WO2015027369A1 - 一种抗草甘膦融合蛋白及其编码基因、产生方法与应用 - Google Patents

一种抗草甘膦融合蛋白及其编码基因、产生方法与应用 Download PDF

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WO2015027369A1
WO2015027369A1 PCT/CN2013/082238 CN2013082238W WO2015027369A1 WO 2015027369 A1 WO2015027369 A1 WO 2015027369A1 CN 2013082238 W CN2013082238 W CN 2013082238W WO 2015027369 A1 WO2015027369 A1 WO 2015027369A1
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glyphosate
gene
transgenic
epsps
seq
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PCT/CN2013/082238
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English (en)
French (fr)
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崔洪志
何云蔚
王建胜
孙超
王君丹
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创世纪种业有限公司
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Priority to PCT/CN2013/082238 priority Critical patent/WO2015027369A1/zh
Priority to CN201380008110.6A priority patent/CN104684934B/zh
Publication of WO2015027369A1 publication Critical patent/WO2015027369A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • 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
    • C12N15/8275—Glyphosate
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1085—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5)
    • C12N9/1092—3-Phosphoshikimate 1-carboxyvinyltransferase (2.5.1.19), i.e. 5-enolpyruvylshikimate-3-phosphate synthase
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • Glyphosate-resistant fusion protein and coding gene thereof production method and application thereof
  • the present invention relates to fusion proteins and coding genes thereof and applications thereof, and in particular to a fusion 5-enolpyruvylshikimate-3 -phosphate synthase (EPSPS) and a coding gene thereof
  • EPSPS fusion 5-enolpyruvylshikimate-3 -phosphate synthase
  • Glyphosate is a non-selective herbicide with the advantages of stable physical and chemical properties, high efficiency, broad spectrum, low toxicity, low residue, easy decomposition by microorganisms, no damage to the soil environment, and has been widely used in agricultural production. It has become the most productive pesticide variety in the world.
  • the mechanism of action of glyphosate is mainly to competitively inhibit the activity of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in the shikimate pathway.
  • EPSPS 5-enolpyruvylshikimate-3-phosphate synthase
  • the enzyme is a key enzyme in the biosynthesis of aromatic amino acids (including tryptophan, tyrosine, phenylalanine) in fungi, bacteria, algae and higher plants, which consists of two conserved subunits. Dumbbell structure.
  • Glyphosate is an analogue of phosphoenolpyruvate (PEP), a competitive EPSPS inhibitor that combines glyphosate, EPSPS and shikimic acid trisphosphate (S3P) to form an EPSPS-S3P-glyphosate complex (this)
  • PEP phosphoenolpyruvate
  • S3P shikimic acid trisphosphate
  • the complex is very stable), inhibiting the activity of EPSPS leads to hindered choline acid synthesis, blocking the biosynthesis of aromatic amino acids and some aromatic compounds, and ultimately leading to metabolic disorders of some hormones and key metabolites such as flavonoids, lignin and phenolic compounds. , thereby disturbing the normal nitrogen metabolism of the organism and causing it to die.
  • the first is to overexpress EPSPS in plants and thereby antagonize the competitive inhibition of glyphosate.
  • Amrhem et al. isolated a glyphosate-tolerant petunia cell line by gradually increasing the concentration of glyphosate and increasing the selection pressure of glyphosate.
  • Analysis of the EPSPS gene in this cell line revealed that the copy number of the ⁇ S3 ⁇ 4 gene in the genome of the cell line was amplified 20-fold, which greatly increased the yield of the enzyme (Amrhein N, Johanning D, et al. Biochemical basis For glyphosate-tolerance in a bacterium and plant tissue culture. FEBS Lett., 1983, 157: 191-196).
  • the second is to mutate EPSPS to reduce the affinity for glyphosate while maintaining catalytic activity, even without affinity.
  • Stalker et al. isolated a strain resistant to glyphosate from Salmonella typhimurium. It was found that the 101st proline residue of EPSPS became a serine residue, and the transgene encoding gene could be transgenic into tobacco. Tobacco has increased resistance to glyphosate (Stalker DM, Hiatt WR, et al. A amino acid substitution in the enzyme 5 -enolpyruvylshikimate-3 - phosphate synthase confers resistance to the herbicide glyphosate.
  • Padgette et al. mutated the glycine residue at position 96 of EPSPS encoded by the aroA gene of Escherichia coli to an alanine residue, and found that the binding activity of the mutant protein to glyphosate was significantly reduced, and the mutation was encoded.
  • the gene of the protein can significantly increase its resistance to glyphosate after expression in petunia (Padgette SR, Re DB, Gasser CS, et al. Site-directed mutagenesis of a conserved region of the 5 -enolpyruvylshikimate-3 - Phosphate synthase active site.
  • the applicant has site-directed mutagenesis of the 101th threonine residue from cotton EPS to an isoleucine residue, and the 105th proline residue has been changed to a serine residue to obtain a certain glyphosate resistance.
  • the EPSPS mutant gene (PCT/CN2010/078327).
  • mutant fusion proteins were obtained by different EPSPS homology and contrast complementary design ideas, and further analysis of glyphosate resistance was performed to screen for mutant fusion proteins with significantly improved functions.
  • One of the designs implements the content of the present invention.
  • the inventors found a deletion or redundancy of small amino acids in different regions of MC-EPSPS by homologous analysis of the amino acid sequences of MC-EPSPS and another EPSPS protein G2-aroA (Chinese Patent 03826892). (figure 1 ) . Accordingly, the present inventors have obtained an EPSPS mutant fusion protein (hereinafter referred to as MC2-EPSPS) using a combination of protein engineering and genetic engineering, and the fusion protein is G2-fused at the N-terminus of the MC-EPSPS protein. 26 amino acid residues at the N-terminus of the aroA protein. Through prokaryotic expression and functional identification of transgenic plants, the glyphosate level of the fusion protein MC2-EPSPS of the present invention was significantly higher than that of MC-EPSPS and G2-aroA.
  • MC2-EPSPS EPSPS mutant fusion protein
  • the present invention adopts the EPSPS fusion protein screening scheme for the first time, and obtains an EPSPS mutant fusion protein (MC2-EPSPS) with significantly improved glyphosate resistance and a gene encoding the MC2-EPSPS fusion protein, which enables expression of an exogenous EPSPS gene.
  • MC2-EPSPS EPSPS mutant fusion protein
  • a glyphosate-resistant transgenic plant is obtained, thereby providing a preferred protocol for obtaining a glyphosate-tolerant herbicide-transgenic plant.
  • a first aspect of the invention provides a fusion protein having the sequence of SEQ ID NO: 2.
  • a second aspect of the invention provides a nucleotide sequence encoding the fusion protein of the first aspect of the invention; preferably, the nucleotide sequence has the nucleotide sequence of SEQ ID NO: 1.
  • a third aspect of the invention provides a recombinant expression vector comprising the nucleotide sequence of the second aspect of the invention, wherein the nucleotide sequence is operably linked to an expression control sequence of the expression vector.
  • the expression vector is pET28b ; in another preferred embodiment, the expression vector is pCAMBIA2300.
  • a fourth aspect of the invention provides a recombinant cell comprising the nucleotide sequence of the second aspect of the invention or the recombinant expression vector of the third aspect of the invention; in a preferred embodiment, the recombination
  • the cells are recombinant E. coli cells; in another preferred embodiment, the recombinant cells are recombinant Agrobacterium cells.
  • a fifth aspect of the invention provides a method for improving glyphosate resistance of a plant, comprising: introducing the nucleotide sequence of the second aspect of the invention or the recombinant expression vector of the third aspect of the invention into a plant cell, The tissue, organ or plant and expresses the nucleotide sequence; in a preferred embodiment, the plant is tobacco; in another preferred embodiment, the plant is cotton.
  • a sixth aspect of the invention provides a method for producing a fusion protein, which comprises inserting the nucleotide sequence of the second aspect of the invention into an expression vector and expressing the nucleotide sequence and the expression control sequence of the expression vector Operably ligating, and then introducing the resulting recombinant expression vector into an organism to express the gene; in a preferred embodiment, the organism is the large intestine Bacillus; In another preferred embodiment, the organism is tobacco; in another preferred embodiment, the organism is cotton.
  • a seventh aspect of the invention provides the fusion protein of the first aspect of the invention, the nucleotide sequence of the second aspect of the invention, the recombinant expression vector of the third aspect of the invention or the fourth aspect of the invention
  • the recombinant cells are used to improve plant glyphosate resistance and for plant breeding; in a preferred embodiment, the plant is tobacco; in another preferred embodiment, the plant is cotton.
  • BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 : Results of homologous analysis of the amino acid sequence of cotton MC-EPSPS protein and EPSPS protein G2-aroA.
  • Figure 2 Plasmid map of the prokaryotic expression vector pET28b-MC2.
  • Figure 3 Growth curve of BL21 (DE3) PlysS transformants containing pET28b-MC2, pET28b-MC, pET28b-G2 or pET28b in liquid M9 basal medium containing 150 mM glyphosate.
  • Figure 4 Expression of proteins in BL21 (pET28b-MC), BL21 (pET28b-MC2) and BL21 (pET28b-G2).
  • the first lane is protein Marker (name: Unstained Protein Molecular Weight Marker; purchased from Shenzhen Yanshun Biotechnology Co., Ltd.);
  • the second and third lanes are crude extracts of two clones of BL21 (pET28b-G2), the first 4 and 5 lanes are BL21 (pET28b-MC) crude extracts of two clones, lanes 6 and 7 are BL21 (pET28b-MC2) two clone protein crude extracts, lanes 8 and 9 are BL21 (pET28b) Two crude extracts of cloned proteins.
  • Figure 5 Plasmid map of pBI121-ATP-G2.
  • Figure 6 Plasmid map of pBI121-PTP-G2.
  • Figure 7 Plasmid map of pBI121-CTP-G2.
  • Figure 8 shows the construction process of pCAMBIA2300-2;
  • Figure 8b and Figure 8c show the construction process of pCAMBIA-35 S-PTP-MC2.
  • Figure 9 Randomly arranged non-transgenic tobacco seedlings and TO-transgenic tobacco seedlings at 6 to 7 leaf stage, after 15 days of spraying 2000 ppm glyphosate, the results were observed after spraying 3000 ppm of glyphosate for another 7 days.
