CN114525292A - Application of gat3 gene and mutant thereof in cultivation of glyphosate-resistant crops - Google Patents
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Abstract
The invention disclosesgat3The gene and the application of the mutant thereof in culturing glyphosate-resistant crops. The describedgat3The nucleotide sequence of the gene is shown in a sequence table SEQ ID No: 1 is shown in the specification; the mutants comprise gat3-17, gat3-35, gat3-38, gat3-53, gat3-77 and gat 3-93. The invention screens a high glyphosate resistance from Pfam databasegat3Genes, and pairsgat3The protein is subjected to point mutation to further obtain the glyphosate-resistant proteingat3Protein processThe variant lays a foundation for cultivating crops with high glyphosate resistance.
Description
Technical Field
The invention belongs to the technical field of plant biology, and particularly relates to a plant biological enzyme inhibitorgat3The gene and the application of the mutant thereof in culturing glyphosate-resistant crops.
Background
The glyphosate is the only one which can effectively inhibit the activity of phosphoenolpyruvylshikimate-3-phosphate synthase (EPSPS) in the plant shikimate metabolic pathway, thereby blocking the biosynthesis of aromatic amino acids and finally leading the plant to die. Plants with natural resistance to glyphosate exist in nature, researchers find different resistance mechanisms from the plants, and use the resistance genes to obtain transgenic crops with high glyphosate tolerance. Among the existing strategies against glyphosate, there are roughly two major categories of mechanisms: target resistance and non-target resistance. The target resistance is mainly directed to relevant mechanisms of EPSPS, such as overexpression or mutation. Non-target resistance can be divided into chemical modification, oxidative degradation, transport and other related mechanisms.
In 2004, Castle et al first published a research result on N-acetyltransferase modification of Glyphosate, a group of which isolated a Glyphosate N-acetyltransferase (GAT) from the soil microorganism Bacillus licheniformis, and obtained GAT enzyme of high catalytic efficiency through 11 rounds of DNA shuffling. The enzyme belongs to the GNAT superfamily, can transfer acetyl to secondary amine of glyphosate, no longer has inhibition effect on EPSP synthase, and thus loses herbicide activity. The present invention is expected to find GAT enzyme with high natural activity and replace the better amino acid residue to change the activity of GAT enzyme, and meanwhile, the 11 th round GAT enzyme in Castle team is used as positive control in the experiment and is temporarily named as GATC11。
Disclosure of Invention
The object of the present invention is to providegat3The gene and the application of the mutant thereof in culturing glyphosate-resistant crops.
A kind ofgat3A gene ofgatThe polynucleotide of the gene is represented by (a), (b), (c) or (d):
(a) as shown in a sequence table SEQ ID No: 1; or
(b) And SEQ ID No: 1 under stringent hybridization conditions, and the protein encoded by the polynucleotide still has glyphosate resistance activity;
(c) and SEQ ID No: 1 or a polynucleotide having a homology of at least 90% or more; or
(d) In SEQ ID No: 1 by deletion, substitution or insertion of one or more bases, and the protein coded by the polynucleotide mutant still has glyphosate resistant activity.
A gat3 protein, wherein the amino acid sequence of the gat3 protein is shown in (a), (b) or (c):
(a) as shown in a sequence table SEQ ID No: 2; or
(b) And SEQ ID No: 2, an amino acid having at least 90% or more homology with the amino acid shown in the formula (I); or
(c) In SEQ ID No: 2, and the protein mutant is obtained by deleting, substituting or inserting one or more amino acids on the basis of the protein shown in the formula 2, and the protein still has glyphosate resistant activity.
A mutant gat3 protein, which comprises gat3-17, gat3-35, gat3-38, gat3-53, gat3-77, gat 3-93; the 17 th amino acid residue of the amino acid sequence of the mutant gat3-17, which is the gat3 protein, is mutated from L to I; the 35 th amino acid residue of the amino acid sequence of the mutant gat3-35, which is the gat3 protein, is mutated from S to G; the 38 th amino acid residue of the amino acid sequence of the mutant gat3-38, which is the gat3 protein, is mutated from M to D; the 53 th amino acid residue of the amino acid sequence of the mutant gat3-53, which is the gat3 protein, is mutated from I to V; the 77 th amino acid residue of the amino acid sequence of the mutant gat3-77 gat3 protein is mutated from V to M; the 93 th amino acid residue of the amino acid sequence of the mutant gat3-93, which is the gat3 protein, is mutated from V to I.
