CN116789780A - Chloroplast transit peptide for glyphosate-resistant herbicide gene and application thereof - Google Patents
Chloroplast transit peptide for glyphosate-resistant herbicide gene and application thereof Download PDFInfo
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
- Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants
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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention belongs to the technical field of plant biology, and particularly discloses a cotton chloroplast transit peptide, a coding gene GhCTP1 thereof and application thereof in cultivation of glyphosate-resistant herbicide plants. The invention also discloses a fusion gene consisting of GhCTP1 and a glyphosate-resistant herbicide gene and a fusion protein encoded by the fusion gene. The chloroplast transit peptide GhCTP1 has high transit efficiency, can be fused with any herbicide EPSPS gene for expression, and can enhance the tolerance of plants such as cotton to glyphosate herbicide. The invention has important significance for reducing weed harm, reducing artificial weeding investment, reducing production cost and improving yield in the production process of plants such as cotton and the like.
Description
Technical Field
The invention belongs to the technical field of plant biology, and particularly relates to a chloroplast transit peptide for a glyphosate-resistant herbicide gene; fusion genes and fusion proteins containing the chloroplast transit peptide, and uses thereof are also described.
Background
Glyphosate is the main active ingredient of herbicide noda (Roundup) produced by the company Mengshan, U.S. and has the advantages of high efficiency, broad spectrum, low toxicity, easy decomposition by microorganisms, etc. Glyphosate is used as a systemic conduction herbicide, can be absorbed by plants after being sprayed on stems and leaves of the plants, and can be rapidly conducted to the whole plants and roots thereof, thereby eliminating weeds. However, glyphosate is a non-selective herbicide, and has the same effect of killing weeds and killing crops, so that the application range and the application time of the glyphosate are greatly limited.
5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) is a key enzyme of shikimate pathway in plants, and is also an action target of glyphosate, and the glyphosate tolerance of plants can be effectively improved by introducing EPSPS genes insensitive to glyphosate or over-expressing the EPSPS genes in transgenic plants. Along with the continuous discovery of the glyphosate-resistant EPSPS gene, the transgenic technology is used for transforming the glyphosate-resistant EPSPS gene into crops so as to obtain the crops resistant to glyphosate herbicide, which has become an effective means for solving the problem of non-selectivity of the glyphosate herbicide. The glyphosate-resistant herbicide genes which are currently applied to cotton are mainly the CP4-EPSPS gene of Monsanto corporation of America and the GR79-EPSPS gene (ZL 2014102047036) of the institute of biotechnology of national academy of agricultural sciences.
Since EPSPS is located in chloroplasts in plant cells, a chloroplast transit peptide (Chloroplast Transpit Peptide, abbreviated as CTP, also called chloroplast leader peptide or leader peptide, etc.) having a high efficiency targeting effect is required in addition to the EPSPS herbicide resistance gene itself, and further, EPSPS enzymes are transported into chloroplasts to function. Therefore, chloroplast transit peptides are critical to whether EPSPS can function effectively in chloroplasts.
The current commercialized glyphosate herbicide resistant transgenic cotton (Monsanto Co. Of the United states) is mainly obtained by fusion and introduction of a chloroplast transit peptide AtCTP derived from an Arabidopsis thaliana EPSPS gene and a CP4EPSPS gene derived from Agrobacterium into cotton. Because the chloroplast transit peptide AtCTP is derived from Arabidopsis thaliana, belongs to a heterologous gene, and has a lower transit function after expression in cotton than the transit efficiency of the chloroplast transit peptide in the cotton. Therefore, if the chloroplast transit peptide of cotton itself and herbicide-resistant genes are fused and introduced into cotton, the efficiency of transferring glyphosate-resistant herbicide EPSPS proteins into cotton chloroplasts can be greatly improved, thereby improving the resistance of transgenic cotton to glyphosate herbicides.
At present, no report on cotton chloroplast transit peptide for transferring glyphosate-resistant herbicide proteins has been found.
Disclosure of Invention
The invention aims to provide a chloroplast transit peptide.
Another object of the present invention is to provide a gene encoding the chloroplast transit peptide described above.
The third object of the present invention is to provide a fusion gene comprising the chloroplast transit peptide gene described above.
The fourth object of the present invention is to provide a fusion protein encoded by the above fusion gene.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
a chloroplast transit peptide has an amino acid sequence shown in SEQ ID NO. 1.
