WO2012031421A1 - Gene of cotton epsp synthetase variant and uses thereof - Google Patents

Abstract

A cotton EPSP synthetase variant and artificial mutant gene MC-EPSPS encoding the EPSP synthetase variant are provided. The mutant gene encoding the variant with two mutant amino acid sites and anti-glyphosate property is obtained by designing mutant sites according to documents and analysis of protein structure and then conducting PCR amplification by using the EPSP synthetase gene of Gossypium hirsutum as template, which utilizes gene homologous recombination and multiple-site mutations of DNA sequence. The binding efficiency of the EPSP synthetase encoded by the mutant gene to glyphosate is decreased dominantly, and the EPSP synthetase has certain enzyme catalysis efficiency. The gene encoding the EPSP synthetase variant can be used to cultivating new breeds of transgenic plants with anti-glyphosate property.

Description

 Cotton EPSP synthase mutant gene and application thereof

 Technical field

 The invention belongs to the field of plant genetic engineering research, and particularly relates to cotton EPSP Modification of synthetase gene and construction of expression vector, and application in the development of glyphosate-tolerant transgenic plants.

 Background technique

Glyphosate is a non-selective herbicide with the advantages of broad-spectrum weeding and rapid degradation in soil. It is one of the safest herbicides for humans and animals because it is not present in animals. Glyphosate has the advantages of stable physical and chemical properties, high efficiency, broad spectrum, low toxicity, low residue, easy to be decomposed by microorganisms, and does not damage the soil environment. It has been widely used in agricultural production and has become the most abundant pesticide variety in the world. since Glyphosate herbicide, Monsanto, USA, 1976 - Roundup Since the successful development and wide application, the glyphosate-to-glycan transgenic research has become a hot spot in the research of herbicide resistance genetic engineering. With the development of glyphosate-resistant gene clones, glyphosate-tolerant GM crops have also been introduced and widely applied.

 The mechanism of action of glyphosate is mainly competitive inhibition of the shikimate pathway 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) Activity. The enzyme is a key enzyme in the biosynthesis of aromatic amino acids (including tryptophan, tyrosine, phenylalanine) in fungi, bacteria, algae and higher plants. Glyphosate is a dilute phosphate pyruvate An analog of (PEP), a competitive inhibitor of EPSPS, which combines glyphosate, EPSPS, and shikimic acid trisphosphate (S3P) to form an EPSPS-S3P-glyphosate complex ( This complex is very stable), suppress EPSPS The activity of the branched acid is blocked, blocking the biosynthesis of aromatic amino acids and some aromatic compounds, which eventually leads to the metabolic imbalance of some hormones and key metabolites such as flavonoids, lignin and phenolic compounds, thus disturbing the normality of the organism. Nitrogen metabolism causes it to die.

 At present, there are four methods for plant glyphosate transgenic engineering research: 1) Select EPSPS that is not sensitive to glyphosate Or obtain a resistant EPSPS gene by amino acid sequence alteration; 2) over-expression of the encoded EPSPS in the plant; 3) introduction of a gene that degrades glyphosate (eg, encoding a glyphosate oxidoreductase) Gox gene ) 4) Introduction of nitrogen acetyltransferase (GAT gene) to acetylate glyphosate, etc. (Barry et al., 1992; Padgette et A1. , 1996 ; Dill , 2005 ; Tan et a1 . , 2006 ; Castle et a1 . , 2004) . Among them, the most widely used is the overexpression of EPSPS genes that are insensitive or mutated to glyphosate in plants.

