicide resistance genes, such as EPSPS genes, so as to obtain simultaneous resistance to several herbicides including glyphosate. Furthermore, the gene of the present invention may be introduced into transgenic plants simultaneously with the insect-resistant gene, thereby obtaining transgenic plants resistant to both herbicide and insect.
By using the herbicide-resistant GST gene provided in the present invention, other homologous herbicide-resistant genes can be obtained by existing methods. For example, one of ordinary skill in the art can obtain these homologous genes by Southern hybridization methods or PCR methods. Further, one skilled in the art can use existing techniques to obtain variants of this gene, for example, including but not limited to: 1) Different nucleotide sequences obtained using different codons of the same amino acid, which sequences encode a protein polypeptide of the same activity; 2) By introducing variations in the nucleotide sequence but still encoding a protein having herbicide resistance. Such variations may be random variations, targeted point variations, or insertion or deletion variations. One of ordinary skill in the art will be able to make such variations by molecular biological methods. Thus, GST genes according to the invention also include genes which encode proteins which are at least 80% identical to SEQ ID NO. 3 or SEQ ID NO. 4 and which are resistant to herbicides, the herbicide resistance of these genes being verified by the herbicide resistance of the transgenic plants. Transgenic plants into which these genes are introduced are resistant to at least 1 herbicide: including but not limited to quizalofop-p-ethyl, haloxyfop-methyl, fenoxaprop-p-ethyl, clodinafop-propargyl, metamifop, cyhalofop-butyl, and the like.
The invention also provides a method for modifying plants, which comprises the following steps: comprising the steps of functionally connecting the herbicide resistant gene, a promoter and a terminator by using a plant gene transformation technology to form an expression frame, and expressing the herbicide resistant gene in plant cells. The obtained plants have herbicide resistance. Construction of an expression cassette capable of expression in plants has been a common technique. The plant may be rice, maize, cotton, wheat, soybean, turf or pasture.
The invention also provides a system for rapidly detecting the toxic and metabolic effects of the recombinant expression protein on herbicides IN vitro, wherein the nucleotide sequences SEQ ID NO. 1 and SEQ ID NO. 2 are subjected to homologous recombination with a linearized pET-28a vector subjected to double digestion by EcoRI and hindIII through an IN FUSION (Takara, company) kit. The resulting recombinant plasmid was transformed into highly expressed strain E.coli BL21 (DE 3) (Optimaceae, inc.). Recombinant expression strain BL21 (DE 3) was induced at 28℃for 12h with IPTG at a final concentration of 1mM, and expressed in large amounts to carry HIS 6 The tag GST1 protein was further subjected to affinity chromatography using a His tag protein purification kit (Coolaber, inc.) to obtain a purified GST1 protein. And the optimal elution conditions for GST1 protein were determined. Enzymatic degradation experiments are performed by using purified GST1 protein, and it is found that CDNB and NBD-CI can be used as substrates of GST1 enzyme, wherein NBD-CI can inhibit the activity of GST1 enzyme on CDNB at high concentration, and quizalofop-p-ethyl main metabolite quizalofop-ethyl can compete with CDNB for the coupling activity of GST1 enzyme and glutathione, and the characteristics can provide possibility for the cultivation of transgenic crops. The system provides a basis for rapidly identifying the metabolic effect of exogenous genes on herbicides.
The invention also provides a system for rapidly detecting the antioxidant enzyme activity of the in-vitro recombinant expressed glutathione-S-transferase. The glutathione-S-transferase sequence was homologous recombined with the linearized pET-28a vector after double digestion with EcoRI and hindIII by means of an IN FUSION (Takara, inc.) kit. The resulting recombinant plasmid was transformed into highly expressed strain E.coli BL21 (DE 3) (Optimaceae, inc.). 0.2mL of transformed E.coli BL21 (DE 3) (pET-GST) was cultured in LB liquid medium until OD600 = 1, and spread evenly on a medium containing kanamycin (50 mg L) -1 ) And IPTG (1 mmol L) -1 ) On LB solid medium plates, the plates were transferred to an incubator at 37℃for 1h. The sterilizing filters (diameter 5 mM) were then soaked in 0, 40, 80, 160 and 320mM CHP (CHP dry powder dissolved in acetone), respectively. Placing the soaked sterilizing filter sheet on the pre-cultured large intestine rodBacterial E.coli BL21 (DE 3) (pET-GST) and pET-28a were empty on LB plates. After 48h incubation at 37 ℃, CHP inhibition loop size was recorded and the antioxidant activity of the protein of interest was judged.