  • Figure 9a shows two tobacco plants transgenic with the PTP-MC2-EPSPS gene, and the remaining resistant plants are similar thereto, not shown here;
  • Figure 9b shows two tobacco plants transgenic with the 7?- 1 ⁇ -£ ⁇ 3 ⁇ 43 ⁇ 4 gene, and the rest The plants are similar thereto and are not shown here;
  • Fig. 9c is two transgenic tobacco plants, the other plants are similar thereto, not shown here;
  • Fig. 9d is a non-transgenic control plant.
  • FIG. 10 Electrophoresis results for RT-PCR detection of transgenic tobacco and non-transgenic tobacco.
  • 1-3 are transgenic tobacco plants resistant to 3000 ppm glyphosate TO transgenic tobacco plants
  • 4-6 are transgenic tobacco plants transgenic with PTP-MC-EPSPS gene that are not resistant to 3000 ppm glyphosate T0 generation
  • 7-9 is transgenic ( ⁇ - The gene is not resistant to 3000 ppm glyphosate glyphosate TO transgenic tobacco plants
  • 10-12 is transgenic 7
  • 1 ⁇ 2-£ ⁇ 3 ⁇ 43 ⁇ 4 gene is not resistant to 3000 1 11 glyphosate T0 transgenic tobacco plants
  • 13 is a non-transgenic control tobacco.
  • the internal reference is the tobacco endogenous Actm gene (http: ⁇ www. ncbi.Blm. mh. ov/nuccore/ AB 158612.1).
  • Figure 11 Randomly arranged non-transgenic cotton seedlings of 4 to 5 leaf stage and TO transgenic cotton seedlings, and sprayed 3000 ppm of glyphosate to saturation (50 ml per square meter) for 10 days.
  • Figure 11a shows two turns? ? - ⁇ ⁇ gene-resistant cotton plants, the remaining resistant plants are similar thereto, not shown here;
  • Figure l ib is two transgenic cotton plants with resistance to 7?-1 ⁇ -£ ⁇ »3 ⁇ 4 gene, the remaining resistance The sex plants are similar thereto and are not shown here;
  • Fig. 11c is two transgenic cotton plants, and the other resistant plants are similar thereto, not shown here;
  • Fig. 1 id is a non-transgenic control plant .
  • FIG. 12 Electrophoresis results of RT-PCR detection of non-transgenic control cotton of transgenic cotton machine.
  • 1-2 is a glyphosate-resistant 0-transgenic cotton plant with a transformation of 7?- ⁇ -£ ⁇ »3 ⁇ 4 gene
  • 3-5 is a non-resistance to the PTP-MC-EPSPS gene.
  • Phospho-TO transgenic cotton plants, 6-8 are G-tolerant glyphosate-resistant TO cottons
  • 9-11 are non-glyphosate-tolerant PTP-MC2-EPSPS genes in TO cotton, 12 For non-transgenic control cotton.
  • the endogenous Actin gene http://'www.ncbi.nlni.nih.gov/niiccore/ 32186889) is an internal reference.
  • Figure 13 is a plasmid map of pCAMBIA-35 S-PTP-MC2.
  • BEST MODE FOR CARRYING OUT THE INVENTION The following examples are provided to facilitate a better understanding of the present invention by those skilled in the art. The examples are for illustrative purposes only and are not intended to limit the scope of the invention. The compounds or reagents used in the examples are commercially available or can be prepared by conventional methods known to those skilled in the art; the experimental apparatus used is commercially available.
  • Total RNA reverse transcription in cotton leaves In a 0.2 ml EP tube, add 2.0 ⁇ total RA (0.1 ⁇ / ⁇ 1) and 2.0 ⁇ 01igo (dT12-18) (2 M), mix, and bath at 65 °C for 10 minutes. Immediately ice bath for 5 minutes, then add 2.0 ⁇ lOxRT buffer, 2.0 ⁇ 250 ⁇ dNTP mix, 2.0 ⁇ 100 mM DTT, 9.8 ⁇ DEPC H 2 0, 0.2 ⁇ 200 U / ⁇ SuperScriptlll ( Invitrogen ), 50 °C after mixing The reaction was carried out for 60 minutes; then inactivated at 70 ° C for 15 minutes; stored at -20 ° C.
  • EPSPS coding gene of cotton Using the cotton cDNA obtained above as a template, the 5'-end untranslated region (5'UTR), chloroplast-derived peptide (CTP), EPSPS-encoding gene and 3' end of the cotton EPSPS-encoding gene were amplified. Genomic sequence within the untranslated region (3'UTR).
  • the reaction system is as follows:
  • PCR reaction system 10 ⁇ 5 xPS Buffer, 3 ⁇ 2.5 mM dNTP, 2.0 ⁇ cDNA, 1.0 ⁇ PrimeSTAR HS DNA polymerase (purchased from TAKARA), 10 ⁇ primers SEQ ID NO: 3 and SEQ ID NO: 4 each 2.0 ⁇ (for subsequent construction, introduce coR I, Sac I recognition sites at both ends of the primer), and 30 ⁇ double distilled water.
  • PCR reaction conditions pre-denaturation 5 mm at 94 °C, 5 cycles (denaturation at 94 °C for 45 s, annealing at 56 °C for 45 s, extension at 72 °C for 2 min), 25 cycles (94 °C denaturation for 45 s, 60 °) C is annealed for 45 s, extended at 72 ° C for 2 min), and extended at 72 ° C for 7 min.
  • the resulting amplified product was digested with EcoR I and P Sac I and inserted into the cloning vector pBluescript II SK (-) (hereinafter referred to as pBluescript vector, purchased from Shenzhen Yanshun Biotechnology Co., Ltd.), £coR I and S «c.
  • pBluescript vector purchased from Shenzhen Yanshun Biotechnology Co., Ltd.
  • the ligation reaction system and the reaction conditions are as follows: 3 ⁇ 1 of the PCR product after digestion, 5 ⁇ l of 2xT4 DNA ligase buffer, 1 ⁇ 1 of the pBluescript vector after digestion, ⁇ 4 DNA ligase 1 11, connected overnight at 4 °C.
  • the resulting vector was named pBluescript-C-EPSPS.
  • the clones were picked and PCR was performed separately (the reaction system and conditions were the same as above).
  • the PCR positive clones were sent to Yingji Jieji (Shanghai) Trading Co., Ltd. for sequencing.
  • the primers used for sequencing were BcaBEST TM Sequencing Primer RV-M (SEQ ID NO: 5), BcaBESTTM Sequencing Primer M13-47 (SEQ ID NO: 6), specific primer (SEQ ID NO: 7), and the sequence obtained by sequencing is SEQ ID NO: 8.
  • SEQ ID NO: 8 is 7th 1st - 184 1 ⁇ is 5 '1711 region; 185 bp - 418 bp is a chloroplast-derived peptide nucleotide sequence; 419 bp - 1750 bp is a nucleotide encoding EPSPS protein
  • the sequence (designated C-EPSPS'), the amino acid sequence corresponding to C-£3 ⁇ 4 3 ⁇ 4 is SEQ ID NO: 9 (which is the amino acid sequence of the EPSPS protein after removal of the leader peptide and addition of methionine at the N-terminus) ; 1751-2028 bp It is 3'UTR.
  • the site-directed mutagenesis process is as follows:
  • the primer phosphorylation reaction process is as follows:
  • T4 DNA Polynucleotide Kinase purchased from TAKARA: 1 ⁇
  • Site-directed mutagenesis The cloning vector pBluescript-C-EPSPS containing the C-£3 ⁇ 4 ⁇ 4 gene was used as a template, and the reaction procedure was carried out using the QuikChange Multi Mutant Kit of STRATAGENE and referring to the instructions (in a 25 ⁇ M reaction system, add about 70 Ng template plasmid, 2.5 ⁇ lO Quickchange Multi Buffer, 3.0 ⁇ phosphorylated primer mix, 1.0 ⁇ dNTP Mix, 1.0 ⁇ Multi enzyme blend, add water to 25 ⁇ l.
  • Reaction conditions pre-denaturation at 95.0 °C for 1 min; 30 cycles ( Denaturation at 95.0 °C for 1 min; annealing at 55 °C for 1 min; extension at 65 °C for 10 min), after completion of the reaction, the temperature was lowered to 37.0 °C by ice bath, 1 ⁇ , 37.0 °C for 2 h, 2 ⁇
  • the digestion product is directly transformed into E. coli).
  • the resulting mutated vector was named pBl ueSC ript -MC-EPSPS.
  • the 3 mutated plasmid was transformed into E. coli JM109 competent cells for cloning and sequencing (reaction system and method are the same as above).
  • the clones with the target mutation were verified by PCR and sequencing and stored for later use.
  • the primers SEQ ID NO: 13 and SEQ ID NO: 14 were designed to amplify the cotton EPSPS coding region sequence except for the leader peptide sequence, using the pBluescript-MC-EPSPS plasmid which has been verified to have the target mutation as a template, and the 5' end thereof was increased.
  • the start codon ATG is used for subsequent construction.
  • the reaction system and reaction conditions are as follows:
  • PCR reaction system 10 ⁇ 5 ⁇ PS Buffer, 3 ⁇ 2.5 mM dNTP, 2.0 ⁇ pBluescript-MC-EPSPS plasmid, 1.0 ⁇ PrimeSTAR HS DNA polymerase (purchased from TAKARA), 10 ⁇ primer SEQ ID NO: 13 and SEQ ID NO: 14 2.0 ⁇ l each, and 30 ⁇ of double distilled water.
  • PCR reaction conditions pre-denaturation at 94 °C for 5 min, 33 cycles (denaturation at 94 °C for 45 s, annealing at 56 °C for 45 s, extension at 72 °C for 2 min), extension at 72 °C for 7 mintician
  • Nco I and 3 ⁇ 4o I sites were designed at the ends of the two primers, and the amplified products were digested with Nco I and 3 ⁇ 4o I and inserted into the Nco I of pET28b and the 3 ⁇ 4o I multiple cloning site to obtain the recombinant expression vector pET28b. -MC.
  • the reaction product was ligated and transformed into 100 L E. coli JM109 competent cells (method as above).