 The selection of the resistant EPSPS gene is a key factor in obtaining transgenic crops. Change EPSPS The amino acid sequence of the functional region reduces its affinity for glyphosate while maintaining the catalytic activity of the enzyme. Stalker DM, etc. (1985 The chemical mutagen was used to treat Salmonella typhimurium, and the aroA gene mutant was cloned from the glyphosate resistant mutant. The 101st Ser in the mutant was confirmed by DNA and protein sequence analysis. The pro-gene was transferred into E. coli to obtain glyphosate-resistant properties. Sost D et al. (1990) cloned and sequenced wild-type Klebsiella. AroA of pneumoniae and glyphosate-resistant mutant Klebsiella pneumoniae K1 (which encodes a glyphosate-insensitive EPSPS) The gene, which shows a single base difference, causes the 96-position Gly to become Ala in the amino acid sequence. Baerson SR et al found a glyphosate-resistant goosegrass in the Malay Islands (Eleusine indica), which has a 2-4 fold higher LD50 than other glyphosate-sensitive species in the area, by comparing the EPSPS of resistant Goosegrass and Sensual Goosegrass The sequence was found to have two amino acids, one of which was an intentional mutation, ie the 106th Pro of the resistant Goosegrass EPSPS was replaced by Ser. China's Xiang Wensheng et al (2001 ), compared with the glyphosate-resistant bean, the 513 position of the resistant bean is Gln, and the point of the sensitive bean is Glu. He Ming et al (2002 The EPSPS gene of Salmonella typhimurium and Escherichia coli with improved resistance was obtained, and two mutants replaced Thr at 42 Met. Monsanto The company replaced Gly in 80-120 of the EPSPS gene from Petunia, tomato, Arabidopsis, soybean, maize, and Salmonella typhimurium with Ala, in 170-210 Replace Ala with Thr to obtain glyphosate-resistant soybeans, corn, canola, etc.

 Overexpression of the EPSPS gene in plants significantly enhanced the glyphosate resistance of the plants. Amrhein A screen-to-glyphosate petunia cell line was screened by increasing the selection pressure of glyphosate. The EPSPS gene of the cell line was analyzed and the copy number of the target gene was increased by about 20 times. Use 35S The promoter initiates the gene expression in a large number of transgenic plants, and the transgenic plants are tolerant to glyphosate application for more than four times the amount of non-transgenic plants. Todd A The study on the long-term use of the long-growth in the glyphosate field showed that the EPSPS activity in the glyphosate-resistant plants was consistent with the sensitivity, but the gene copy number increased by 5 to 160. After further analysis, it was found that the level of resistance was positively correlated with protein expression and gene copy number.

 EPSPS Only transport to the chloroplast can play a role. Therefore, when constructing a plant expression vector, a peptide sequence needs to be added before the gene to form a fusion expression, which facilitates the targeted transfer of the target protein into the chloroplast. When constructing a plant expression vector, the present invention selects cotton The chloroplast-derived peptide (CTP) of the EPSPS gene serves as a fusion expression leader peptide of the gene of interest.

 In the vector construction, a OK (Omega & Kozak) sequence was added to the promoter and added after the gene stop codon. PS ( Processing & Splicing sequence ) (already in ZL 95 119563.8 Fully disclosed) can promote efficient transcription and translation of foreign genes in plants.

 Summary of the invention

 The object of the present invention is to provide a cotton 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) The nucleotide sequence of the mutant gene is shown as SEQ ID NO: 1.

 A second object of the present invention is to provide an amino acid sequence of a cotton EPSPS mutant, such as SEQ ID NO: 2 Shown.

 A third object of the present invention is to provide a nucleotide sequence of a cotton EPSPS mutant fusion gene, such as SEQ ID NO : 3 is shown.

 A fourth object of the present invention is to provide an amino acid sequence of a cotton EPSPS mutant fusion gene, such as SEQ ID NO : 4 is shown.

 A fifth object of the present invention is to provide a plant expression vector comprising the cotton EPSPS mutant gene or a fusion gene thereof.

 A sixth object of the present invention is to provide a plant cell, tissue or plant transformed with the plant expression vector.

 A seventh object of the present invention is to use the cotton EPSPS mutant gene or a fusion gene thereof in a glyphosate resistant plant variety.

 The invention is achieved by the following technical solutions:

1) Total RNA was extracted from Upland cotton (Euyang 11F1), and cDNA was obtained by reverse transcription of Oligo dT. The primers of NCPS were used to design primers to amplify cotton EPSPS and its fusion peptide sequences, which were named C- EPSPS , CTP.

2) The C- EPSPS gene was modified by multi-point mutation technology to obtain the mutant gene MC- EPSPS .

3) Identification of prokaryotic expression product glyphosate-resistant function: construct prokaryotic expression vector of MC- EPSPS gene, transform expression plasmid BL21 (DE3) PlySs , culture under selective pressure of glyphosate, observe its growth, and turn The expression bacteria of C -EPSPS and empty vector were used as controls. The expression of MC -EPSPS and C- EPSPS under normal induction conditions was analyzed.

4) Enzymatic Activity Assay: C -EPSPS each containing MC -EPSPS and prokaryotic expression vectors were transformed into strain ER2799 EPSPS deficient in growth observed on M9 medium.