The invention also provides a nucleotide sequence molecule using the herbicide-resistant gene, which is used as a screening mark in plant transgenic cell culture. The herbicide resistant artificial gene capable of being expressed in plant cells may be constructed on the same DNA transformation fragment of the same plant transformation plasmid as the gene expression cassette of interest. The gene of interest may be any gene of value. The plant transformation plasmid can be introduced into plant tissue by gene gun, agrobacterium method or other methods, and a medium containing a suitable concentration of herbicide (e.g., quizalofop-p-ethyl) can selectively kill plant cells not introduced with the DNA transformation fragment, thereby selecting plant cells containing the gene of interest.
In summary, the gene of the present invention can be used for expression in plants to obtain resistance to at least 1 herbicide, so that weeds can be selectively killed by the herbicide, and the present invention can also be used for crop breeding and screening for plant cell culture. The invention also provides a system for rapidly detecting the metabolism of the exogenous gene on the herbicide.
The endogenous resistance sites of major herbicide target genes such as EPSPS, ACCase, ALS, HPPD and the like are known to be protected by patents of foreign commercial companies. More serious is the fact that these nationwide companies have been making prolonged protection of their core patents by means of minor alterations and modifications to the gene sequences, making them "new" patents, effectively circumventing the so-called 20-year patent validity period, trying to achieve the goal of monopolizing these functional genes and transformation technologies. In recent years, part of ACCase gene patents are granted (Chinese patent 109371000B and 109082416A), but most of the patents are based on foreign core patents, and if the purpose of industrialization of transgenes is to realize foreign control, the foreign mastered patent right is a past-free bank.
The invention clarifies the resistance mechanism of weeds to herbicide, discovers a new herbicide resistance gene resource glutathione-S-transferase gene GST1 with complete independent intellectual property rights, cultures herbicide resistant crop germplasm materials with complete independent intellectual property rights based on new functional genes, and provides a guarantee for the culture of new varieties of herbicide resistant transgenic crops in China.
Drawings
The following describes the embodiments of the present invention in further detail with reference to the accompanying drawings.
FIG. 1 is a graph showing the growth status of resistant (R) and susceptible (S) club head populations at the same dose of quizalofop-p-ethyl;
FIG. 2 is a schematic construction diagram of a plant expression vector, the expression cassette (a) consisting of a promoter, a club head herbicide resistance gene GST1 and a terminator;
FIG. 3 is a test result of the tolerance of the transgenic grass herbicide resistant gene GST1 rice callus to quizalofop-p-ethyl;
FIG. 4 is a test result of tolerance of the crop plant herbicide resistance gene GST1 rice callus to haloxyfop-R-methyl;
FIG. 5 is a schematic diagram showing the construction of GST1 protein recombinant expression vector expressed in E.coli BL21 (DE 3);
FIG. 6 is an electrophoresis chart of SDS-PAGE detection of a target protein;
FIG. 7 is a graph showing the enzymatic kinetic analysis of GST1 protein for substrates CDNB and NBD-CI, and the concentration of quizalofop-p-ethyl in inhibition of GST1 protein (LC 50 )；
FIG. 8 shows that the disc inhibition zone method pET-GST1 induces expression in E.coli BL21 (DE 3) to affect the sensitivity to CHP.
Detailed Description
The invention will be further illustrated with reference to specific examples, but the scope of the invention is not limited thereto. It should be understood that these examples are merely illustrative of the method of the present invention and are not intended to limit the scope of the present invention. The experimental methods, which are not explicitly stated or are not specifically stated, are conventional conditions and conventional methods well known to those skilled in the art.