  • the bacterial culture solution after 200 L culture was applied to an LB solid culture plate containing 50 g/mL kanamycin (purchased from Beijing Bayerdi Biotechnology Co., Ltd.), and incubated at 37 ° C for 18 hours.
  • the normally growing colonies were selected for sequencing verification, and the resulting mutated gene sequence was SEQ ID ⁇ : 15, and it was named MC-£3 ⁇ 43 ⁇ 4.
  • Primers SEQ ID NO: 17 and SEQ ID NO: 18 were designed to amplify the 78 nucleotide sequence at the 5' end of the gene with the Nco I site at the 5' end and the Nde I site at the 3' end. PCR was carried out using TAKARA's PrimeSTAR HS DNA polymerase using the above pUC57-G2 plasmid as a template.
  • PCR reaction system 10 ⁇ 5 ⁇ PS Buffer, 3 ⁇ 2.5 mM dNTP, 2.0 ⁇ pUC57-G2 plasmid, 1.0 ⁇ PrimeSTAR HS DNA polymerase, 10 ⁇ primers SEQ ID NO: 17 and SEQ ID NO: 18 each 2.0 11, and 30 ⁇ of double distilled water.
  • PCR reaction conditions pre-denaturation at 94 ° C for 5 min, 33 cycles (denaturation at 94 ° C for 30 s, annealing at 58 ° C for 30 s, extension at 72 ° C for 10 s), extension at 72 ° C for 10 min.
  • PCR amplification product plus A tail The PCR product was added with 2.5 volumes of absolute ethanol, placed at -20 ° C for 10 minutes, centrifuged, de-cleared, air-dried, and dissolved in 21 ⁇ l of double distilled water. Add 2.5 ⁇ ⁇ Buffer, 0.5 ⁇ 5 mM dATP, 2.5 ⁇ Ex Taq. Ex Taq and ⁇ Buffer were purchased from TAKARA. Reaction conditions: The reaction was carried out at 70 ° C for 30 minutes.
  • the obtained DNA fragment of about 100 bp was recovered (using a recovery kit purchased from Omega), ligated into pGEM T-easy vector (purchased from Promega), and then transformed into JM109 competent cells ((purchased from TAKARA) (The ligation system and transformation method were the same as above. Randomly pick 10 white colonies in LB liquid medium containing 50 g/mL ampicillin, and incubate at 37 °C overnight, add glycerol to a final concentration of 20%, and store at -80 °C. After the Nco I and N ⁇ I double digestion, two positive clones were sent to Yingjie Jieji (Shanghai) Trading Co., Ltd. for sequencing, and the correct clones were retained and named pGEM-G2 (78).
  • the primers SEQ ID ⁇ :19 and SEQ ID ⁇ :14 were used to amplify the MC-£3 ⁇ 43 ⁇ 4 gene using the above-mentioned sequence-proven pET28b-MC plasmid as a template, such that the 5' end carries the NI site and the 3' end band; 3 ⁇ 4o I site, PCR reaction conditions: pre-denaturation at 94 °C for 5 min, 33 cycles (denaturation at 94 °C for 30 s, annealing at 57 °C for 30 s, extension at 72 °C for 2 min), extension at 72 °C for 10 min.
  • the resulting PCR product was ligated into the pGEM T-easy vector (purchased from Promega), and then transformed into JM109 competent cells for PCR sequencing (reaction system and method as above). After Nde I and 3 ⁇ 4o I double-enzyme digestion were identified, two positive clones were sent to Yingji Jieji (Shanghai) Trading Co., Ltd. for sequencing, and the correct clones were retained and named pGEM-MC.
  • pET28b was digested with Nco l ⁇ WXhol, and the vector fragment was recovered; pGEM-G2 (78) was digested with Nco I and Nde I, and a fragment of about 78 bp was recovered; pGEM-MC was digested with NI and 3 ⁇ 4oI. The 1300 bp fragment was recovered, and the above three fragments were ligated into three fragments to obtain the recombinant plasmid pET28b-MC2 (see Fig. 2).
  • the fusion gene consisting of the 78 bp plus MC-£3 ⁇ 43 ⁇ 4 gene at the 5' end was named MC2-£3 ⁇ 43 ⁇ 4. .
  • prokaryotic expression vector Referring to the construction strategy and procedure of the above pET28b-MC2 vector, primers SEQ ID ⁇ :17 and SEQ ID ⁇ :20 were used to introduce Nco l ⁇ WXhol sites at both ends of the G2 gene, respectively, and passed Nco l ⁇ WXhol The G2 gene was inserted into the pET28b vector by restriction enzyme digestion to obtain the prokaryotic expression vector pET28b-G2.
  • Liquid M9 basal medium (containing 48 mM Na 2 P0 4 -7H 2 0, 22 mM KH 2 P0 4 , 8.5 mM Nacl, 19 mM NH 4 C1, 2 mM MgS0 4 , 0.1 mM CaCl 2 and 0.4% glucose), After overnight activation at 37 ° C, it was diluted 1:100 (V/V). 300 dilutions of the bacterial solution were inoculated into 30 mL of liquid M9 basal medium (containing kanamycin 50 g/mL), and cultured at 200 rpm in an air bath at 37 °C.
  • IPTG isopropyl- ⁇ -D-thiogalactoside
  • Glyphosate purchased from Shandong Binzhou Jiu'an Chemical Co., Ltd.
  • the precipitated cells were separately centrifuged, and the cells were resuspended in 2x SDS loading buffer, and boiled in boiling water for 5 mm to obtain crude protein extract, 12000.
  • the rpm was centrifuged at 1 mm, and 15 supernatants were separately subjected to SDS-PAGE electrophoresis analysis.
  • the protein was separated by 10% SDS-PAGE electrophoresis according to the method of Sambrook et al. (1989), and stained with 0.25 % Coomassie Brilliant Blue R-250.
  • the results are shown in Figure 4.
  • the first lane is Protein Marker (name: Unstained Protein Molecular Weight Marker; purchased from Shenzhen Yanshun Biotechnology Co., Ltd.);
  • Lanes 2 and 3 are BL21 (pET28b-G2) two clones.
  • the crude protein extract, the 4th and 5th lanes are the crude protein extracts of two clones of BL21 (pET28b-MC), and the 6th and 6th lanes are the crude extracts of two cloned proteins of BL21 (pET28b-MC2).
  • Lanes 8 and 9 are crude extracts of two cloned proteins of BL21 (pET28b).
  • the IPTG-inducible clones carrying the MC-£3 ⁇ 4 3 ⁇ 4 and MC2-EPSPS G2 genes can express the target protein of about 47 kDa, and the MC2-EPSPS protein is slightly larger than MC-EPSPS and G2 protein. This indicates that under the same expression conditions, all three can obtain effective expression, and the electrophoresis results show that there is no significant difference in the protein expression levels of the three.
  • MC2-EPSPS glyphosate resistance is not due to overexpression of protein, but due to changes in protein structure.
  • the obtained sequences were SEQ ID NO: 21 (ATP) and SEQ ID ⁇ : 22 ( ⁇ 7?), respectively.
  • the three sequences were separately cloned into pUC57-T In the vector, the resulting recombinant plasmids were named pUC57-ATP, pUC57-PTP and pUC57-CTP, respectively.
  • the primers SEQ ID NO: 17 and SEQ ID NO: 24 were used to carry out a PCR reaction using pUK57-G2 plasmid using TAKARA's PrimeSTAR HS DNA polymerase as a template to amplify the gene at the 5' end with Nco I site, 3 'End with S «c I site.
  • PCR reaction system 10 ⁇ 5 ⁇ PS Buffer, 3 ⁇ 2.5 mM dNTP, 2.0 ⁇ pUC57-G2 plasmid, 1.0 ⁇ PrimeSTAR HS DNA polymerase, 10 ⁇ primers SEQ ID NO: 17 and SEQ ID NO: 24 2.0 11, and 30 ⁇ of double distilled water.
  • PCR reaction conditions pre-denaturation at 94 °C for 5 min, 33 cycles (denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 90 s), extension at 72 °C for 10 mintician
  • PCR amplification product plus A tail The PCR product was added with 2.5 volumes of absolute ethanol, placed at -20 ° C for 10 minutes, centrifuged, de-cleared, air-dried, and dissolved in 21 ⁇ l of double distilled water. Add 2.5 ⁇ ⁇ Buffer, 0.5 ⁇ 5 mM dATP, 2.5 ⁇ Ex Taq. Ex Taq and ⁇ Buffer were purchased from TAKARA. Reaction conditions: The reaction was carried out at 70 ° C for 30 minutes.
  • a DNA fragment of about 1300 bp was recovered (using a recovery kit purchased from Omega), ligated into the pGEM T-easy vector (purchased from Promega), and then transformed into JM109 competent cells (ligation system and transformation method as above) 10 white colonies were randomly picked and inoculated in LB liquid medium containing 50 g/mL ampicillin, and cultured overnight at 37 ° C, glycerol was added to a final concentration of 20%, and stored at -80 ° C until use. After identification by Nco I and S «c I double digestion, two positive clones were sent to Yingjie Jieji (Shanghai) Trading Co., Ltd. for sequencing, and the correct clones were retained and named pGEM-G2.
  • the plasmid pUC57-ATP was digested with ⁇ I and Nco I to recover the ATP fragment; the pGEM-G2 was digested with Nco I and Sac I, the fragment was recovered, and the plasmid pBI121 was digested with ⁇ «»3 ⁇ 4 ⁇ ⁇ and S «cl ( Purchased from Beijing Huaxia Yuanyang Technology Co., Ltd., the vector fragment was recovered, and the above-recovered ATP and G2 fragments were simultaneously inserted into pBI121 by three-fragment ligation to obtain plant expression vector pBI121-ATP-G2.
  • pBI121-PTP-G2 and pBI121-CTP-G2 were constructed, and the spectra are shown in Figures 5 and 6, respectively.
  • the above plasmids were transformed into Agrobacterium LBA4404 by electric shock (purchased from Invitrogen, the transformation method was referred to ⁇ Xiaoying et al. The electroporation transformation conditions affecting Agrobacterium tumefaciens. Journal of Food Science and Biotechnology. 2005, 04), and obtained by antibiotic and PCR screening. Positive transformed clones.