5) Construction of a plant expression vector containing MC -EPSPS , the method comprising: selecting a 35S promoter containing a double enhancer and Tnos as a promoter and terminator of the MC- EPSPS gene, and adding an OK sequence after the promoter, The stop codon of the gene is followed by a PS sequence. The chloroplast-derived peptide sequence (CTP) of the cotton EPSPS gene was added to the 5' end of the gene of interest to obtain fusion expression in plants. The entire expression cassette was inserted into the modified binary expression vector pBI121 to obtain a glyphosate resistant plant expression vector pBI-MC- EPSPS . Transformation of tobacco and cotton by Agrobacterium-mediated transformation.

 6) Identification of glyphosate-resistant function of transgenic plants: smearing or spraying 0.2% on the leaves of transgenic plants positive by PCR Glyphosate, observed after 7 days.

 DRAWINGS

Figure 1: Growth curves of transformants containing MC- EPSPS- pET30a, C- EPSPS- pET30a and pET30a in M9 medium containing 100 mM glyphosate

Figure 2: Growth curve of BL21 (DE3) PlysS transformants containing MC- EPSPS- pET30a, C- EPSPS- pET30a and pET30a in M9 liquid medium containing 150 mM glyphosate

FIG 3: A growth is EPSPS deficient strain ER2799 containing plasmid MC -EPSPS -pET30a on M9 solid medium; B is EPSPS deficient strain ER2799 containing plasmid C -EPSPS -pET30a on M9 solid medium Growth condition; C is the growth of EPSPS-deficient strain ER2799 containing pET30a plasmid in M9 solid medium

Figure 4: Protein expression of BL21 (DE3) PlysS transformants containing MC- EPSPS -pET30a, C- EPSPS -pET30a and pET30a, respectively

Figure 5: Roadmap for constructing plant expression vector pBI-MC- EPSPS

 Figure 6: A is the result of tobacco application to the MC-EPSPS gene after 0.2% glyphosate 7 days; B Non-GMO tobacco smeared 0.2% glyphosate results after 7 days

 Figure 7: A is the result of spraying 0.2% glyphosate on cotton for MC-EPSPS gene for 7 days; B Is the result of non-GM cotton spraying 0.2% glyphosate after 7 days

 detailed description

 The following preferred embodiments may further illustrate the technical route of the present invention, but do not constitute a limitation of the present invention.

 Example 1 Acquisition of the MC-EPSPS gene:

 1. Extraction of total RNA from cotton

 A. After taking 0.1g cotton leaf liquid nitrogen, add 0.5ml plant RNA extract (purchased from invitrogen ), shake until thoroughly mixed.

 B. Leave at room temperature for 5 minutes.

 Centrifuge at 12,000 rpm for 1 minute at C.4 °C. Transfer the supernatant to a new RNase-free centrifuge tube.

 D. Add 0.1 ml of 5 M NaCl and mix gently.

 E. Add 0.3 ml of chloroform and mix upside down.

 Centrifuge at 12,000 rpm for 10 minutes at F.4 °C and transfer the upper aqueous phase to a new RNase-free centrifuge tube.

 G. Add equal volume of isopropanol to the obtained water, mix and let stand for 10 minutes at room temperature.

 Centrifuge at 12,000 rpm for 10 minutes at H.4 °C. Discard the supernatant and take care not to pour out the sediment. Add 1ml 75% Ethanol.

 Centrifuge at 5,000 rpm for 3 minutes at I.4 °C. Pour out the liquid, and the remaining small amount of liquid is briefly centrifuged, then sucked out with a pipette and dried at room temperature. 2-3 minutes.

 J. Add 50μl of RNase-free water, repeatedly blow and mix to fully dissolve RNA.

 2. Removal of DNA contamination in RNA samples

 A. Add 16 μl of total RNA, 2 in RNase-free Eppendorf tube m l10×Buffer , 1 m l RnaseOUT , 1 m l RNase-free DNaseI ( 2U/ m l );

 B. Leave at room temperature for 15 min ;

 C. Add 2 ml of 25 mM EDTA and incubate at 65 °C for 15 min.

 3, reverse transcription

 A. In the 0.2ml tube, add the following ingredients:

 Total RNA (0.1μg/μl) 2.0μl

 Oligo (dT12-18) (2μM) 2.0μl

 B. Water bath at 70 °C for 10 minutes. Immediately placed in an ice bath;

 C. Add the following ingredients:

 2.0 μl 10×RT buffer;