The invention also provides a gene for encoding the chloroplast transit peptide, which is named as: the nucleotide sequence of the GhCTP1 gene is shown as SEQ ID NO. 2.
The invention also provides application of the chloroplast transit peptide or gene in transferring glyphosate-resistant herbicide proteins.
The glyphosate-resistant herbicide protein in the application refers to a CP4EPSPS protein or a GR79EPSPS protein and the like.
The invention also provides application of the chloroplast transit peptide or the gene in cultivation of glyphosate-resistant herbicide plants.
The plant refers to cotton, rape or soybean, etc.
The cotton refers to upland cotton (Gossypium hirsutum Linn.) or sea island cotton (Gossypium barbadense Linn.), such as R15, WC, SU12, etc.
The invention also provides an expression vector, an expression cassette, a transgenic cell line or recombinant bacteria containing the gene.
The invention also provides application of the expression vector, the expression cassette, the transgenic cell line or the recombinant bacteria and the like in cultivation of glyphosate-resistant herbicide plants.
The plant refers to cotton, rape or soybean, etc.
The cotton refers to upland cotton (Gossypium hirsutum Linn.) or sea island cotton (Gossypium barbadense Linn.), such as R15, WC, SU12, etc.
The invention also provides a fusion gene, which consists of the chloroplast transit peptide gene and a glyphosate herbicide resistant gene; the nucleotide sequence of the chloroplast transit peptide gene is shown as SEQ ID NO. 2.
The glyphosate-resistant herbicide gene in the fusion gene refers to GR79EPSPS gene, CP4EPSPS gene and the like.
The nucleotide sequence of the GR79EPSPS gene is shown as SEQ ID NO. 5.
The nucleotide sequence of the fusion gene is shown as SEQ ID NO. 6.
The invention also provides a fusion protein encoded by the fusion gene, and the fusion protein consists of the chloroplast transit peptide and glyphosate herbicide resistant protein.
The amino acid sequence of the chloroplast transit peptide in the fusion protein is shown as SEQ ID NO. 1.
The glyphosate-resistant herbicide protein in the fusion protein refers to GR79EPSPS protein, CP4EPSPS protein and the like.
The amino acid sequence of the GR79EPSPS protein is shown in SEQ ID NO. 7.
The amino acid sequence of the fusion protein is shown as SEQ ID NO. 8.
The fusion gene or the fusion protein is applied to cultivation of glyphosate-resistant herbicide plants.
The plant refers to cotton, rape or soybean, etc.
The cotton refers to upland cotton (Gossypium hirsutum Linn.) and island cotton (Gossypium barbadense Linn.), such as R15, WC, SU12, etc.
The invention also provides an expression vector, an expression cassette, a transgenic cell line or recombinant bacteria containing the fusion gene.
The expression cassette comprises a CaMV 35S promoter, a NOS terminator and the fusion gene.
The expression vector refers to a plant expression vector pBI121-GhCTP1-GR79 EPSPS for resisting herbicide, which is obtained by connecting the expression cassette to a pBI121 vector.
The application of the expression vector, the expression cassette, the transgenic cell line or the recombinant bacteria and the like in cultivating glyphosate-resistant herbicide plants.
The plant refers to cotton, rape or soybean, etc.
The cotton refers to upland cotton (Gossypium hirsutum Linn.) and island cotton (Gossypium barbadense Linn.); cotton lines such as R15, WC or SU 12.
The invention also provides a cultivation method for cultivating the glyphosate herbicide-resistant cotton variety, which comprises the steps of introducing the fusion gene containing chloroplast transit peptide and GR79EPSPS into cotton by a transgenic method, and then screening offspring with tolerance to the glyphosate herbicide.
The invention also provides a method for improving the glyphosate herbicide resistance of cotton, which comprises the steps of introducing a plant expression vector containing chloroplast transit peptide and GR79EPSPS fusion gene into cotton by a transgenic method, and screening transgenic progeny materials of the glyphosate herbicide resistance.
The nucleotide sequence of the chloroplast transit peptide in the plant expression vector is shown as SEQ ID No. 2.