  • the Agrobacterium single colony containing the plasmid was inoculated into YEB liquid medium (containing 0.1%) containing 50 mg/L kanamycin and 25 mg/L rifampicin (purchased from Shenzhen Kailian Biotechnology Co., Ltd.).
  • Yeast extract 0.5% beef extract, 0.5% peptone, 0.5% sucrose, 0.05% MgSO 4 .7H 2 O).
  • the dark culture was shaken overnight at 28 ° C, 200 rpm to log phase (OD 6 . . value 0.8-1.2) for plant transformation.
  • the sterile seedling leaves were cut into 5 mmx5 mm leaf discs, and the Agrobacterium tumefaciens LBA4404 containing the expression vector pBI121-ATP-G2, pBI121-PTP-G2 or pBI121-CTP-G2 plasmid in the logarithmic growth phase were stained with leaves. Plate 10 mm, blot the bacterial solution, and co-culture for 2 days in the dark (MS solid medium).
  • the leaves were transferred to differentiation medium (MS+1 mg L cytokinin (BA, purchased from Shanghai Jiafeng Horticultural Products Co., Ltd.) + 0.1 mg/L naphthaleneacetic acid (NAA, purchased from Shanghai Jiafeng Gardening Products Co., Ltd.) + 50 mg L kanamycin + 500 mg L cephalosporin (purchased from Shenzhen Kailian Biotechnology Co., Ltd.)), cultured for about 45 days under light conditions, and then transferred to rooting medium after bud growth. MS+50 mg L kanamycin + 500 mg L cephalosporin) was cultured for about 30 days. After the root system was developed, the seedlings were transferred to MS medium containing 500 mg/L cephalosporin for number storage.
  • MS+1 mg L cytokinin BA, purchased from Shanghai Jiafeng Horticultural Products Co., Ltd.
  • NAA purchased from Shanghai Jiafeng Gardening Products Co., Ltd.
  • 50 mg L kanamycin + 500 mg L cephalosporin
  • PCR reaction conditions pre-denaturation at 94 ° C for 5 min, 33 cycles (94 ° C denaturation 30 s 55 ° C annealing 30 s 72 ° C extension 2 min), and the plants identified as positive by PCR were kept for later use.
  • the above three plasmids obtained about 30 transformation events, respectively.
  • the plant binary expression vector PCAMBIA2300 (purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd.) was selected as a plant expression vector, and the 35S promoter containing the double enhancer of the ⁇ gene was replaced with the Pnos promoter to reduce the expression of prion protein in plants. .
  • the 35S promoter containing the double enhancer and the Tnos terminator were selected as promoters and terminators of the MC2-£3 ⁇ 4 ⁇ 4 gene, respectively.
  • the plant expression vector pBI121 (purchased from Beijing Huaxia Ocean Technology Co., Ltd.) was used as a template to amplify the Pnos promoter with its coRL g/II restriction sites.
  • PCR reaction conditions 94 °C pre-denaturation for 5 min, 33 cycles (94 °C denaturation 30 s 56 °C annealing 30 s 72 ° C extension 30 s 72 ° C extension 10 minheim
  • the resulting PCR product was digested with EcoR I Bgl II into pCAMBIA2300 (purchased from Promega, T4 ligase cassette) to obtain pCAMBIA2300-l
  • the Tnos terminator was amplified using the pBI121 plasmid as a template, with cl coRI cleavage sites at both ends.
  • PCR reaction conditions pre-denaturation at 94 ° C for 5 min, 33 cycles (94 °C denaturation 30 s 58 ° C annealing 30 s 72 ° C extension 30 s 72 ° C extension 10 min. by cl £coR I
  • the obtained PCR product was inserted into pCAMBIA2300-1 to obtain pCAMBIA2300-2, and the specific construction procedure is shown in Fig. 8a.
  • the 35S promoter containing the double enhancer was amplified using the vector pCAMBIA2300 as a template.
  • the 5' ends of the two primers were digested with H «d III and BamH I PCR products, and inserted into pUC18 vector (purchased from TAKARA) to obtain recombinant plasmid pUC18-35S.
  • the recombinant plasmid pUC18-35S-OK-PS was obtained by inserting OK-i3 ⁇ 4 l-Xho I-PS into the recombinant plasmid pUC18-35S by digestion with BamH I and Sac I.
  • Primers SEQ ID NO: 32 and SEQ ID NO: 33 were designed, and the PTP sequence was amplified with plasmid pUC57-PTP as a template, and the Pst I restriction site was added to the 5' end, and the Nco I restriction site was added to the 3' end.
  • the recombinant plasmid pUC18-35S-OK-PTP-MC-PS and pUC18-35S-OK-PTP-G2.PS were obtained by the same procedure as the pCAMBIA-35S-PTP-MC2 vector construction.
  • the 35S to PS fragments (SEQ ID NO: 35, SEQ ID NO: 36, respectively) of the two plasmids were inserted between the Hz' «d III and Sac I polyclonal cleavage sites of pCAMBIA2300-2 and cloned.
  • the tobacco leaf disc was infiltrated with Agrobacterium LBA4404 containing the expression vector pCAMBIA-35S-PTP-MC2, pCAMBIA-35S-PTP-MC or pCAMBIA-35S-PTP-G2 plasmid in the logarithmic growth phase obtained in Example 4,
  • the transformation method and the negative control tobacco obtaining method were as in Example 3.
  • the obtained transgenic tobacco leaves were extracted, DNA was extracted (method refer to Molecular Cloning (3rd edition), primers SEQ ID NO: 37 and SEQ ID NO: 38 (50 ⁇ ⁇ reaction system: 5 ⁇ lOxEx Buffer, 3 ⁇ ) 2.5 mM dNTP, 2.0 ⁇ DNA, 1.0 ⁇ Ex Taq, 10 ⁇ primers SEQ ID NO: 37 and SEQ ID NO: 38 each 2.0 ⁇ l, and 35 ⁇ double distilled water.
  • PCR reaction conditions 94 °C pre-denaturation 5 min, 33 cycles (denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 2 min), extension at 72 °C for 10 min), and the plants with positive results were kept for later use.
  • the transgene of each of the plasmids obtained about 80 transformation events, respectively.
  • Non-transgenic tobacco regeneration seedlings of 6 ⁇ 7 leaf stage and T0 generation transgenic tobacco seedlings were randomly arranged and sprayed with 2000 ppm glyphosate
  • FIG. 9b is two transfer PTP-MC-EPSPS
  • Reverse transcription was carried out according to the method provided by the Invitrogen Reverse Transcriptase (1 total RNA as a template and the reverse transcription primers are SEQ ID NO: 38, respectively).
  • PTP was amplified using primers SEQ ID NO: 37 and SEQ ID NO: 38, and the relative expression of the fusion protein was detected and expressed in fusion with the £ ⁇ »3 ⁇ 4 gene, and the detected transcription level represents the transcription level of the gene).
  • 50 ⁇ PCR reaction system 50 ⁇ PCR reaction system: 5 ⁇ ⁇ Buffer, 3 ⁇ 2.5 mM dNTP, 2.0 ⁇ cDNA, 1.0 ⁇ Ex Taq ⁇ 10 ⁇ primers SEQ ID NO: 37 and SEQ ID NO: 38 2.0 ⁇ l, and 35 ⁇ double distilled water.
  • PCR reaction conditions pre-denaturation at 94 °C for 5 min, 33 cycles (denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 2 min), extension at 72 °C for 10 min.
  • the above transgenic samples were subjected to RT-PCR detection using the endogenous Actin gene of tobacco (http://www.ncbi.nlm.nih.gOv/nuccore/AB158612.l) as an internal reference.
  • the reverse transcription primer is SEQ ID NO: 40; the PCR primers are SEQ ID NO: 39 and SEQ ID NO: 40.
  • the reverse transcription and PCR systems and conditions are the same as above.
  • PCR product electrophoresis The results of PCR product electrophoresis are shown in Figure 10: 1-3 is a transgenic tobacco plant resistant to 3000 ppm glyphosate T0 generation, and 4-6 is a transgenic P7P-MC-PSPS gene that is not resistant to 3000 ppm glyphosate T0 transgenic Tobacco plants, 7-9 are transgenic tobacco plants that are not resistant to 3000 ppm glyphosate glyphosate T0 generation, and 10-12 are T0 transgenic tobacco plants that are not transgenic to 3000 ppm glyphosate, 13 is non- Transgenic control tobacco.
  • Cotton seeds are removed from concentrated sulphuric acid (H 2 S0 4 ), and the surface of the seeds is washed with tap water. After drying, the seeds are surface-sterilized by soaking with 70% ethanol for 1 mm, and then 10% 15%. Hydrogen peroxide (H 2 0 2 ) treatment for 24 h, rinse with sterile water for 2 ⁇ 3 times;
  • hypocotyl of the sterile seedling was cut into 0.5-0.6 cm sections with a scalpel and immersed in the Agrobacterium liquid (OD 6 . 0.81.2) containing the expression vector pCAMBIA-35S-PTP-MC2. Mm, then remove the hypocotyl segments, 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 on the surface of the medium), sealed with a parafilm. Co-cultivation was carried out for 2 days in the dark at 22 ° C and 25 °C.
  • the co-cultured hypocotyl segments were placed in callus induction medium (MS + 2,4-D 0.1 mg/L + KT 0.1 mg/L).
  • the induced resistant callus was introduced into a proliferation medium (MS medium + MgCl 2 0.91 g/L + deacetylated gellan gum 2.0 g/L + glucose 30 g/L, pH 5.8), 25 ° C culture, subculture every other month until callus differentiation. After the first and second transfer into 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 increased.
  • 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.
  • transgenic cotton leaves were extracted and DNA was extracted (the method is referred to "Molecular Cloning” (third edition)), and as in Example 4, the transgenic seedlings were identified by PCR, and the plants identified as positive were retained.
  • Non-transgenic cotton seedlings of 4 to 5 leaf stage and TO transgenic cotton seedlings were randomly arranged, and sprayed with 3000 ppm glyphosate (trade name: Nongda, purchased from Shenzhen Shijia Trading Co., Ltd.) to saturation (50 per square meter) Ml) After 10 days of spraying, observe the test results (see figure la-l ld) and find that the non-transgenic control plants were severely wilting or even dead (Fig. 1 id), among the 31 cottons transgenic with P7?-MC2-£3 ⁇ 4 3 ⁇ 4 gene Except for 8 leaves with mild yellowing and wilting, the other 23 plants all grew normally (Fig.