 2.0 μl 250 μM dNTP mix ;

 2.0 μl 100 mM DTT;

 9.8 μl DEPC H2O;

 0.2μl 200Uμ/l SuperScriptIII;

 D. The following reactions were carried out: 42 °C for 90 minutes; 70 °C for 15 minutes; -20 °C for storage.

 4, cotton EPSPS gene acquisition

Using the cDNA as a template, the genomic sequence including the 5-terminal untranslated region of cotton EPSPS gene, chloroplast peptide (CTP), EPSPS gene and 3-terminal untranslated region was amplified. The EPSPS gene of cotton was named C- EPSPS .

 The amplification primer sequence is as in Appendix SEQ ID No: 5, SEQ ID No: 6:

 PCR conditions: 94 °C 5min, 94 °C 45s, 56 °C 45s, 72 °C 4min , 5 cycles; 94 °C 45s, 60 °C 45s, 72 °C 4min, 25 cycles; 72 °C 7min.

The amplified product was digested with EcoR I and Sac I and constructed into the cloning vector pBulescript. The vector was named pBulescript-C- EPSPS .

 5, fixed point mutation

Site-directed mutagenesis of the C-EPSP synthase gene, changing the threonine in the third alpha helix at the N-terminus to isoleucine (as indicated by amino acid 102 in SEQ ID No: 2), proline Change to serine (as indicated by amino acid 106 in SEQ ID No: 2). To construct expression vectors needs, the C- EPSPS first 208 and second 406 of the proline-alanine synonymous mutations, has removed the genes present in the Nde I (CATATG mutated to CTTATG) and Nco I ( CCATGG mutation to CTATGG) The effect of two restriction sites on subsequent construction. The mutant gene was named MC- EPSPS. Mutant primers are shown in Appendix SEQ ID No: 7, SEQ ID No: 8, and SEQ ID No: 9, respectively.

 Phosphorylation of the 5' end of the primer facilitates the ligation and cyclization of subsequent PCR products. The phosphorylation process is as follows:

 reaction system:

 Primer (10μM) : 5μl

 10×T4 DNA Polynucleotide Kinase Buffer : 3μl

 T4 DNA Polynucleotide Kinase : 1 μ l

 ATP (10mM) : 3μl

 Hydration to 30μl

 Reaction conditions: 37 ° C reaction for 45 min.

The multi-point mutation was carried out using the cloning vector pBulescript-C- EPSPS containing the cotton EPSP synthase gene as a template, and the reaction procedure was carried out in accordance with the instructions in the QuikChange Multi Mutation Kit of STRATAGENE. The vector after the mutation was named pBulescript-MC- EPSPS .

 The mutant nucleotide sequence is as in Appendix SEQ ID No: 1, and the amino acid sequence is as in Appendix SEQ ID No: 2 The nucleotide homology with the original gene is 99.55%, and only the amino acids at the 102nd and 106th positions are changed at the amino acid level, and the homology is also 99.55%. Mutant clones by PCR And after sequencing and correct identification, save and reserve.

 Example 2 Construction of prokaryotic expression of MC-EPSPS gene:

The primers were designed to amplify the MC-EPSPS gene, and the Nde I restriction site was added to the 5' end, and the Sac I restriction site was added to the 3' end. The mutated pBulescript-MC- EPSPS was used as a template, and the primer sequence is shown in Appendix SEQ ID. No: 10, SEQ ID No: 11. PCR conditions: 94 ° C 5 min, 94 ° C 45 s, 54 ° C 45 s, 72 ° C 4 min, 30 cycles; 72 ° C 7 min. The PCR product was digested with Nde I and Sac I and constructed into prokaryotic expression vector pET30a. The recombinant expression vector MC- EPSPS- pET30a was obtained and transformed into prokaryotic expression strain BL21 (DE3) PlysS .