Example 1 resistance assay against quizalofop-p-ethyl club head grass:
the grass weeds (Polypogonfugax) belong to the Gramineae, are annual weeds, and are one of the most serious weeds in winter rape and winter wheat fields in China. To determine the level of quizalofop-p-ethyl resistance of the lolium clavatum, the sensitivity level of the collected lolium clavatum organisms to quizalofop-p-ethyl was studied using the whole plant assay. The results show that the collected resistant club head grass biological R can normally grow under the recommended dose of quizalofop-p-ethyl field, and the fresh weight inhibition medium dose (GD 50 ) The value is 57g a.i./ha; whereas the control sensitive club head grass biological S GD 50 The value was 9.6g a.i./ha, the relative fold resistance was up to 6 times (FIG. 1). In addition, the resistance of R population to other ACCase inhibitor herbicides is also detected, and the R population is found to have resistance to haloxyfop-R-methyl with the resistance multiple reaching 6.08 times. Thus, resistant club head grass organisms may contain genes for quizalofop-p-ethyl and haloxyfop-methyl.
Example 2 cloning of resistance genes:
in nature, a clear evidence of plant quizalofop-p-ethyl resistance is that the quizalofop-ethyl resistance target enzyme ACCase gene is possessed, namely, the target enzyme ACCase related site mutation reduces the sensitivity of quizalofop-ethyl to the quizalofop-ethyl so as to obtain the resistance. The research of the resistance mechanism of the target enzyme is carried out on the resistant lolium grass seed group, 9 strains are selected from the resistant and sensitive populations respectively, cloning of the quizalofop-p-ethyl target enzyme gene acetyl coenzyme A carboxylase (ACCase) fragment is carried out, and the cloned gene sequences are compared, so that the R population has no target enzyme mutation compared with the S population, and the non-target enzyme resistance mechanism of the population is illustrated.
Transcriptome data for 2 groups of lolium clavatum (R, S) were obtained by transcriptome sequencing techniques. When the quality value of sequencing is statistically evaluated, the base Q30 of the sequencing is found to be more than 90%, which indicates that the sequencing quality is very reliable and successful. Further analysis of the amount of gene expression after assembly revealed that there were 102 genes differentially expressed between R and S, of which there were 1 gene involved in non-target resistance. Fluorescent quantitative PCR validation of the 1 non-target resistance-related differential genes found using transcriptome sequenced RNA samples revealed that: compared with the sensitive population S, the glutathione-S-transferase gene (GST 1) in R in the resistant population is always highly expressed. The total RNA of the extracted grass of Leptoradix was used as a template and was inverted into cDNA using the HiScript II cDNA first strand synthesis kit (Vazyme, inc.). Two specific primers (F: ATGGCGCCGGTGAAGGTGTT, R: TCAAGCTTTGGGCGGAACCATGCTG) were designed based on the CDS sequence of the GST1 gene predicted by the transcriptome, see SEQ ID No.5-6; full length amplification was performed using high fidelity 2 x PrimeSTAR Max Premix (TaKaRa, inc.) under the following conditions:
conditions are as follows:
and purifying, recovering, cloning and sequencing the target PCR product to finally obtain two variants (SEQ ID NO:1 and SEQ ID NO: 2) of the club head grass GST1 gene successfully, wherein the nucleotide sequence has the identity of 89.88 percent and the amino acids (SEQ ID NO:3 and SEQ ID NO: 4) have the identity of 86.76 percent.
Example 3 construction of GST1 Gene overexpression vector and obtaining of transgenic herbicide-resistant Rice
1) And constructing a GST1 gene overexpression vector.
Designing a specific primer (F: TGTTACTTCTGCAGGATGGCGCCGGTGAAGGTGT, R: CGGATCCATAACGCGTCAAGCTTTGGGCGGAACCA is shown IN SEQ ID NO. 7-8), amplifying the full-length sequence of a target gene from a T clone sequencing vector, combining a polyclonal enzyme cutting site of an expression vector POX (purchased from Wohabo biotechnology Co., ltd.), uniformly mixing with the linearized expression vector POX, carrying out ligation reaction by using an IN FUSION (Takara, inc.) kit, transforming competent cells of escherichia coli after the ligation reaction is finished, carrying out sequencing on a plasmid capable of amplifying a desired target fragment, finally obtaining a recombinant expression vector (figure 2), and transferring the recombinant expression vector into an agrobacterium competent EHA105 (Coolaber, inc.) by an electric shock method.