  • FIG. 11a are two transgenic P-MC2-£3 ⁇ 4 3 ⁇ 4 gene resistant cotton plants, and the other resistant plants are similar, not shown here.
  • FIG. 1 ib are two cotton plants resistant to ⁇ - ⁇ - ⁇ , and the remaining resistant plants are similar.
  • Shown and 29 transgenic cottons (Fig. 11c are two transgenic cotton plants, the other resistant plants are similar, not shown here), showing varying degrees of yellowing and wilting , treated at the glyphosate concentration Resistant plants were not screened.
  • FIG. 12 The electrophoresis results of the amplified products are shown in Figure 12: 1-2 is a glyphosate-tolerant transgenic cotton plant transgenic with ??- 1 ⁇ 2-£ ⁇ »3 ⁇ 4 gene, and 3-5 is a transgenic PTP-MC-EPSPS gene.
  • Non-glyphosate-resistant TO-transgenic cotton plants, 6-8 are G-toxins resistant to glyphosate-transformed cotton with PTP-G2 gene, and 9- 1 is not resistant to glyphosate 7?- ⁇ £ TO»3 ⁇ 4 gene TO-generation cotton, 12 is non-transgenic control cotton.

Abstract

提供了一种5-烯醇丙酮莽草酸-3-磷酸合成酶（EPSPS）融合蛋白，其编码基因，和获得该融合蛋白的方法。还提供了该5-烯醇丙酮莽草酸-3-磷酸合成酶融合蛋白在培育抗草甘膦植物中的应用。

Description

种抗草甘膦融合蛋白及其编码基因、 产生方法与应用
技术领域 本发明涉及融合蛋白及其编码基因与应用， 特别是涉及一种融合的 5-烯醇丙酮莽草酸 -3-磷酸 合成酶 （ 5 -enolpyruvylshikimate-3 -phosphate synthase, EPSPS ) 及其编码基因、 获得该融合蛋白的方 法及其在培育抗草甘膦植物中的应用。 背景技术 草甘膦是一种非选择性除草剂， 具有理化性质稳定、 高效、 广谱、 低毒、 低残留、 易于被微生物 分解、 不破坏土壤环境等优点， 已广泛应用于农业生产中， 成为目前世界上生产量最大的农药品种。 草甘膦的作用机理主要是竞争性抑制莽草酸途径中 5-烯醇丙酮莽草酸 -3-磷酸合成酶 (EPSPS)的活性。 该酶是真菌、 细菌、 藻类和高等植物体内芳香族氨基酸 (包括色氨酸、 酪氨酸、 苯丙氨酸)生物合成过 程中的一个关键酶， 其具有由两个保守的亚基组成的哑铃状结构。 草甘膦是磷酸烯醇式丙酮酸 (PEP) 的类似物， 是竞争性 EPSPS抑制剂， 草甘膦、 EPSPS和三磷酸莽草酸 (S3P)结合形成 EPSPS-S3P-草甘 膦复合体 (此复合体非常稳定)， 抑制 EPSPS的活性导致分支酸合成受阻， 阻断芳香族氨基酸和一些芳 香化合物的生物合成， 最终导致一些激素和关键性代谢物如类黄酮、 木质素和酚类化合物代谢失调， 从而扰乱生物体正常的氮代谢而使其死亡。
1976年， 美国孟山都公司的草甘膦类除草剂 -农达 (Roundup)研制成功并得到广泛应用。 随着植物 基因工程技术的发展和应用， 抗草甘膦除草剂的转基因作物研究已成为热点。 目前， 转 £^¾¾基因 的抗草甘膦除草剂转基因作物已经在多个国家广泛应用。
目前利用 EPSPS基因开展植物抗草甘膦研究主要有两种方法：
第一种， 是在植物中过量表达 EPSPS, 并以此拮抗草甘膦的竞争性抑制。 Amrhem等通过逐渐增 加草甘膦的浓度， 加大草甘膦的选择压力， 分离获得了一个耐草甘膦的矮牵牛细胞系。 分析这个细胞 系中的 EPSPS基因，发现该细胞系的基因组中^ S ¾基因的拷贝数扩增了 20倍，使该酶的产量大大 增力口 (Amrhein N, Johanning D, et al. Biochemical basis for glyphosate-tolerance in a bacterium and plant tissue culture. FEBS Lett., 1983, 157: 191-196 ) 。
第二种， 是使 EPSPS发生突变， 使其在保持催化活性的同时降低对草甘膦的亲和性， 甚至不亲 禾口。 Stalker等从鼠伤寒沙门氏菌中分离到对草甘膦表现抗性的菌株， 研究发现 EPSPS的第 101位的 脯氨酸残基变为丝氨酸残基,将突变体编码基因转入烟草后可使转基因烟草对草甘膦的抗性提高 (Stalker DM, Hiatt WR, et al. A amino acid substitution in the enzyme 5 -enolpyruvylshikimate-3 - phosphate synthase confers resistance to the herbicide glyphosate. J Biol Chem, 1985, 260:4724-4728)； Padgette等将来自大肠杆菌的 aroA基因编码的 EPSPS的第 96位的甘氨酸残基突变为丙氨酸残基，结 果发现突变蛋白对草甘膦的结合活性显著降低，将编码该突变蛋白的基因在矮牵牛中表达后可显著提 高其对草甘膦的抗性 （Padgette SR, Re DB, Gasser CS, et al. Site-directed mutagenesis of a conserved region of the 5 -enolpyruvylshikimate-3 -phosphate synthase active site. J Biol Chem, 1991, 266:22364-22369 ) ； 朱桢等 （2005， 专利申请号： 200510002739.7) 通过易错 PCR(Error-Prone PCR) 方法获得水稻的抗草甘膦突变体， 其 EPSPS的第 106位氨基酸残基由脯氨酸残基变为亮氨酸残基； Michael Spnecer等将玉米 EPSPS第 102位的苏氨酸残基突变为异亮氨酸残基， 第 106位脯氨酸残基 突变为丝氨酸残基， 使含有该突变的 EPSPS 的编码基因的植株获得抗除草剂特性 （美国专利号： 6040497 ) 。 本申请人曾将来自棉花的 EPSPS第 101位苏氨酸残基定点突变为异亮氨酸残基， 第 105 位脯氨酸残基改变为丝氨酸残基， 获得可提供一定草甘膦抗性的 EPSPS 突变基因 ( PCT/CN2010/078327 ) 。
某些外源基因 （例如 EPSPS编码基因） 在植物中过量表达会增加植物的草甘膦抗性， 但是外源 基因的过量表达在一定程度上会影响受体的正常生长发育。 因此， 获得新的 EPSPS编码基因， 使其 在同样的表达强度下实现更高的草甘膦抗性水平， 是开展植物草甘膦抗性基因工程的优选方案。 目前 为止， 所有的研究报道均是在不改变 EPSPS长度的情况下， 利用对功能域氨基酸残基的定点突变， 增加酶对草甘膦的抗性。 发明内容 本发明的思路是以微减棉花 EPSPS 蛋白突变体 BHR2 ( PCT/CN2010/078327,下文称为
MC-EPSPS ) 的活性中心容积为目的,采用不同 EPSPS 同源对比差异互补的设计思路获得一批突变体 融合蛋白， 并进一步进行草甘膦抗性鉴定分析， 筛选功能显著改善的突变融合蛋白。其中的一个设计 实现了本发明的内容。
根据上述发明思路， 发明人通过对 MC-EPSPS 和另外一种 EPSPS 蛋白 G2-aroA (中国专利 03826892.2 )的氨基酸序列进行同源对比分析， 发现 MC-EPSPS在不同区域存在小段氨基酸的缺 失或冗余（图 1 ) 。据此发现， 本发明采用蛋白质工程和基因工程相结合的技术， 获得了一个 EPSPS 突变体融合蛋白 （下文称为 MC2-EPSPS ) ， 该融合蛋白是在 MC-EPSPS蛋白的 N端融合了 G2-aroA 蛋白 N端的 26个氨基酸残基。通过原核表达及转基因植物功能鉴定发现，本发明融合蛋白 MC2-EPSPS 的抗草甘膦水平较 MC-EPSPS和 G2-aroA均有显著提高。
本发明首次采用 EPSPS融合蛋白筛选方案， 获得了草甘膦抗性显著提高的 EPSPS突变体融合蛋 白 （MC2-EPSPS ) 及编码所述 MC2-EPSPS融合蛋白的基因， 这使得可在外源 EPSPS基因表达水平 相同的情况下， 获得草甘膦抗性更强的转基因植物， 从而提供了一种获得抗草甘膦除草剂转基因植物 的优选方案。
本发明的第一方面提供一种融合蛋白， 其序列为 SEQ ID NO : 2。
本发明的第二方面提供编码本发明第一方面所述融合蛋白的核苷酸序列； 优选地， 所述核 苷酸序列具有 SEQ ID NO : 1所示的核苷酸序列。
本发明的第三方面提供一种重组表达载体， 其包含本发明第二方面所述的核苷酸序列， 其 中所述核苷酸序列与表达载体的表达控制序列可操作地连接。
在一个优选的实施方案中， 所述表达载体为 pET28b ; 在另一个优选的实施方案中， 所述表 达载体为 pCAMBIA2300。