 Example 3 Analysis of glyphosate resistance of prokaryotic expression products of MC-EPSPS gene

Glyphosate-resistant functional analysis of MC-EPSPS gene: Invertants BL21 (MC- EPSPS -pET30a), BL21 (C- EPSPS -pET30a) and BL21 (pET30a) were inoculated into liquid M9 basal medium (including Cannamycin) 50 μg/mL), after activation at 37 °C overnight, dilute 1:100, inoculate 300 μL into 30 mL liquid M9 basal medium (containing kanamycin 50 μg/mL) at 200 rpm , 37 ° C air bath conditions. Incubate to an OD ₆₀₀ = 0.100, add IPTG to a final concentration of 1 mmol/L, add glyphosate to a final concentration of 100 or 150 mM, incubate at 200 rpm, 37 ° C air bath, and culture until 14 or 16 hours. The culture solution OD600 was measured every 2 hours, and the growth was recorded. The measurement results are shown in Fig. 1 and Fig. 2 in the drawing. As can be seen from the growth curves of Fig. 1 and Fig. 2, the transformants carrying the mutant gene survived on the M9 minimal medium containing 150 mM glyphosate, while the transformants carrying the wild type gene contained 150 mM of grass. Growth on the M9 minimal medium of glycophosphorus has been severely inhibited. Thus, the glyphosate resistance of the mutant was significantly improved compared to the wild type.

Analysis of prokaryotic expression: Single colonies containing BL21 (MC- EPSPS -pET30a), BL21 (C- EPSPS -pET30a) and BL21 (pET30a) were inoculated into liquid LB (containing kanamycin 50 μg/mL). After overnight activation at 37 °C, dilute 1:100, inoculate 50 μL into 5 mL liquid LB (containing kanamycin 50 μg/mL), incubate at 37 °C until OD ₆₀₀ = 0.6, add IPTG The concentration was 1 mmol/L, and the expression was induced by shaking at 37 °C for 3 h. The bacterial solution was collected by centrifugation, and the cells were resuspended in 0.2 volumes of gel loading buffer, boiled in boiling water for 5 min, and 15 μL was applied for loading. SDS-PAGE electrophoresis analysis was performed according to Sambrook et al. (1989), 10% SDS. Protein was separated by electrophoresis on -PAGE, stained with 0.25 % Coomassie Brilliant Blue R-250. The electrophoresis results are shown in Figure 3 of the accompanying drawings. Lanes 1 to 3 are crude protein extracts of three clones of BL21 (C- EPSPS -pET30a), and lanes 4 to 6 are BL21 (MC- EPSPS -pET30a). Crude extracts of the clones, Lanes 7 and 8 are BL21 (pET30a) crude extracts of two clone proteins. It can be seen from the electropherogram that the IPTG-inducible clone carrying the EPSPS gene can express the target protein of about 47 kDa in size. It was shown that both wild-type and mutant genes were efficiently expressed under the same expression conditions, and there was no significant difference in expression levels.

 From the above results, MC-EPSPS The increase in resistance is not due to overexpression of the gene, but is due to changes in protein structure.

 Example 4 Identification of prokaryotic expression products of MC-EPSPS gene

To further validate the enzymatic activity of the mutant gene, transformants BL21 (MC -EPSPS -pET30a), BL21 (C- EPSPS -pET30a) and BL21 (pET30a) were inoculated into each inoculated into a liquid LB medium, 37 [deg.] C, Incubate for 12 h at 200 rpm. The plasmid was extracted by alkaline lysis method, and 0.1 μg of the plasmid was transformed into EPSP synthase-deficient strain Escherichia coli ER2799 . The transformants were cultured on M9 solid medium containing 1 mM IPTG and kanamycin 50 ug/ml for 36 h. The growth situation is shown in Fig. 4A, Fig. 4B, and Fig. 4C. It can be seen from the growth of the transformants in Fig. 4A, Fig. 4B and Fig. 4C that the number and size of ER2799 (MC- EPSPS -pET30a) and ER2799 (C- EPSPS -pET30a) transformants are basically the same, while ER2799 ( pET30a) No transformants appeared. It is indicated that the mutant protein still has certain enzymatic activity.