Comprising the followinggat3A vector for the gene.
Comprising the followinggat3Engineering bacteria of gene carrier.
Detecting thegat3Primers for any fragment of the gene.
The use of the gat3 protein or the mutant of the gat3 protein in the cultivation of glyphosate-resistant crops.
The invention has the beneficial effects that: the invention screens a high glyphosate resistance from Pfam databasegat3Genes, and pairsgat3The protein is subjected to point mutation to further obtain the glyphosate-resistant proteingat3The protein mutant lays a foundation for cultivating crops with high glyphosate resistance.
Drawings
FIG. 1 shows pET28a-gat1Vector mapping.
FIG. 2 shows the qualitative screening of resistant strains on LK plates containing 100 mM glyphosate.
FIG. 3 shows pET28a-gat NMIC values of transformed strains.
FIG. 4 shows pET28a-gat Growth curves of the N transformed strains.
FIG. 5 shows the qualitative screening of resistant strains on LK plates containing 100 mM glyphosate.
FIG. 6 shows pET28a-mgat3-NMIC values of transformed strains.
Detailed Description
In order that the invention may be more fully understood, reference will now be made to the following description. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Example 1 vector construction
gatSelection of resistance genes: the information of the PF00583 (Pfam: Family: Acetyltransf-1 (PF00583) (xfam. org)) protein Family was downloaded in the Pfam database (http:// Pfam. xfam. org /), and 3043 sequences were searched in the Uniparc database using hmmsearch.Performing cluster analysis on the sequences according to 90 percent of similarity, classifying the sequences into 1196 classes, sequencing the sequences from large to small according to the numerical value of the class center sequence, and selecting 18 sequences (cluster number) from the sequences>20) Are sequentially named asgat 1 - gat 18。
18 sequences andgat c11 (results of round 11) sequence fragments were optimized and synthesized by Shimeji Kogyo according to E.coli codons. The BamH I/Sac I double enzyme digestion synthesis vector and pET28a + (R) vector are used, the former is used for recovering synthetic fragments, the latter is used for providing a vector framework, each synthetic fragment is respectively connected with the vector framework to form pET28a-gat1And (5) screening the carrier.
The 18 constructed screening vectors were transformed into Trans1-T1 strain for storage, and at the same time, transformed into BL21 (DE 3) strain for subsequent screening experiments. All strains were kept in one portion at-20 ℃ and one portion at-80 ℃. The vector skeleton and gene fragment are linearized by digestion and then treated with T4The DNA is formed by connection of DNA enzymes, so that the constructed vector needs to be identified again by an enzyme digestion method. The single clones cultured after plating were picked up in 5 mL LK broth and cultured overnight in a shaker at 37 ℃ and 200 rpm. Extracting plasmid from 2 mL bacterial liquid, digesting with BamH I and Sac I, if electrophoresis band can show more than 5kb and correspondinggatTwo bands of fragment size are the screening vectors for accurate ligation.
Example 2 selection of resistance of transformed strains
Strain activation: 50 μ L of the deposited strain was shake-cultured in 5 mL LK broth at 37 ℃ for 8-10 h at 200 rpm. Burning the inoculating loop on the outer flame of an alcohol lamp for sterilization, drawing lines on an LK solid plate after cooling to room temperature and dipping the bacterium liquid, sealing the mark, and then placing the mark upside down in an incubator at 37 ℃ for overnight culture.
5 mL LK broth was added to the tubes for use, a 10. mu.L tip was clamped with sterilized forceps, and 3 single colonies of appropriate size were picked up on the plate in 3 tubes and shake-cultured at 37 ℃ at 200 rpm. If the bacterial liquid OD600 reaches 1.0, the culture is carried out for 12-13 h. If the OD600 of the bacterial liquid is 0.6, the bacterial liquid needs to be cultured for 6-8 h.
Plate qualitative screening: in plate screening, the bacterial solution needs to be activated until the OD600 reaches about 0.60. And (3) sucking 200 mu L of bacterial liquid in each test tube, measuring the OD600 value of the bacterial liquid by using an enzyme-labeling instrument, selecting the bacterial liquid with the value closest to 0.60, diluting the bacterial liquid, and adjusting the bacterial liquid to be between 0.590 and 0.610.