2) Screening of resistant callus and determination of sensitivity to herbicides.
Rice seeds are used as materials, after glume is removed, the rice seeds are sterilized by 75% ethanol and placed on a N6D culture medium for dark culture at 28 ℃, and dense callus particles are selected for transformation after 3 weeks. Transferring the recombinant plasmid into competent cells by using agrobacterium strain, to obtain the positive agrobacterium with expression vector POX-GST1 (Optimum, company) and culturing fresh agrobacterium solution. The rice callus is immersed in the bacterial liquid and is transferred to a selection culture medium for screening the resistant callus.
Determination of herbicide resistance of transgenic calli. The co-cultured calli were placed on screening medium containing Hygromycin, dark cultured for 14 days, and transferred to freshly prepared screening medium for further screening for 14 days. From the resistant calli grown after two rounds of screening, the milky yellow dense resistant calli were selected for herbicide tolerance experiments. The transgenic rice callus with POX-GFP over expression is used as a control, and the resistance of the transgenic rice callus with POX-GST1 over expression to 2 AOPP herbicides (quizalofop-p-ethyl and haloxyfop-methyl) is observed. The result shows that: 1) Compared with GFP, GST1 over-expressed rice callus grew better at quizalofop-p-ethyl 10, 20, 40nM concentrations. However, the GST1-2 variant grew better than GST1-1, indicating that the GST1-2 variant confers a higher level of resistance to quizalofop-p-ethyl on rice (FIG. 3). 2) Compared with GFP, GST1-2 variant type over-expression rice callus has better growth state under the high-efficiency haloxyfop-R-methyl 50, 100 and 200nM concentration. However, there was no significant difference in GST1-1 variant (FIG. 4). It was shown that the GST1-2 variant confers resistance to haloxyfop-R-methyl on rice, whereas the GST1-1 variant does not.
EXAMPLE 4 efficient in vitro expression and purification of GST1 Gene
1) Construction of GST1 gene recombinant expression vector. Specific primers containing the homology arm of pET-28a were designed (F: TGGGTCGCGGATCCGAAATGGCGCCGGTGAAGGTGT, R: GTGCGGCCGCAAGCTTGTCAAGCTTTGGGCGGAACCA, SEQ ID NO. 9-10;) the target fragment was amplified from the T cloning vector, purified and recovered, and the expression vector pET28a (from St. On the next hand, inc.) was recovered by EcoRI and hindIII cleavage gel. The amount of both the target fragment and linearized pET28a after recovery was 1:1 by the IN FUSION (Takara, inc.) kit ligation, suggesting 50ng-100ng each.
And (3) connecting a reaction system:
after the ligation reaction is finished, E.coli competent cells are transformed, the sequence of the plasmid capable of amplifying the expected target fragment is determined, and finally the obtained recombinant expression vector (figure 5) is transferred into E.coli BL21 (DE 3) strain by a heat shock method. Is coated on a liquid containing 50mg L -1 Plates of kanamycin, recombinant transformants E.coli BL21 (DE 3) (pET-GST 1) were obtained.
2) Expression and purification of GST1 proteins
Culturing recombinant strain E.coli BL21 (DE 3) (pET-GST 1) in LB medium to OD 600 After induction at 28℃for 12h with IPTG at a final concentration of 1mM added to the medium, cells were collected by centrifugation. The obtained cells were washed with pre-chilled PBS (50 mm, ph=8.0) and resuspended, then sonicated, and the supernatant was centrifuged. Because the constructed recombinant strain E.coli BL21 (DE 3) (pET-GST 1) has a label composed of 6 histidine residues at the N-terminal of GST1 protein induced and expressed by IPTG, the recombinant protein can be eluted and purified by a His-tagged protein purification kit (Coolaber, company), and detailed purification steps and required solutions are shown in the kit attached with instructions. Finally, SDS-PAGE electrophoresis is performed to detect the purity of the target protein, and the pure enzyme of GST1 is finally obtained (FIG. 6), and the optimal elution conditions are determined as the eluent (20 mM Tris-HCI,200mM imidazole, 500mM NaCI). Protein concentration was determined using Bradford method, bovine serum albumin as standard protein.