The invention also provides a method for cultivating a cotton variety resistant to glyphosate herbicide by using the chloroplast transit peptide, which comprises the following steps:
(1) Artificial synthesis of chloroplast transit peptide sequence: the nucleotide sequence of the GhCTP1 gene is artificially synthesized according to the sequence shown in SEQ ID NO. 2; then, a BamHI enzyme cutting site and a PstI enzyme cutting site are respectively added at the 5 'end and the 3' end of the sequence shown in SEQ ID NO.2 to respectively obtain a sense strand shown in SEQ ID NO.3 and an antisense strand shown in SEQ ID NO. 4; annealing the sense strand shown in SEQ ID NO.3 and the antisense strand shown in SEQ ID NO.4 in a water bath kettle to form a chloroplast transit peptide double-stranded DNA fragment containing a sticky end;
(2) Construction of a plant expression vector: double-restriction pBI121 plant expression vectors are cut by BamH I and Pst I; inserting the chloroplast transit peptide double-stranded DNA fragment (GhCTP 1 sequence) in the step (1) between a CaMV 35S promoter and a T-NOS terminator of the pBI121 plant expression vector by using a T4 DNA ligase to obtain a pBI121-GhCTP1 vector; then, the GR79EPSPS gene is inserted between the GhCTP1 and the T-NOS terminator in the pBI121-GhCTP1 vector by using double enzyme digestion of PstI and XhoI, and a plant expression vector for resisting glyphosate herbicide is obtained and named as follows: pBI121-GhCTP1-GR79 EPSPS;
(3) Transgenic herbicide-resistant cotton creation: introducing the plant expression vector obtained in the step (2) into agrobacterium, and then introducing the plant expression vector into cotton by using an agrobacterium-mediated cotton genetic transformation method to obtain transgenic cotton containing GhCTP1 and GR79EPSPS fusion genes;
(4) And (3) carrying out molecular identification and glyphosate herbicide resistance identification on the transgenic plant obtained in the step (3), and selecting a cotton plant resistant to the glyphosate herbicide.
The invention also provides a breeding method of the herbicide-resistant cotton variety, which comprises the steps of taking the glyphosate-resistant herbicide-transgenic cotton obtained by the method as one of parents, selecting plants with strong herbicide resistance in offspring through a hybridization or backcross method, and continuously selecting 4-5 generations or backcross 4-5 generations to obtain the glyphosate-resistant herbicide-resistant cotton variety.
Compared with the prior art, the invention has the advantages and beneficial technical effects that: the chloroplast transit peptide GhCTP1 has high transit efficiency, can be fused with any herbicide EPSPS gene for expression, and can enhance the tolerance of plants such as cotton to glyphosate herbicide. The invention has important significance for reducing weed harm, reducing artificial weeding investment, reducing production cost and improving yield in the production process of plants such as cotton and the like.
Drawings
FIG. 1 schematic diagram of pBI121-GhCTP1-GR79 EPSPS expression vector.
FIG. 2. PCR identification of transgenic cotton T0 generation plants GhCTP1-GR79 EPSPS fusion gene electropherograms; wherein M is DNA standard molecular weight; "-" is a negative control; 1-7 are transgenic individual plant materials G1, G2, G5, G6, G7, G9 and G10 respectively.
FIG. 3 is a comparison photograph of the identification of glyphosate herbicide tolerance of transgenic cotton plants; wherein 1 is wild-type material WC;2 is a single plant of the T1 generation of the transgenic material G2.
Detailed Description
Unless otherwise specified, the experimental methods used in the following examples are all conventional. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
The discovery process of chloroplast transit peptide comprises the following steps: plant EPSPS enzymes are key proteins for the synthesis of plant aromatic amino acids, which are transported into the chloroplasts by their own chloroplast transit peptide. Thus, it is possible to isolate chloroplast transit peptides derived from the cotton EPSPS gene. The inventor refers to EPSPS gene design primer in upland cotton TM-1 genome sequence, uses upland cotton strain SU12 leaf cDNA as template to make PCR amplification to obtain EPSPS gene 5' end cDNA sequence in SU12 cotton strain, predicts the amino acid sequence coded by said sequence in SignalIP-6.0 software, and finds out a signal peptide composed of 70 amino acids, at the same time refers to CTP sequence of Arabidopsis thaliana AtEPSPS gene, and determines the protein sequence of cotton EPSPS gene chloroplast transit peptide as 73 amino acids (see sequence table SEQ ID NO. 1); the nucleotide sequence of the coding gene is shown as SEQ ID NO.2, and the gene is named as: ghCTP1.