本发明的第四方面提供一种重组细胞，其包含本发明第二方面所述的核苷酸序列或者本发明第三 方面所述的重组表达载体； 在一个优选的实施方案中， 所述重组细胞为重组大肠杆菌细胞； 在另一个 优选的实施方案中， 所述重组细胞为重组农杆菌细胞。
本发明的第五方面提供一种改善植物草甘膦抗性的方法， 包括： 将本发明第二方面所述的 核苷酸序列或者本发明第三方面所述的重组表达载体导入植物细胞、 组织、 器官或植株并使所 述核苷酸序列表达； 在一个优选的实施方案中， 所述植物为烟草； 在另一个优选的实施方案中， 所述植物为棉花。
本发明的第六方面提供一种制备融合蛋白的方法， 其包括将本发明第二方面所述的核苷酸 序列插入表达载体并使所述核苷酸序列与所述表达载体的表达调控序列可操作地连接， 然后将 所得重组表达载体导入生物体使所述基因表达； 在一个优选的实施方案中， 所述生物体是大肠 杆菌； 在另一个优选的实施方案中， 所述生物体为烟草； 在另一个优选的实施方案中， 所述生 物体为棉花。
本发明的第七方面提供本发明第一方面所述的融合蛋白、 本发明第二方面所述的核苷酸序 列、 本发明第三方面所述的重组表达载体或本发明第四方面所述的重组细胞用于改善植物草甘 膦抗性以及用于植物育种的用途； 在一个优选的实施方案中， 所述植物为烟草； 在另一个优选 的实施方案中， 所述植物为棉花。 附图说明 图 1 : 棉花 MC-EPSPS蛋白与 EPSPS蛋白 G2-aroA的氨基酸序列进行同源对比分析的结果。 图 2 : 原核表达载体 pET28b-MC2的质粒图。
图 3 : 分别含 pET28b-MC2、 pET28b-MC、 pET28b-G2或 pET28b的 BL21 (DE3 ) PlysS转化子 在含 150 mM草甘膦的液体 M9基础培养基中的生长曲线图。
图 4: BL21(pET28b-MC)、 BL21(pET28b-MC2)和 BL21(pET28b-G2)中蛋白的表达情况。其中第 1 泳道为蛋白 Marker (名称： Unstained Protein Molecular Weight Marker; 购自深圳研顺生物科技有限公 司）；第 2和 3泳道为 BL21(pET28b-G2)两个克隆子的蛋白粗提物，第 4禾口 5泳道为 BL21(pET28b-MC) 两个克隆子的蛋白粗提物， 第 6和 7泳道为 BL21(pET28b-MC2)两个克隆子蛋白粗提物， 第 8和 9泳 道为 BL21(pET28b)两个克隆子蛋白粗提物。
图 5 : pBI121-ATP-G2的质粒图。
图 6 : pBI121-PTP-G2的质粒图。
图 7: pBI121-CTP-G2的质粒图。
图 8 : 图 8a为 pCAMBIA2300-2的构建流程； 图 8b禾卩 8c为 pCAMBIA-35 S-PTP-MC2的构 建流程。
图 9 : 将 6〜7叶期的非转基因烟草再生幼苗和 TO代转基因烟草幼苗随机排列， 喷施 2000 ppm 草甘膦 15天后， 再次喷施 3000 ppm草甘膦 7天后观察到的结果。 图 9a为两个转 PTP-MC2-EPSPS 基因的烟草植物， 其余抗性植物与其类似， 在此未示出； 图 9b为两个转 7?- 1^-£^¾¾基因的 烟草植株， 其余植株与其类似， 在此未示出； 图 9c为两个转 ^?- (^基因烟草植株， 其余植株与 其类似， 在此未示出； 图 9d为非转基因对照植株。
图 10 : 对转基因烟草和非转基因烟草进行 RT-PCR 检测的电泳结果。 1-3 为转 基因的抗 3000 ppm草甘膦 TO代转基因烟草植株， 4-6为转 PTP-MC-EPSPS基 因的不抗 3000 ppm草甘膦 T0代转基因烟草植株， 7-9为转 (^- 基因的不抗 3000 ppm 草甘膦草甘膦 TO代转基因烟草植株， 10-12为转 7 )- 1^2-£^¾¾基因不抗3000 111草甘膦的 T0 代转基因烟草植株， 13 为非转基因对照烟草。 内参为烟草内源 Actm 基因 ( http：〃 www. ncbi.Blm. mh. ov/nuccore/ AB 158612.1 ) 。
图 11 : 将 4〜5叶期的非转基因棉花幼苗和 TO代转基因棉花幼苗随机排列， 喷施 3000 ppm草甘 膦至饱和 （每平米喷施 50 ml) 10天后所观察到的结果。 图 11a为两个转^？？- ^ ^^^基因抗 性棉花植株， 其余抗性植株与其类似， 在此未示出； 图 l ib为两个转^7?- 1^-£^»¾基因抗性棉 花植株， 其余抗性植株与其类似， 在此未示出； 图 11c为两个转 ^7?- (^基因抗性棉花植株， 其 余抗性植株与其类似， 在此未示出； 图 l id为非转基因对照植株。
图 12 : 对转基因棉花机非转基因对照棉花进行 RT-PCR 检测的电泳结果。 1-2 为转 Ρ7?-Μ -£Λ»¾基因的抗草甘膦 Τ0代转基因棉花植株， 3-5为 PTP-MC-EPSPS基因的不抗草甘 膦 TO代转基因棉花植株， 6-8为转 PTP-G2基因的不抗草甘膦的 TO代棉花， 9- 11为不抗草甘膦 的转 PTP-MC2-EPSPS基因的 TO 代棉花， 12 为非转基因对照棉花。 陆地棉内源 Actin 基因 ( http://'www. ncbi.nlni.nih.gov/niiccore/ 32186889 ) 为内参。
图 13为 pCAMBIA-35 S-PTP-MC2的质粒图。 具体实施方式 提供以下实施例， 以方便本领域技术人员更好地理解本发明。 所述实施例仅出于示例性目的， 并 非意在限制本发明的范围。实施例中使用的化合物或试剂可通过商业途径购得， 或者通过本领域技术 人员已知的常规方法制备得到； 所使用的实验仪器可通过商业途径购得。 实施例 1 MC2-EPSPS编码基因的获得
1. 棉花 5-烯醇丙酮莽草酸 -3-磷酸合成酶基因 C-EPSPS的克隆
取鄂杂棉 11F1 (创世纪转基因技术有限公司销售） 叶片 0.5 g， 按植物 R A提取试剂盒 （购自 Invitrogen) 说明书操作提取棉花叶片的总 RNA。
棉花叶片总 RNA 逆转录： 在 0.2 ml EP 管中， 加入 2.0 μΐ 总 R A(0.1 μ§/μ1)和 2.0 μΐ 01igo(dT12-18)(2 M)， 混匀， 65 °C水浴 10分钟， 立即冰浴 5分钟， 然后加入 2.0 μΐ lOxRT buffer, 2.0 μΐ 250 μΜ dNTP mix, 2.0 μΐ 100 mM DTT， 9.8 μΐ DEPC H20， 0.2 μΐ 200 U /μΐ SuperScriptlll ( Invitrogen ) , 混匀后 50°C反应 60分钟； 然后在 70°C失活 15分钟； -20°C保存。
棉花 EPSPS编码基因的获得： 以上述所得棉花 cDNA为模板， 扩增得到包括棉花 EPSPS编码基 因的 5 '端非翻译区 （5 'UTR)、 叶绿体导肽 （CTP)、 EPSPS编码基因和 3 '端非翻译区 （3'UTR) 在内 的基因组序列。 反应体系如下：
50 μΐ PCR反应体系： 10 μΐ 5 xPS Buffer, 3 μΐ 2.5 mM的 dNTP， 2.0 μΐ cDNA, 1.0 μΐ PrimeSTAR HS DNA聚合酶（购自 TAKARA)、 10 μΜ的引物 SEQ ID NO:3和 SEQ ID NO:4各 2.0 μΐ (便于后续构建， 在引物两端分别引入 coR I ,Sac I识别位点），以及 30 μΐ的双蒸水。 PCR反应条件： 94 °C预变性 5 mm、 5个循环（94°C变性 45 s、 56°C退火 45 s、 72°C延伸 2 min) , 25个循环（94°C变性 45s、 60°C退火 45s、 72°C延伸 2 min)， 72°C延伸 7 min。
所得的扩增产物经 EcoR I 禾 P Sac I 酶切后插入到克隆载体 pBluescript II SK (-) (下文称为 pBluescript载体， 购自深圳研顺生物技术有限公司） 的 £coR I和 S«c I多克隆酶切位点之间， 连接反 应体系及反应条件如下：酶切后的 PCR产物 3 μ1， 2xT4 DNA连接酶缓冲液 5 μ1，酶切后的 pBluescript 载体 1 μ1， Τ4 DNA连接酶 1 μ1， 于 4°C连接过夜。 所得载体命名为 pBluescript-C-EPSPS。
取 10 连接反应产物，加入到 100 L大肠杆菌 JM109感受态细胞（购自 TAKARA)中并混匀， 冰浴 30 min, 42°C热休克 60 s、冰浴 2 min, 另加 250 L LB培养液（含 1%胰蛋白胨， 购自 OXOID ; 0.5%酵母提取物， 购自 OXOID ; l% NaCl, 购自国药）后置于 37°C摇床中， 以 225 r/min振荡培养 30 mm。取 200 培养后的菌液涂布于含 50 g/mL氨苄青霉素（购自北京拜尔迪）的 LB固体培养平板 (含 1%胰蛋白胨、 0.5%酵母提取物、 l% NaCl、 1.5%琼脂） 上， 37°C培育 18 h。
挑取克隆子并分别进行 PCR (反应体系及条件同上） 验证， 将 PCR阳性克隆送英潍捷基 （上海） 贸易有限公司测序， 测序所用引物为 BcaBESTTM Sequencing Primer RV-M(SEQ ID NO:5), BcaBEST™ Sequencing Primer M13-47(SEQ ID NO:6),特异性引物 (SEQ ID NO:7), 测序所得序列为 SEQ ID NO:8。
8 SEQ ID NO:8中第 7 1^—184 1^为5 '1711区； 第 185 bp— 418 bp为叶绿体导肽核苷酸序列； 第 419 bp- 1750 bp为编码 EPSPS蛋白的核苷酸序列 (命名为 C-EPSPS') , 所述 C-£ ¾ ¾对应的氨基酸序列为 SEQ ID NO:9 (其为去除导肽并在 N端增加甲硫氨酸后的 EPSPS蛋白的氨基酸序列）； 第 1751— 2028 bp 为 3'UTR。