EXAMPLE 5GST1 protease assay
Enzymatic analysis is an enzymatic kinetic experiment by measuring the reaction of GST1 protein catalyzed Glutathione (GSH) with 1-chloro-2, 4-dinitrobenzene (CDNB), 4-chloro-7-nitrobenzo-2-oxa-1, 3-diazole (NBD-CI) substrates. GST1 protein was tested for enzymatic kinetics on known substrates (CDNB and NBD-CI) for GSTs enzymes.
1) Enzymatic reaction System with substrate CDNB (200. Mu.L):
GSH (final concentration 1 mM) 50. Mu.L
CDNB (final concentration 0.25-3 mM) 100. Mu.L
Purification of GST1 recombinant protein (1 mg/ml) 50ul
After addition of the recombinant protein, the reaction was started, carried out at 30℃and performed in 96-well plates by means of a microplate reader (Tecan Infinite M200Pro,austria) was measured at a wavelength of 340nm, once every 1min for a total of 3min. The results show (FIG. 7), V of both GST1-1 and GST1-2 variants against the substrate CDNB max 15.93mM and 3.41mM, respectively. K (K) m 0.45mM and 1.56mM, respectively. The GST1 protein has better coupling affinity to the substrate CDNB, but the coupling affinity of the GST1-1 protein to the CDNB is obviously higher than that of the GST1-2 protein.
2) Enzymatic reaction System with substrate NBD-CI (200. Mu.L):
GSH (final concentration 1 mM) 50. Mu.L
NBD-CI (final concentration 0.025-0.25 mM) 100. Mu.L
Purification of GST1 recombinant protein (1 mg/ml) 50ul
After addition of the recombinant protein, the reaction was started, the absorbance at 419nm was measured in 96-well plates at 30℃and detected every 1min for a total of 3min. The results show (FIG. 7), V of both GST1-1 and GST1-2 variants against substrate NBD-CI max 15.79mM and 3.12mM, respectively. K (K) m 0.31mM and 0.26mM, respectively. The GST1 protein has better coupling affinity to the substrate NBD-CI, but the coupling affinity of the GST1-1 protein to CDNB is obviously higher than that of the GST1-2 protein. In addition, it was observed that NBD-CI at a final concentration of greater than 0.25mM inhibited the enzymatic activity of GST1 protein.
3) Quizalofop-p-ethyl and quizalofop-ethyl serving as main metabolite have middle concentration (150 mu L of reaction system) for inhibiting GST1 protease activity
GSH (final concentration 1 mM) 50. Mu.L
Quizalofop-p-ethyl or quizalofop-p-ethyl acid (final concentration 3.125-800. Mu.M) 50. Mu.L
Purification of GST1 recombinant protein (1 mg/ml) 50ul
After addition of the recombinant protein, the reaction was started and was allowed to react at 37℃for 2 hours. GST was detected by adding 100ul of CDNB solution at a concentration of 2mM. The change in absorbance was measured at a wavelength of 340nm for 3min. The results showed (FIG. 7) that quizalofop-p (3.125-800. Mu.M) did not alter the coupling activity of GST1 protein to the substrate CDNB, with the quizalofop-p-main metabolite quizalofop-p-ethyl having a median inhibitory concentration of 656.15. Mu.M for GST1-1 protein and 49.99. Mu.M for GST1-2 protein. This indicates that quizalofop-p-ethyl, the main metabolite quizalofop-p-ethyl, competes for the coupling activity of GST1 protein to the substrate CDNB, and that quizalofop-ethyl is more suitable as a substrate for variant protein GST1-2, and quizalofop-p-ethyl cannot be used as a substrate for GST1 protein. This is probably the mechanism by which GST1 mediates the metabolism of club head grass against quizalofop-p-ethyl in example 1.