Example 1 construction of expression vectors containing chloroplast transit peptides GhCTP1 and GR79EPSPS genes
The method is carried out according to the following steps:
(1) Artificial synthesis of chloroplast transit peptide sequence: the nucleotide sequence of the GhCTP1 gene is artificially synthesized according to the sequence shown in SEQ ID NO. 2; then, a BamH I enzyme cutting site and a PstI enzyme cutting site are respectively added at the 5 'end and the 3' end of the sequence shown in SEQ ID NO.2 to respectively obtain a sense strand shown in SEQ ID NO.3 and an antisense strand shown in SEQ ID NO. 4;
(2) Single-stranded DNA was synthesized from bioengineering (Shanghai) Co., ltd according to the nucleotide sequences shown in SEQ ID No.3 and SEQ ID No.4, and then annealed in a water bath to form double-stranded DNA fragments. The annealing method is as follows: dissolving the synthesized products of SEQ ID No.3 and SEQ ID No.4 to 30 mu M by using ultrapure water, placing 10 mu L of each into a 1.5mL centrifuge tube, fully and uniformly mixing, then placing into a water bath kettle at 98 ℃ for heating for 2min, naturally cooling to room temperature, and finally obtaining the chloroplast transit peptide double-stranded DNA fragment containing the sticky end.
(3) And (3) cutting the pBI121 plant expression vector by utilizing BamH I and Pst I, and inserting the chloroplast transit peptide double-stranded DNA fragment obtained in the step (2) between the CaMV 35S promoter and the T-NOS terminator of the pBI121 plant expression vector to obtain the pBI121-GhCTP1 vector. The reaction system is as follows: 1. Mu.L (5 ng) of chloroplast transit peptide double-stranded DNA fragment, 1. Mu.L (10 ng) of double-digested pBI121 vector, 1. Mu.L of T4 DNA ligase (NEB), 1. Mu.L of T4 DNA ligase Buffer, ddH 2 O6. Mu.L. After overnight ligation at 16℃the ligation products were transformed into E.coli and screened for the correct pBI121-GhCTP1 vector.
(3) The pBI121-GhCTP1 vector and GR79EPSPS gene (ZL 2014102047036, sequence shown in SEQ ID NO. 5) were digested with PstI and XhoI, and the GR79EPSPS gene was inserted between the chloroplast transit peptide GhCTP1 and the T-NOS terminator of the pBI121-GhCTP1 plant expression vector to obtain a plant expression vector (see FIG. 1) containing the GhCTP1 and GR79EPSPS fusion gene (see SEQ ID NO. 6), which was designated as follows: pBI121-GhCTP1-GR79 EPSPS. Wherein the reaction system is as follows: 1. Mu.L (5 ng) of double-stranded DNA fragment of GR79EPSPS gene, 1. Mu.L (10 ng) of double-digested pBI121-GhCTP1 vector, 1. Mu.L of T4 DNA ligase (NEB), 1. Mu.L of T4 DNA ligase Buffer and 6. Mu.L of ddH 2O. After overnight ligation at 16℃E.coli was transformed with the ligation product and the correct pBI121-GhCTP1-GR79 EPSPS expression vector was obtained by sequencing and screening.
Example 2 test of transgenic cotton for glyphosate tolerant herbicide Using the fusion Gene of the invention containing GhCTP1
The method is carried out according to the following steps:
(1) Plant expression vector pBI121-GhCTP1-GR79 EPSPS transformed agrobacterium
2-5 mu g of pBI121-GhCTP1-GR79 EPSPS expression vector obtained in example 1 and 100 mu L of Agrobacterium competent cell GV3101 were mixed uniformly and placed on ice for 5min; then, the mixture was added to a 2mm electric shock cup, electric shock was applied at 2500V, 800. Mu.L of LB liquid medium was rapidly added after electric shock, and transferred to a 1.5mL centrifuge tube, and the mixture was subjected to activation culture at 28℃and 180rpm for 5 hours. 100uL of the bacterial liquid was taken out and spread on LB solid medium plates containing kanamycin and rifampicin (both at a concentration of 50. Mu.g/mL), and cultured at 28℃for 48 hours. Randomly picking 5 resistant clones, delivering to a biological engineering (Shanghai) stock company for sequencing verification, selecting one clone with correct expression vector sequence, and storing for later use.