The strains are diluted in LK liquid culture medium in 10 times to form a gradient and the gradient is 10 times5And (4) doubling. Taking 1 mL of bacterial liquid into a 2 mL centrifuge tube, adding 900 mu L of LK liquid culture medium into 5 dilution gradient centrifuge tubes, sucking 100 mu L of bacterial liquid into a first dilution gradient centrifuge tube, namely diluting the original bacterial liquid to 101Multiple, marked as "10" on centrifuge tube lid1", then, 100. mu.L of diluted bacteria liquid is sucked from the tube and is diluted to 102Multiple, marked as "102", and in turn, completing the dilution gradient.
On LK square solid plates containing 100 mM glyphosate, 8. mu.L of each gradiental bacterium was pipetted down to the center of each cell. Wherein, each horizontal row is taken as the dilution gradient of one strain, and each vertical row is respectively taken as a positive control from top to bottomgatc11The transformation strain, 4 experimental groups of transformation strains and a blank vector transformation strain. Each strain had two parallel replicate plates. And sealing each plate, then placing the plate upside down in an incubator at 37 ℃ for culturing for 40h, photographing and recording the growth condition of the strains.
The experimental results are shown in FIG. 2, which are positive control, 18 experimental groups and transformed strains of blank vector from top to bottom. Wherein the content of the first and second substances,gat2andgat3the growth conditions of each diluted gradient bacterial liquid of the transformed strain are better than those of other strains.
And (3) MIC quantitative screening:
when the MIC value of each strain is measured, the OD600 of the bacterial liquid needs to reach about 1.0. And (3) sucking 200 mu L of bacterial liquid in each test tube, measuring the OD600 value of the bacterial liquid by using an enzyme-labeling instrument, selecting the bacterial liquid with the value closest to 1.0, diluting the bacterial liquid, and adjusting the bacterial liquid to be between 0.990 and 1.010.
In a 96-well plate (12 × 8), each column was a concentration gradient, and from left to right, 990 μ L of M9 liquid medium was sequentially added from the addition well by a discharging gun to M9 liquid medium containing 0 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, and 110 mM glyphosate. Repeating 4 groups of 12 gradients of each strain in 4 transverse rows of the 96 deep-well plate, sucking 10 mu L of bacterial liquid from a sample adding groove by using a discharge gun, inoculating the bacterial liquid into a liquid culture medium by 1 percent of inoculation amount, sealing the opening, and placing the opening on a shaker at 200 rpm and 37 ℃ for 24 hours. And (3) sucking 200 mu L of bacterial liquid in each plate into the ELISA plate by using a row gun, measuring the OD600 value of the bacterial liquid and analyzing the MIC value of each strain.
As shown in FIG. 3, the growth of most strains was already inhibited by 10 mM glyphosate, and thus 10 mM was considered as a limiting concentration for glyphosate resistance. In resistant strains, glyphosate pairsgat1The minimum inhibitory concentration of the transformed strain is 60 mM, and the minimum MIC value of the high-resistance strain is also the minimum MIC value, so that 50 mM can be considered as the screening concentration of the high-resistance strain. Wherein the positive controlgat c11 The MIC value of the transformed strain was 90 mM,gat1the MIC value of the transformed strain was 60 mM,gat2the MIC value of the transformed strain was 70 mM,gat3the MIC value of the transformed strain was 100 mM. Namely thatgat1、gat2Andgat3the transformed strains show high resistance to glyphosate.
And (3) measuring a growth curve:
when the MIC value of each strain is measured, the OD600 of the bacterial liquid needs to reach about 1.0. And (3) sucking 200 mu L of bacterial liquid in each test tube, measuring the OD600 value of the bacterial liquid by using an enzyme-labeling instrument, selecting the bacterial liquid with the value closest to 1.0, diluting the bacterial liquid, and adjusting the bacterial liquid to be between 0.990 and 1.010.
mu.L of M9 liquid medium containing 50 mM glyphosate was added to the honeycomb plate, and 2. mu.L of the bacterial suspension was inoculated thereto. The honeycomb plate was placed in a full-automatic growth curve measuring apparatus, and the growth curve of each strain was observed for 80 hours.
The results of the experiment are shown in FIG. 4, althoughgat1The growth curve has a plateau OD600 value slightly higher than that of other strains, butgat2Andgat3the growth conditions of (2) are obviously better.
Example 3 mutant site screening
Primarily selecting mutation sites:
according to 18 turnsThe experimental data of the strains are changed, and 18 strains are obtainedgatThe sequences were ranked as shown in Table 1, andgat3each amino acid position within the gene was scored and 12 mutation positions were selected as shown in table 2 below.