 Example 5 MC-EPSPS gene Construction of plant expression vector

Selecting binary vector pBI121 as a plant expression vector, to amplify the 35S promoter with double enhancer from pCAMBIA2300 vector, both ends with Hin d III and Bam HI, a primer sequence such as SEQ ID No: 12 and SEQ ID No: 13 As shown, the PCR product was digested and cloned into pUC18. The OK- Pst I- Xho I - PS fragment was synthesized according to the published OK (Omega & Kozak) sequence and the PS (Processing & Splicing sequence) sequence (both fully disclosed in ZL 95 119563.8) with Bam HI and The Sac I restriction site is ligated between the OK and PS by Pst I and Xho I restriction sites, and the protection bases are inserted between the two restriction sites to facilitate subsequent restriction enzyme construction, using Bam HI and Sac I. The OK- Pst I- Xho I - PS was cloned into the recombinant plasmid 35S-pUC18 to obtain the recombinant plasmid 35S-OK-PS-pUC18. The CTP - MC- EPSPS fragment was amplified by using pBulescript-MC- EPSPS as a template. The gene sequence is shown in SEQ ID No: 3, and the Pst I and Xho I restriction sites were added to the ends of the fusion sequence, and the primer sequence was SEQ ID. As shown in No:14 and SEQ ID No: 15, CTP-MC- EPSPS was ligated into 35S-OK-PS-pUC18 using the restriction sites at both ends to obtain recombinant plasmid 35S-OK-CTP-MC-PS-pUC18. . The 35S-PS fragment of 35S-OK-CTP-MC-PS-pUC18 was cloned into pBI121 by using Hin d III and Sac I, and the original 35S - GUS was replaced to obtain the recombinant plant expression vector pBI-MC- EPSPS . The process is shown in Figure 5 of the accompanying drawings.

 Example 6 Obtaining glyphosate resistant tobacco using Agrobacterium-mediated transformation

 Soak the tobacco seeds with 75% alcohol for 30s, then soak them with 0.1% liters of mercury for 8min. , surface disinfection. The sterile tobacco seeds were placed in MS medium (30 g/L sucrose) and sterilely germinated to prepare sterile seedlings. Take the sterile seedlings and cut them into 5mm×5mm The leaf discs of the size were inoculated with the Agrobacterium containing the expression vector in the logarithmic growth phase for 10 min, and the bacterial cells were aspirated and co-cultured for 2 days in the dark (MS medium). Transfer the leaves to the differentiation medium ( MS+1mg/L BA+0.1mg/L NAA+50mg/L kanamycin +500mg/L cephalosporin), culture under light conditions 45 After about days, when the buds grow up, they are cut and transferred to rooting medium (MS+50mg/L kanamycin +500mg/L cephalosporin) for about 30 days. After the root system is developed, the seedlings are transferred to only Number storage was performed on MS medium of 500 mg/L cephalosporin.

The obtained transgenic tobacco leaves were extracted and identified by PCR. The tobacco leaves which were positive by 0.2% glyphosate spray PCR were observed. After 7 days, the experimental results were observed. As shown in Figures 6A and 6B in the accompanying drawings, the MC was transferred to MC- The EPSPS gene transgenic tobacco grew normally (Fig. 6A), while the non-transgenic tobacco growth was significantly inhibited, and the leaves were yellow and wilting (Fig. 6B).

The above results indicate that the tobacco transfected with the pBI-MC- EPSPS plasmid has better glyphosate resistance.

 Example 7 Transformation of cotton with Agrobacterium-mediated transformation to obtain herbicide-tolerant transgenic cotton

 Agrobacterium-mediated transformation is a plant genetic transformation method well known to researchers in the field. The specific operating procedures are:

 1 . Strain culture

The constructed pBI-MC- EPSPS plasmid was electroporated into Agrobacterium strain LBA4404 , and Agrobacterium single colony was inoculated into LB or YEB liquid medium containing kanamycin 50 mg/L and rifampicin 25 mg/L. in. The dark culture was shaken overnight at 28 ° C until the logarithmic phase of bacterial growth. Dilute the bacterial solution with LB or YEB liquid medium, shake it for 4-6 hours, and dilute the bacterial solution to an OD600 value of 0.3 to 0.35.

 2 . Aseptic seedling preparation

(1) The cotton seeds are desulfurized with sulfuric acid (H ₂ SO ₄ ), the sulfuric acid on the surface of the seeds is washed away with tap water, dried, and the seeds are surface-sterilized with 70% ethanol for 1 min, and then peroxidized with 10% to 15%. Hydrogen (H ₂ O ₂ ) treatment for 2 ~ 4 h, rinse with sterile water for 2 ~ 3 times;

 (2) Soak in sterile water for 18 ~ 24 h, leave the seeds white, then peel off the seed coat under sterile conditions, and plant seed culture medium. (1/2 MS + agar 6g/L, pH 6.8);

 (3) 25 ° C ~ 28 ° C light culture for 3 ~ 5 d when used.