(2) Agrobacterium-mediated genetic transformation of cotton and acquisition of transgenic plants
Taking a upland cotton strain WC (provided by cotton research institute of the national academy of agricultural sciences of Shanxi) as a receptor, infecting a cotton hypocotyl with the agrobacterium obtained in the step (1), and then obtaining a transgenic plant through tissue culture, wherein the specific steps are as follows:
(a) Sterile seedling preparation: surface-sterilizing seeds with 70% ethanol for 30s, using 30% H 2 O 2 Soaking for sterilizing for 2 hr, washing with sterile distilled water for three times, and removing residual H on the surface of seed 2 O 2 . Soaking the sterilized seeds in sterile water at 28deg.C overnight, removing seed coat after the seeds germinate and are exposed to white, and sowing in 1/2Murashige&Skoog (MS) medium. The sown seeds were cultured at 28℃in the dark for 4 days, then at 16 hours light/8 hours dark for about 2 days, and the seedling hypocotyls were cut and divided into 0.5cm lengths for callus-induced explants.
(b) Genetic transformation and regeneration plant obtaining: agrobacterium containing pBI121-GhCTP1-GR79 EPSPS gene expression vector was inoculated into 100mL of LB liquid medium (containing 50mg/L kanamycin and rifampicin), and cultured at 28℃and 250rpm for 8 to 10h. Then centrifuging at 4000rpm for 10 min, collecting the cultured Agrobacterium cells, and re-suspending the Agrobacterium cells to OD by using MS liquid medium 600 At 0.4, the segmented explant from step (a) was immersed in the resuspension for 30 minutes. Subsequently, the infected explants were dried on sterile filter paper and transferred to co-culture medium (MSB 5 solid medium containing 0.05mg/L KT, 2.5 mg/L2, 4-D) and co-cultured at 24℃for 2 days under light-shielding conditions. The infected explant is transferred to CIM callus induction medium (MSB 5 solid medium containing 100mg/L kanamycin, 500mg/L cephalosporin, 0.05mg/L KT and 2.5 mg/L2, 4-D) and cultured for 2-3 months under the condition of 28 ℃ and 16 hours illumination/8 hours darkness to induce callus.
The callus cells which grow actively are selected and transferred to EIM embryo induction culture medium (MSB 5 solid culture medium containing 100mg/L kanamycin and 500mg/L cephalosporin) to be cultured for 2-3 months under the conditions of 28 ℃ and 16 hours of illumination/8 hours of darkness. The green healthy callus in the callus cells was then transferred to an EIM without kanamycin to induce embryo development to form shoots. Selecting shoots with normal appearance development, transferring the shoots into MS culture medium containing 200mg/L IAA, culturing and inducing rooting under the conditions of 28 ℃ and 16 hours of light/8 hours of darkness, and finally developing into transgenic regenerated plants.
(3) PCR identification of transgenic plants
Transplanting the obtained transgenic plants in a field, respectively extracting genome DNA of each plant, and then carrying out PCR amplification by taking specific primers GhCTP1-GR79-Fp and GhCTP1-GR79-Rp designed according to fusion gene sequences of GhCTP1 and GR79EPSPS as primers; the primer is as follows:
GhCTP1-GR79-Fp：5’-GGATCCATGGCAACGCAGTTTGGC-3’(SEQ ID NO.9)；
GhCTP1-GR79-Rp：5’-GTTGCTTGTTGGGGTGACCAGGGC-3’(SEQ ID NO.10)。
as a result (see FIG. 2), the size of the PCR product was 309bp. Finally, 7 positive T0 generation plants were obtained, which were respectively named: g1, G2, G5, G6, G7, G9 and G10.
EXAMPLE 3 identification of the glyphosate herbicide resistance of transgenic cotton according to the invention
The method is carried out according to the following steps:
(1) The 7 positive T0 plants obtained in example 2 were transplanted to field and cultivated while 2 times the production concentration (4 mg/L) of the nodap (Roundup) glyphosate herbicide was sprayed, and finally a single plant with no growth inhibition, no difference in yield and no difference in control was obtained in the T0 plant designated G2, and the T1 cotton seeds of G2 were harvested.
(2) And sowing the T1 generation cotton seeds of G2 into a nutrition pot to culture seedlings, and obtaining 50T 1 generation seedlings in total. When the cotton seedlings grow to 3 true leaves, the pesticide of the agricultural chemical Dada (Roundup) is sprayed, and the concentration of the glyphosate herbicide is about 2mg/L. The seedling phenotype was observed after 5 days of spraying.
Results (see fig. 3) 50T 1-generation transgenic plants derived from G2 all grew normally, while wild-type receptor WC plants died from drying out. The cotton chloroplast transit peptide GhCTP1 has high transit efficiency, can efficiently transit the glyphosate-resistant herbicide GR79EPSPS protein into chloroplasts, and has strong glyphosate-resistant herbicide capability; meanwhile, the glyphosate-resistant herbicide GR79EPSPS protein transported by the chloroplast transit peptide GhCTP1 can be efficiently expressed in chloroplasts, and the obtained transgenic plant has strong glyphosate-resistant herbicide capability. The method has great potential application value in culturing new herbicide-resistant varieties by transgenic technology.