TABLE 1gatGene fractionation
Selected from Table 2gat3Mutation site in gene
Construction of Single mutation vectors:
by pET28a-gat3The plasmid was used as a template, and PCR amplification was carried out using the sequences shown in Table 3 as primers. Then using the recovered PCR product as a large fragment primer, and using pET28a-gat3The plasmid was used as a template for PCR amplification of the entire plasmid, thereby obtaining a single mutation vector.
TABLE 3 primer information for Single mutation vector construction
The constructed 12 single mutation vectors were transformed into Trans1-T1 strain for storage, and at the same time, transformed into BL21 (DE 3) strain for subsequent screening experiments. All strains were kept in one portion at-20 ℃ and one portion at-80 ℃.
Because the mutation condition can not be detected by self, a single clone of each strain is picked up and cultured in 5 mL LK liquid culture medium in a shaking table at the speed of 200 rpm and the temperature of 37 ℃ overnight, the bacterial liquid is sent for sequencing verification, and the sequencing primer sequence is shown in Table 4.
TABLE 4 sequencing primers for Single mutant vectors
Plate qualitative screening:
the experimental method is the same as that of example 2, and the experimental results are shown in fig. 5, which is a positive control, a wild type, 12 experimental groups and a transformed strain of a blank vector from top to bottom. Wherein the content of the first and second substances,mGAT3-17、mGAT3-35、mGAT3-38、mGAT3-53、mGAT3- 77、mGAT3-93the growth conditions of each diluted gradient bacterial liquid of the transformed strain are better than those of other strains.
And (3) MIC quantitative screening:
the experimental method was the same as example 2, and the experimental results are shown in FIG. 6, where it was difficult to analyze the differences among the respective strains by MIC values. Thus, the OD600 value of each strain at 50 mM was compared with that of the wild typegat3The ratios of the transformed strains were compared for differences between strains, and the results are shown in Table 5,mgat3-17、mgat3-35、mgat3-38、mgat3-40、mgat3-46、mgat3-53、mgat3-62、mgat3-77andmgat3-93the growth of the transformed strain under these conditions is better than that of the wild typegat3。
TABLE 5 comparison of glyphosate at 50 mM withgat3OD600 ratio of the transformed Strain
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Sequence listing
<110> institute of biotechnology of Chinese academy of agricultural sciences
Application of <120> gat3 gene and mutant thereof in culturing glyphosate-resistant crops
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 447
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
atggcagcca ttgaagttaa accgattaat gcagaagata cctatgatct gcgtcatcgt 60
gtgctgcgtc cgaatcagcc gattgaagcc tgtatgtttg atagtgatct gatgcgcagt 120
gcctttcatc tgggtggctt ttatggcggc aaactgatta gcgtggcaag ctttcatcag 180
gccgaacata ccgaactgca gggtcagaaa cagtatcagc tgcgcggtgt ggcaaccctg 240
gaaggttatc gcgaacagaa agccggtagt agcctggtta aacatgccga agaaattctg 300
cgtaaacgcg gtgttgatct gctgtggtgt aatgcacgta ccagtgccag tggctattat 360
aaaaaactgg gctttagtga acagggcgaa gtgtttgata ccccgccggt tggtccgcat 420
attctgatgt ataaacgcat tacctaa 447
<210> 3
<211> 148
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 3
Met Ala Ala Ile Glu Val Lys Pro Ile Asn Ala Glu Asp Thr Tyr Asp
1 5 10 15
Leu Arg His Arg Val Leu Arg Pro Asn Gln Pro Ile Glu Ala Cys Met
20 25 30
Phe Asp Ser Asp Leu Met Arg Ser Ala Phe His Leu Gly Gly Phe Tyr
35 40 45
Gly Gly Lys Leu Ile Ser Val Ala Ser Phe His Gln Ala Glu His Thr
50 55 60
Glu Leu Gln Gly Gln Lys Gln Tyr Gln Leu Arg Gly Val Ala Thr Leu
65 70 75 80
Glu Gly Tyr Arg Glu Gln Lys Ala Gly Ser Ser Leu Val Lys His Ala
85 90 95
Glu Glu Ile Leu Arg Lys Arg Gly Val Asp Leu Leu Trp Cys Asn Ala
100 105 110
Arg Thr Ser Ala Ser Gly Tyr Tyr Lys Lys Leu Gly Phe Ser Glu Gln
115 120 125
Gly Glu Val Phe Asp Thr Pro Pro Val Gly Pro His Ile Leu Met Tyr
130 135 140
Lys Arg Ile Thr
145
Claims (7)
1. A kind ofgat3A gene characterized in thatgat3The polynucleotide of the gene is represented by (a), (b), (c) or (d):
(a) as shown in a sequence table SEQ ID No: 1; or
(b) And SEQ ID No: 1 under stringent hybridization conditions, and the protein encoded by the polynucleotide still has glyphosate resistance activity;
(c) and SEQ ID No: 1 or a polynucleotide having at least 90% or more homology thereto; or
(d) In SEQ ID No: 1 by deletion, substitution or insertion of one or more bases, and the protein coded by the polynucleotide mutant still has glyphosate resistant activity.