2. 棉花 EPSPS编码基因 C-EPSPS的定点突变
以上述经过测序验证的 pBluescript-C-EPSPS质粒为模板， 对 C-£¾¾基因进行定点突变， 将其所 编码蛋白 N端第三个 α螺旋中的苏氨酸（SEQ ID ΝΟ:9中第 102位氨基酸）改变为异亮氨酸，脯氨酸（SEQ IDNO:9中第 106位氨基酸） 改变为丝氨酸。 为表达载体构建需要， 将 SEQIDNO:9中第 208位的脯氨 酸及第 406位的丙氨酸进行同义突变， 消除了原基因中存在的 Ntfe I (SEQ ID ΝΟ:8中第 1038 bp处的 "CATATG"突变为 "CTTATG") 和 Nco I(SEQ ID NO:8中第 1632 bp处的" CCATGG"突变为 "CTATGG")两 个酶切位点对后续构建的影响。
所述定点突变过程如下：
1) 使用突变引物 SEQIDNO:10、 SEQ ID ΝΟ:11和 SEQ ID ΝΟ:12， 并将其 5'端磷酸化以利于后续
PCR产物的连接、 环化。
引物磷酸化反应过程如下：
引物 SEQIDNO:10 (50 μΜ): 5 μΐ
引物 SEQIDNO:ll (50 μΜ): 5 μΐ
引物86(3101\[0:12 (50 1^): 5 1
10χΤ4 DNA Polynucleotide Kinase Buffer (购自 TAKARA): 3 μΐ
T4 DNA Polynucleotide Kinase (购自 TAKARA): 1 μΐ
ATP(lOmM): 3 μΐ
补水至 30 μΐ
37°C反应 4511111后置于41终止反应并保存。
定点突变过程： 以所述含 C-£¾¾基因的克隆载体 pBluescript-C-EPSPS为模板， 反应过程使用 STRATAGENE的 QuikChange Multi突变试剂盒并参照其说明书操作 （在 25 μΐ反应体系中， 加入约 70 ng模板质粒， 2.5 μΐ lO Quickchange Multi Buffer, 3.0 μΐ磷酸化的引物混合物， 1.0 μΐ dNTP Mix, 1.0 μΐ Multi enzyme blend,加水至 25 μ1。反应条件： 95.0°C预变性 1 min; 30个循环（95.0°C变性 1 min; 55°C退火 1 min; 65°C延伸 10min)，反应完成后通过冰浴将温度降至 37.0°C以下，加 1 μΐϋρηΐ, 37.0°C 消化 2 h，取 2 μΐ消化产物直接转化大肠杆菌）。将所得的突变后的载体命名为 pBlueSCript-MC-EPSPS。
取 3 突变后的质粒,转化大肠杆菌 JM109感受态细胞进行克隆测序验证（反应体系及方法同上）。 经 PCR及测序验证得到发生目标突变的克隆子后保存备用。
设计引物 SEQ ID NO: 13和 SEQ ID NO: 14， 以上述经验证发生目标突变的 pBluescript-MC-EPSPS 质粒为模板， 扩增除导肽序列以外的棉花 EPSPS编码区序列， 其 5'端增加起始密码子 ATG以便于后 续构建。 反应体系及反应条件如下：
50 μΐ PCR反应体系： 10 μΐ 5xPS Buffer, 3 μΐ 2.5 mM的 dNTP， 2.0 μΐ pBluescript-MC-EPSPS质粒， 1.0 μΐ PrimeSTAR HS DNA聚合酶 （购自 TAKARA)、 10 μΜ的引物 SEQ ID NO:13和 SEQ ID NO:14 各 2.0μ1， 以及 30 μΐ的双蒸水。 PCR反应条件： 94°C预变性 5 min、 33个循环 （94°C变性 45s、 56°C 退火 45 s、 72°C延伸 2 min), 72°C延伸 7 min„
两引物末端分别设计 Nco I和; ¾o I位点， 将扩增产物用 Nco I和; ¾o I酶切后插入至 pET28b的 Nco I和; ¾o I多克隆位点之间， 获得重组表达载体 pET28b-MC。 取 10 连接反应产物， 转化到 100 L大肠杆菌 JM109感受态细胞中（方法同上）。取 200 L培养后的菌液涂布于含 50 g/mL 卡那霉素 （购自北京拜尔迪生物技术有限公司） 的 LB固体培养平板上， 37°C培育 18h。 挑选正 常生长的菌落进行测序验证， 所得突变后的基因序列为 SEQ ID ΝΟ: 15， 并将其命名为 MC-£¾¾。
3. 融合基因 1^2-£^¾¾克隆 根据 G2-aroA蛋白 （专利申请号： 03826892.2) 的氨基酸序列， 在保持其氨基酸不变的前提下， 根据棉花密码子偏好性设计 (^基因， 并由生工生物工程 （上海） 股份有限公司合成 （序列为 SEQ ID NO:16)„ 将合成的 基因 T克隆至 pUC57-T载体中， 所得的重组质粒命名为 pUC57-G2。
设计引物 SEQ IDNO:17和SEQ IDNO:18， 以扩增 基因 5'端的 78个核苷酸序列， 并使其 5' 端带 Nco I位点， 3'端带 Nde I位点。采用 TAKARA的 PrimeSTAR HS DNA聚合酶，以上述 pUC57-G2 质粒为模板进行 PCR反应。
50 μΐ PCR反应体系： 10 μΐ 5xPS Buffer, 3 μΐ 2.5 mM的 dNTP， 2.0 μΐ pUC57-G2质粒， 1.0 μΐ PrimeSTAR HS DNA聚合酶、 10 μΜ的引物 SEQ ID NO: 17和 SEQ ID NO: 18各 2.0μ1， 以及 30 μΐ的 双蒸水。 PCR反应条件： 94°C预变性 5min， 33个循环 （94°C变性 30s， 58°C退火 30s， 72°C延伸 10 s)， 72 °C 延伸 10min。
PCR扩增产物加 A尾： PCR产物加 2.5倍体积的无水乙醇， -20°C放置 10分钟，离心，去上清，晾干， 用 21 μΐ双蒸水溶解。加入 2.5 μΐ ΙΟχΕχ Buffer, 0.5 μΐ 5 mM的 dATP ，2.5 μΐ Ex Taq。Ex Taq及 ΙΟχΕχ Buffer 均购自 TAKARA。 反应条件： 70°C反应 30分钟。 将得到的约 100 bp的 DNA片段回收（使用购自 Omega 的回收试剂盒）， 并将其连接至 pGEM T-easy载体（购自 Promega) ,然后转化 JM109感受态细胞（（购自 TAKARA) (连接体系及转化方法同上)， 随机挑取 10个白色菌落于含有 50 g/mL氨苄青霉素的 LB液体 培养基中培养, 37°C培养过夜后加甘油至终浓度 20%， -80°C保存备用。经 Nco I和 N^ I双酶切鉴定后将 得到 2个阳性克隆送至英潍捷基（上海）贸易有限公司测序，保留测序正确克隆子，命名为 pGEM-G2(78)。
利用引物 SEQ ID ΝΟ:19禾卩 SEQ ID ΝΟ:14, 以上述经测序验证的 pET28b-MC质粒为模板扩增 MC-£¾¾基因， 使其 5'端带 N I位点， 3'端带; ¾o I位点， PCR反应条件为： 94°C预变性 5 min， 33个循环 （94 °C 变性 30s， 57°C退火 30s， 72°C延伸 2min)， 72°C延伸 10min。
将所得 PCR产物连接到 pGEM T-easy载体 （购自 Promega) ,然后转化 JM109感受态细胞进行克 隆测序验证 (反应体系及方法同上)。经 Nde I和; ¾o I双酶切鉴定正确后， 将得到 2个阳性克隆送至英 潍捷基 （上海） 贸易有限公司测序， 保留测序正确克隆子,命名为 pGEM-MC。
用 Nco l^WXhol酶切 pET28b， 并回收载体片段； 用 Nco I和 Nde I双酶切 pGEM-G2(78),并回收 约 78 bp大小的片段； 用 N I和； ¾oI双酶切 pGEM-MC， 回收 1300 bp大小的片段， 将上述三个片 段进行三片段连接， 获得重组质粒 pET28b-MC2 (见图 2)， 5'端的 78bp加 MC-£¾¾基因组成的 融合基因命名为 MC2-£¾¾。经转化 JM109感受态细胞后进行克隆测序验证（方法同上）， 该融合基 因的核苷酸序列为 SEQIDNO:l， 其所对应蛋白质的氨基酸序列为 SEQIDNO:2。 实施例 2 1^2-£^¾¾基因原核表达产物抗草甘膦特性分析
原核表达载体构建： 参照上述 pET28b-MC2载体的构建策略和步骤， 使用引物 SEQ ID ΝΟ:17和 SEQ ID ΝΟ:20在 G2基因两端分别引入 Nco l^WXhol位点， 并通过 Nco l^WXhol酶切将 G2基因插 入到 pET28b载体， 获得原核表达载体 pET28b-G2。
抗草甘膦功能分析：将上述 pET28b-MC、pET28b-MC2和 pET28b-G2表达载体及质粒对照 pET28b 分别转化原核表达菌株 BL21 (DE3) PlysS (购自上海博麦德生物技术有限公司）， 转化方法参照《分 子克隆》（第三版）中的描述。将所得阳性转化子分别命名为 BL21 (pET28b-MC)、 BL21 (pET28b-MC2)、 BL21(pET28b-G2)及 BL21(pET28b)， 并将其分别接种到含有 50 g/mL卡那霉素的液体 M9基础培养 基（含 48 mM Na2P04-7H20, 22 mM KH2P04， 8.5 mM Nacl, 19 mM NH4C1, 2 mM MgS04， 0.1 mM CaCl2 和 0.4%葡萄糖）， 37°C下活化过夜后，按 1:100 (V/V)稀释。分别取 300 稀释后的菌液接种到 30 mL 液体 M9基础培养基（含卡那霉素 50 g/mL) 中， 以 200 rpm, 37°C空气浴的条件培养。 培养到 OD600 = 0.100左右， 加入异丙基 -β-D-硫代半乳糖苷 （IPTG, 购自 TAKARA) 至终浓度为 1 mmol/L， 加入 草甘膦 （购自山东滨州九安化工有限公司） 至终浓度 150 mM， 以 200 rpm, 37°C空气浴的条件培养 14小时， 然后继续培养并每隔 2小时测定一次培养物 OD6。。， 并记录其生长情况。 每组设两个重复， 测定结果见表 1。 将每组的值求平均值后， 绘制生长曲线图 （图 3 )。