 3 . Co-culture of cotton explants with Agrobacterium

 Take the hypocotyl of the sterile seedling, cut it into 0.5 ~ 0.6 cm sections with a scalpel, and immerse it in the diluted bacterial solution. 5 ~ 10 Min , then remove the hypocotyl segment, blot excess of the bacterial solution with sterile filter paper, and place on the co-culture medium (MS + 2.4-D 0.1 mg / L + KT 0.1 mg / L + glucose 30 g/L + acetosyringone 200 mg / L + agar 6 g / L, pH 5.0, a layer of sterile filter paper), sealed with a parafilm. 22 ° C ~ 25 ° C co-culture 2 Day.

 4 . Screening of induced callus and resistant callus

 (1) Callus induction

The co-cultured hypocotyl segments were placed in callus induction medium (MS + 2,4-D 0.1 mg/L + KT 0.1 mg/L + MgCl ₂ 0.91 g/L + Gelrite 2.0 g/L + Kanamycin 50 ~ 100 mg / L + cephalosporin 500 mg / L + glucose 30 g / L, pH 5.8), cultured under normal conditions (25 ° C) for 2 months (change the same medium once a month) ).

 (2) Detection of resistant callus

Under the sterile conditions, the callus was picked a little and the selection marker gene nptII was detected. The callus with positive test results continued to be subcultured, and the non-positive callus was eliminated. By detecting the expression of nptII , the frequency of cotton resistant callus was 50% ~ 76%.

 5 . Proliferation of callus

The induced resistant callus was introduced into a proliferation medium (MS medium + MgCl ₂ 0.91 g/L + Gelrite 2.0 g/L + glucose 30 g/L, pH 5.8), and cultured under normal conditions (25 ° C). , once every other month, until the callus differentiates. After the first and second transfer to the proliferation medium, some of the callus browning died, and the normal callus did not proliferate rapidly. After the second passage, the callus proliferation rate was accelerated.

 6 . Callus differentiation and transgenic seedling transplanting

After the callus was subcultured several times, some callus was transformed into rice granules and transferred to the differentiation medium (no NH ₄ + , and KNO ₃ doubled MS + glutamine 1.0 g / L + Asparagine 0.5 g / L + MgCl ₂ 0.91 ~ 1.35 g / L + Gelrite 2.0 ~ 3.0 g / L + glucose 20 ~ 30 g / L, pH 5.8), further differentiate into embryoid bodies, embryoid body length becomes small After the plants are transferred to a large triangular flask, the seedlings are transplanted after the roots are long. Wash the medium of the root of the regenerated cotton plant, plant it in the sterilized vermiculite, and pour the nutrient solution. The planted regenerated cotton seedlings are placed in an artificial incubator with a temperature control of 22 ° C and a humidity control of 80 to 85% for 5 to 7 days, and then cultured in a greenhouse for 10 to 20 days, and then transplanted into a soil pot or a field.

Using the above method, the herbicide-tolerant gene plant expression vector was introduced into cotton to obtain transgenic cotton. The identification method was as in Example 6. The results were as shown in Figures 7A and 7B in the accompanying drawings. The transgenic cotton transgenic with MC- EPSPS gene was able to grow normally (Fig. 7A), while the growth of non-transgenic tobacco was significantly inhibited, and the leaves were yellow and wilting. (Fig. 7B).

The above results indicate that the cotton transfected with the pBI-MC- EPSPS plasmid has better glyphosate resistance.

Claims (1)

A cotton EPSP synthetase mutant gene having a nucleotide sequence as shown in SEQ ID No: 1. 2. The protein encoded by the cotton EPSP synthase mutant gene of claim 1, having SEQ ID NO: 2 The amino acid sequence shown. 3. A cotton EPSP synthase mutant fusion gene having SEQ ID No: 3 The nucleotide sequence shown. The protein encoded by the cotton EPSP synthase mutant fusion gene according to claim 3, which has SEQ ID NO: 4 The amino acid sequence shown. 5. A plant expression vector comprising the cotton EPSP synthase mutant gene of claim 1 or 3 or a fusion gene thereof. 6. A plant cell, tissue or plant transformed with the plant expression vector of claim 5. 7. The cotton EPSP as claimed in claims 1 and 3. Use of a synthetase mutant gene or a fusion gene thereof in a glyphosate resistant plant variety.

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