2. A gat3 protein, wherein the amino acid sequence of gat3 protein is (a), (b) or (c):
(a) as shown in a sequence table SEQ ID No: 2; or
(b) And SEQ ID No: 2, an amino acid having at least 90% or more homology with the amino acid shown in the formula (I); or
(c) In SEQ ID No: 2, and the protein mutant is obtained by deleting, substituting or inserting one or more amino acids on the basis of the protein shown in the formula 2, and the protein still has glyphosate resistant activity.
3. Mutants of the gat3 protein of claim 2, wherein the mutants comprise gat3-17, gat3-35, gat3-38, gat3-53, gat3-77, gat 3-93; the 17 th amino acid residue of the amino acid sequence of the mutant gat3-17, which is the gat3 protein, is mutated from L to I; the 35 th amino acid residue of the amino acid sequence of the mutant gat3-35, which is the gat3 protein, is mutated from S to G; the 38 th amino acid residue of the amino acid sequence of the mutant gat3-38, which is the gat3 protein, is mutated from M to D; the 53 th amino acid residue of the amino acid sequence of the mutant gat3-53, which is the gat3 protein, is mutated from I to V; the 77 th amino acid residue of the amino acid sequence of the mutant gat3-77 gat3 protein is mutated from V to M; the 93 th amino acid residue of the amino acid sequence of the mutant gat3-93, which is the gat3 protein, is mutated from V to I.
4. Comprising the compound of claim 1gat3A vector for the gene.
5. Comprising the compound of claim 4gat3Engineering bacteria of gene vector.
6. Detecting the compound of claim 1gat3Primers for any fragment of the gene.
7. Use of the gat3 protein of claim 2 or a mutant of the gat3 protein of claim 3 in the cultivation of glyphosate resistant crops.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002036782A2 (en) * | 2000-10-30 | 2002-05-10 | Maxygen, Inc. | Novel glyphosate n-acetyltransferase (gat) genes |
| CL2008002917A1 (en) * | 2008-03-03 | 2009-03-06 | Ms Tech Llc | Specific antibodies for double mutant 5-ennolpyruvylshikimate-3-phosphate synthetase (epsps); producer hybridoma; method of generating said antibodies; method of detection of said double mutant epsps enzyme in a composition; kit to detect the presence of said double mutant epsps in the composition. |
| US20180244732A1 (en) * | 2015-02-13 | 2018-08-30 | Beijing Dabeinong Technology Group Co., Ltd. | Herbicide-resistant protein, encoding gene and use thereof |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002036782A2 (en) * | 2000-10-30 | 2002-05-10 | Maxygen, Inc. | Novel glyphosate n-acetyltransferase (gat) genes |
| US20030083480A1 (en) * | 2000-10-30 | 2003-05-01 | Maxygen, Inc. | Novel glyphosate N-acetyl transferase (GAT) genes |
| CL2008002917A1 (en) * | 2008-03-03 | 2009-03-06 | Ms Tech Llc | Specific antibodies for double mutant 5-ennolpyruvylshikimate-3-phosphate synthetase (epsps); producer hybridoma; method of generating said antibodies; method of detection of said double mutant epsps enzyme in a composition; kit to detect the presence of said double mutant epsps in the composition. |
| US20090220999A1 (en) * | 2008-03-03 | 2009-09-03 | Ms Technologies, Llc | Antibodies immunoreactive with mutant 5-enolpyruvlshikimate-3-phosphate synthase |
| US20180244732A1 (en) * | 2015-02-13 | 2018-08-30 | Beijing Dabeinong Technology Group Co., Ltd. | Herbicide-resistant protein, encoding gene and use thereof |
Non-Patent Citations (1)
| Title |
|---|
| CASTLE,L.A.等: "Sequence 948 from patent US 7405074，GenBank: ACH21301.1", 《GENBANK》 * |
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