Claims (10)
1. The chloroplast transit peptide is characterized in that the amino acid sequence of the chloroplast transit peptide is shown as SEQ ID NO. 1.
2. A gene encoding the chloroplast transit peptide of claim 1, designated: ghCTP1, characterized in that the nucleotide sequence of the gene is shown as SEQ ID NO. 2.
3. Use of the chloroplast transit peptide of claim 1 or the gene of claim 2 for the cultivation of glyphosate herbicide resistant plants.
4. An expression vector, an expression cassette, a transgenic cell line or a recombinant bacterium or the like comprising the gene of claim 2.
5. Use of the expression vector, expression cassette, transgenic cell line or recombinant bacterium of claim 4 for the cultivation of glyphosate herbicide resistant plants.
6. A fusion gene comprising the chloroplast transit peptide gene of claim 2 and a glyphosate herbicide resistant gene; the nucleotide sequence of the chloroplast transit peptide gene is shown as SEQ ID NO. 2; the glyphosate-resistant herbicide gene refers to GR79EPSPS gene, CP4EPSPS gene and the like.
7. The fusion gene according to claim 6, wherein the nucleotide sequence of the fusion gene is shown in SEQ ID NO. 6.
8. Use of the fusion gene of claim 6 for cultivating glyphosate-resistant herbicide plants.
9. The use according to claim 3, 5 or 8, wherein the plant is cotton, canola or soybean.
10. A method of growing a glyphosate herbicide resistant cotton variety utilizing the chloroplast transit peptide of claim 1, comprising the steps of:
(1) Artificial synthesis of chloroplast transit peptide sequence: the nucleotide sequence of the GhCTP1 gene is artificially synthesized according to the sequence shown in SEQ ID NO. 2; then, a BamH I enzyme cutting site and a PstI enzyme cutting site are respectively added at the 5 'end and the 3' end of the sequence shown in SEQ ID NO.2 to respectively obtain a sense strand shown in SEQ ID NO.3 and an antisense strand shown in SEQ ID NO. 4; annealing the sense strand shown in SEQ ID NO.3 and the antisense strand shown in SEQ ID NO.4 in a water bath kettle to form a chloroplast transit peptide double-stranded DNA fragment containing a sticky end;
(2) Construction of a plant expression vector: double-restriction pBI121 plant expression vectors are cut by BamH I and Pst I; inserting the chloroplast transit peptide double-stranded DNA fragment (GhCTP 1 sequence) in the step (1) between a CaMV 35S promoter and a T-NOS terminator of the pBI121 plant expression vector by using a T4 DNA ligase to obtain a pBI121-GhCTP1 vector; then, the GR79EPSPS gene is inserted between the GhCTP1 and the T-NOS terminator in the pBI121-GhCTP1 vector by using double enzyme digestion of PstI and XhoI, and a plant expression vector for resisting glyphosate herbicide is obtained and named as follows: pBI121-GhCTP1-GR79 EPSPS;
(3) Transgenic herbicide-resistant cotton creation: introducing the plant expression vector obtained in the step (2) into agrobacterium, and then introducing the plant expression vector into cotton by using an agrobacterium-mediated cotton genetic transformation method to obtain transgenic cotton containing GhCTP1 and GR79EPSPS fusion genes;
(4) And (3) carrying out molecular identification and glyphosate herbicide resistance identification on the transgenic plant obtained in the step (3), and selecting a cotton plant resistant to the glyphosate herbicide.
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| US20110173716A1 (en) * | 2007-11-09 | 2011-07-14 | Biotechnology Research Institute The Chinese Academy of Agricultural Sciences | EPSP synthase with high glyphosate resistance and its encoded sequence |
| US20130205441A1 (en) * | 2012-02-01 | 2013-08-08 | Dow Agrosciences Llc | Chloroplast transit peptide |
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| US20110173716A1 (en) * | 2007-11-09 | 2011-07-14 | Biotechnology Research Institute The Chinese Academy of Agricultural Sciences | EPSP synthase with high glyphosate resistance and its encoded sequence |
| US20130205441A1 (en) * | 2012-02-01 | 2013-08-08 | Dow Agrosciences Llc | Chloroplast transit peptide |
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