EXAMPLE 6 determination of antioxidant Activity of GST1 protein
Culturing 0.2mL of transformed E.coli BL21 (DE 3) (pET-GST 1) in LB liquid medium to OD 600 =1, uniformly coated on a medium containing kanamycin (50 mg L -1 ) And IPTG (1 mmol L) -1 ) On LB solid medium plates, the plates were transferred to an incubator at 37℃for 1h. The sterilizing filters (diameter 5 mM) were then soaked in 0, 40, 80, 160 and 320mM CHP (CHP dry powder dissolved in acetone), respectively. The soaked sterilizing filters were placed on the surface of pre-cultured LB plates of E.coli BL21 (DE 3) (pET-GST 1) and pET-28a empty. After culturing at 37℃for 48 hours, the CHP inhibition zone size was recorded, and the antioxidant activity of the target protein was judged. The results show (FIG. 8) that both pET-GST1-1 and pET-GST1-2 induced expression in E.coli BL21 (DE 3) significantly increased resistance to CHP. The GST1 gene is shown to have antioxidant enzyme activity, which is also probably one of the reasons why GST1 mediates the quizalofop-p-ethyl resistance of club head grass.
Finally, it should also be noted that the above list is merely a few specific embodiments of the present invention. Obviously, the invention is not limited to the above embodiments, but many variations are possible. It should be noted that modifications and adaptations to the present invention may occur to one skilled in the art without departing from the principles of the present invention and are intended to be comprehended within the scope of the present invention.
SEQ ID NO:1
ATGGCGCCGGTGAAGGTGTTCGGGCCGGCCATGTCGGCGAACGTGGCGCGGGTGCTGGTCTGCCTGGAGGAGGTGGGCGCCGAGTACGAGGTGGTCAACATCAACTTCAAGGTCCTGGAGCACAAGAGCCCCGAGCACCTCGCCAGAAACCCGTTCGGGCAAATCCCTGCTTTCCAGGATGGGGATCTGCTTCTCTTCGAGTCACGCGCGATTTCAAAGTACGTGCTCCGCAAGTACAAGACGGACGAGGTCGACCTCTTGAGGGAAGGCGACCTGAAGGAGGCGGCAATGGTGGACGTGTGGACGGAGGTGGACGCGCACACCTACAACCCGGCCGTCTCTCCCGTCGTGCAACAGTGTATCATAAATCCGCTGATGCGCGGGATCCCCACCGACGAGAAGGTCGTGGCCGAGGCTCTCGAGAAGCTGAAGAAGGTGCTCGAGGTCTACGAGGCGCGACTGGCCCAACACGCATACCTGGCTGGGGACTTTGTGAGCTTCGCCGACCTCAACCACTTCGCTTACACCTTCTACTTCATGAAGACGCCGCACGCGGCGCTGTTCGACTCGTACCCGCACGTCAAGGCCTGGTGGGAGAGGATCATGGCCAGGCCCGCCGTCAAGAAGGTCGCCGCCAGCATGGTTCCGCCCAAAGCTTGA
SEQ ID NO:2
ATGGCGCCGGTGAAGGTGTTCGGGCCGGCGATGTCGACGAACGTGGCGCGGGTGCTGGTGTTCCTGGAGGAGGTGGGCGCTGAGTACGAGGTGGTTAACATGGACTACAAGCTCCAGGAGCAGAAGAGCCCCGAGCACCTCGCCAGAAACCCGTTCGGGCAAATCCCTGCTTTCCAGGACGGGGATCTGCTCCTCTTCGAATCACGCGCGATTTCAAAGTACGTGCTCCGCAAATACAAATCAAACGGGGTGGACCTGCTGAGGGAAGGCAACCTGAAAGAAGCCGCGCTGGTGGACGTGTGGACGGAGGTGGACGCGCACACCTACAACCCGGCCCTCTCCCCGGTCATCTACGAGTGCCTCTTCAACCCGCTGATGCGCGGGATCCCCACCAACGACAACGTCGTCGCCGAGAGCCTGGAGAAGCTGAAGAAGGTGCTGGAGGTGTACGAGGCCCGCCTGTCGAAGCACGAGTACCTGGCCGGGGACTTCGTGAGCTTCGCCGACCTCAACCACTTCCCCTACACCTTCTACTTCATGACGACGCCGCACGCGTCCCTCTTCGACTCGTACCCGCACGTCAAGGCCTGGTGGGAGAGGATCATGGCCAGGCCCGCCGTCAAGAAGGTCGCCGCCAGCATGGTTCCGCCCAAAGCTTGA
SEQ ID NO:3
MAPVKVFGPAMSANVARVLVCLEEVGAEYEVVNINFKVLEHKSPEHLARNPFGQIPAFQDGDLLLFESRAISKYVLRKYKTDEVDLLREGDLKEAAMVDVWTEVDAHTYNPAVSPVVQQCIINPLMRGIPTDEKVVAEALEKLKKVLEVYEARLAQHAYLAGDFVSFADLNHFAYTFYFMKTPHAALFDSYPHVKAWWERIMARPAVKKVAASMVPPKA
SEQ ID NO:4
MAPVKVFGPAMSTNVARVLVFLEEVGAEYEVVNMDYKLQEQKSPEHLARNPFGQIPAFQDGDLLLFESRAISKYVLRKYKSNGVDLLREGNLKEAALVDVWTEVDAHTYNPALSPVIYECLFNPLMRGIPTNDNVVAESLEKLKKVLEVYEARLSKHEYLAGDFVSFADLNHFPYTFYFMTTPHASLFDSYPHVKAWWERIMARPAVKKVAASMVPPKA
Claims (10)
1. A herbicide resistant gene, the nucleotide sequence of which is any one of the following: SEQ ID NO. 1, SEQ ID NO. 2, and functionally identical homologous gene sequences.
2. The herbicide-resistant gene of claim 1, wherein the herbicide is an aryloxy-phenoxy propionate, further comprising at least one of quizalofop-p-ethyl, haloxyfop-methyl, fenoxaprop-p-ethyl, sethoxydim, clodinafop-propargyl, oxazafop-p-ethyl, cyhalofop-butyl.
3. The protein polypeptide encoded by the herbicide resistance gene of claim 1 or 2, the amino acid sequence comprising: SEQ ID NO. 3, SEQ ID NO. 4.
4. A plasmid expressing a herbicide resistance gene comprising the nucleotide sequence of claim 1.
5. A recombinant strain expressing herbicide-resistant genes, wherein the plasmid of claim 4 is transformed into E.coli BL21 (DE 3) to obtain recombinant strain E.coli BL21 (DE 3).
6. The use of the herbicide resistance gene of claim 1 or 2, characterized in that the gene is transferred into a plant to obtain a resistant plant; preferably into plant callus, herbicide resistant plant callus is obtained and resistant plants are differentiated.
7. The use according to claim 6, wherein the plants, including dicotyledonous and monocotyledonous plants, further comprise: gramineous crops; still further, the plant is rice, corn, cotton, wheat, soybean, turf or pasture.
8. A system for rapidly detecting the toxic and metabolic effects of recombinant expressed proteins on herbicides in vitro, comprising the steps of:
(1) Recombining sequences shown in SEQ ID NO. 1 and SEQ ID NO. 2 in a protein expression vector;
(2) Transferring the recombinant expression vector of step (1) into a host cell;
(3) Inducing the host cell to express a plurality of proteins of interest;
(4) Purifying the target protein to detect the metabolism of herbicide.
9. A system for rapidly detecting the antioxidant enzyme activity of in vitro recombinant expression glutathione-S-transferase, which is characterized by comprising the following steps:
(1) Recombining sequences shown in SEQ ID NO. 1 and SEQ ID NO. 2 in a protein expression vector;
(2) Transferring the recombinant expression vector of step (1) into a host cell;
(3) Detecting sensitivity of host cells into which glutathione-S-transferase is transferred to CHP on an induction plate medium;
(4) The size of the inhibition zone is recorded.
10. Use of the herbicide resistance gene of claim 1 or 2 as a selectable marker in transgenic plant culture.
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