表 1 150 mM草甘膦浓度下携带不同基因的 BL21 ( DE3 ) PlysS生长情况表
Figure imgf000009_0001
从表 1和图 3可看出， 在含有 150 mM草甘膦的 M9基本培养基中， 携带 MC2-£ ¾ ¾基因的转 化子 BL21(pET28b-MC2)的生长速度显著快于携带 MC-£ ¾ ¾基因的转化子 BL21(pET28b-MC)或携 带 G2基因的转化子 BL21(pET28b-G2) ; 而只携带 pET28b质粒的转化子在含有 150 mM的草甘膦的 M9基本培养基中的生长已受到严重的抑制。每组的两重复间的结果基本一致。由此可见， MC2-EPSPS 蛋白的抗草甘膦能力比 MC-EPSPS蛋白或 G2蛋白的抗草甘膦能力有显著提高。
原核表达情况分析： 挑取 BL21(pET28b-MC；)、 BL21(pET28b-MC2)和 BL21(pET28b-G2)单菌落分 别接种到液体 LB (含卡那霉素 50 g/mL )中， 37 °C下活化过夜后， 按 1 : 100 (V/V)稀释。各取 50 所述稀释液分别接种到 5 mL液体 LB (含卡那霉素 5(^g/mL )中， 在 37°C、 200 rpm下培养到 OD600 = 0.6左右， 分别加入 IPTG至终浓度为 1 mmol L， 于 37 °C振荡培养诱导表达 3 h。 分别离心并收集 沉淀的菌体，用 2x SDS上样缓冲液重悬菌体，沸水中煮 5 mm得到蛋白粗提物， 12000 rpm离心 lmm， 分别取 15 上清进行 SDS-PAGE电泳分析。 参考 Sambrook等 （1989 ) 的方法， 用 10% SDS-PAGE 电泳分离蛋白， 0.25 %考马斯亮蓝 R-250染色。
结果如图 4所示， 第 1泳道为蛋白 Marker (名称： Unstained Protein Molecular Weight Marker; 购 自深圳研顺生物科技有限公司）； 第 2和 3泳道为 BL21(pET28b-G2)两个克隆子的蛋白粗提物， 第 4 禾口 5泳道为 BL21(pET28b-MC)两个克隆子的蛋白粗提物，第 6禾口 7泳道为 BL21(pET28b-MC2)两个克 隆子蛋白粗提物，第 8和 9泳道为 BL21(pET28b)两个克隆子蛋白粗提物。由电泳图可以看出，经 IPTG 诱导的携带 MC-£ ¾ ¾、 MC2-EPSPS G2基因的克隆子均可表达出大小约为 47 kDa的目的蛋白， 并且 MC2-EPSPS蛋白略大于 MC-EPSPS和 G2蛋白。 这表明， 在相同的表达条件下， 三者均可获得 有效表达， 并且电泳结果显示三者的蛋白表达水平没有显著差异。
综上可知， MC2-EPSPS抗草甘膦能力的提高不是由于蛋白的过量表达引起的，而是由于蛋白结构 的改变引起的。
实施例 3 叶绿体导肽筛选、 鉴定
1. 载体构建
对 NCBI已报道的拟南芥、 矮牵牛和棉花的叶绿体导肽基因 7?、 和 CTP的 DNA序列进行 密码子分析， 经同义替换消除 ATP和 PTP中的稀有密码子， 并在所述 3种导肽的 5'端加 B mH I酶 切位点， 3'端加 Nco l酶切位点， 所得序列分别为 SEQ ID N0:21 (ATP) , SEQ ID ΝΟ:22(Ρ7?)和 SEQ ID NO:23(C7P), 由生工生物工程（上海）股份有限公司合成。将所述三个序列分别 T克隆至 pUC57-T 载体中， 所得重组质粒分别命名为 pUC57-ATP、 pUC57-PTP和 pUC57-CTP。
利用引物 SEQ ID NO: 17和 SEQ ID NO:24 , 采用 TAKARA的 PrimeSTAR HS DNA聚合酶， 以 pUC57-G2质粒为模板进行 PCR反应来扩增 基因， 使其 5 '端带 Nco I位点， 3 '端带 S«c I位点。
50 μΐ PCR反应体系： 10 μΐ 5xPS Buffer, 3 μΐ 2.5 mM的 dNTP， 2.0 μΐ pUC57-G2质粒， 1.0 μΐ PrimeSTAR HS DNA聚合酶、 10 μΜ的引物 SEQ ID NO: 17和 SEQ ID NO: 24各 2.0 μ1， 以及 30 μΐ的 双蒸水。 PCR反应条件： 94°C预变性 5 min， 33个循环 （94°C变性 30 s， 58 °C退火 30 s， 72 °C延伸 90 s)， 72°C延伸 10 min„
PCR扩增产物加 A尾： PCR产物加 2.5倍体积的无水乙醇， -20°C放置 10分钟，离心，去上清，晾干， 用 21 μΐ双蒸水溶解。加入 2.5 μΐ ΙΟχΕχ Buffer, 0.5 μΐ 5 mM的 dATP ，2.5 μΐ Ex Taq。Ex Taq及 ΙΟχΕχ Buffer 均购自 TAKARA。 反应条件： 70°C反应 30分钟。 将得到约 1300 bp的 DNA片段回收 （使用购自 Omega 的回收试剂盒）， 并将其连接至 pGEM T-easy载体（购自 Promega) ,然后转化 JM109感受态细胞 (连接体 系及转化方法同上)， 随机挑取 10个白色菌落分别接种于含有 50 g/mL氨苄青霉素的 LB液体培养基中 培养， 37°C培养过夜后加甘油至终浓度 20%， -80°C保存备用。 经 Nco I和 S«c I双酶切鉴定后将得到 2个 阳性克隆送至英潍捷基 （上海） 贸易有限公司测序， 保留测序正确克隆子， 命名为 pGEM-G2。
用 βωηΐί I和 Nco I双酶切质粒 pUC57-ATP，回收 ATP片段；用 Nco I和 Sac I双酶切 pGEM-G2， 回收 片段， 用 β«»¾Η Ι和 S«c l双酶切质粒 pBI121 (购自北京华夏远洋科技有限公司）， 回收载体 片段， 以三片段连接方式将上述回收的 ATP和 G2 片段同时插入 pBI121 中， 获得植物表达载体 pBI121-ATP-G2。 同理构建 pBI121-PTP-G2和 pBI121-CTP-G2,图谱分别如图 5， 6， 7所示。 将上述质 粒分别通过电击转化农杆菌 LBA4404 (购自 Invitrogen, 转化方法参照匡小婴等.影响根癌农杆菌的电 击转化条件.食品与生物技术学报 .2005年 04期），并通过抗生素及 PCR筛选获得阳性转化克隆。将含 有所述质粒的农杆菌单菌落接种于含 50 mg/L卡那霉素和 25 mg/L利福平 （购自深圳市凯联生物 技术有限公司）的 YEB液体培养基（含 0.1%酵母提取物， 0.5%牛肉膏， 0.5%蛋白胨， 0.5%蔗糖， 0.05%MgSO4.7H2O ) 中。 28°C、 200 rpm振荡暗培养过夜至对数期 （ OD6。。值 0.8- 1.2 ) 用于植物 转化。
2. 转化烟草
用 75%酒精浸泡烟草种子 cotiana tabacum L.， 国家烟草中期库， 获取单位： 中国农科院烟 草所， 库编号 I5A00660 ) 30 s， 用灭菌双蒸水洗两次。 再用 0.1%升汞浸泡 8 mm， 用灭菌双蒸水洗两 次， 完成表面灭菌。 将表面灭菌的烟草种子置于 MS固体培养基 （含 18.78 mM ΚΝ03， 1.25 mM KH2P04， 20.6 mM ΝΗ4ΝΟ3, 1.5 mM MgS04， 3.0 mM CaCl2， 50 μΜ KI, 100 μΜ Η3ΒΟ3， 100 μΜ MnS04, 30 μΜ ZnS04, 1 μΜ Na2Mo04, 0.1 μΜ CoCl2, 100 μΜ Na2EDTA， 100 μΜ FeS04, 7.4 g/L琼脂， 蔗糖 30 g L ) 上于 无菌条件下发芽， 制备无菌苗。 取无菌苗叶片剪成 5 mmx5 mm大小的叶盘， 将处于对数生长期的分 别含表达载体 pBI121-ATP-G2、 pBI121-PTP-G2或 pBI121-CTP-G2质粒的农杆菌 LBA4404浸染叶盘 10 mm，吸干菌液，在黑暗条件下共培养 2天（MS固体培养基）。将叶片转到分化培养基（MS+1 mg L 细胞分裂素（BA， 购自上海稼丰园艺用品有限公司） +0.1 mg/L萘乙酸（NAA， 购自上海稼丰园艺用 品有限公司） +50 mg L卡那霉素 +500 mg L头孢霉素（购自深圳市凯联生物技术有限公司））上， 光 照条件下培养 45天左右， 待芽长大后切下转移到生根培养基（MS+50 mg L卡那霉素 +500 mg L头孢 霉素） 中培养 30天左右， 待根系发达后将小苗转入含有 500 mg/L头孢霉素的 MS培养基上进行编号 保存。 在转化过程中部分叶盘不作浸染， 让其再生为正常小苗， 在后续实验中作为阴性对照。 取获得的转基因烟草叶片，提取 DNA (方法参照《分子克隆》（第三版）），用 SEQ ID NO: 17和 SEQ ID NO:24作为检测引物。 50 μΐ PCR反应体系： 5 μΐ ΙΟχΕχ Buffer, 3 μΐ 2.5 mM的 dNTP 2.0 μΐ DNA, 1.0 μΙΕχΤα ^ 10 μΜ的引物 SEQ ID NO: 17和 SEQ ID NO: 24各 2.0 μ1， 以及 35 μΐ的双蒸水。 PCR反应条 件： 94°C预变性 5 min, 33个循环 （94°C变性 30 s 55°C退火 30 s 72°C延伸 2 min), 并将 PCR鉴定为 阳性的植株保存备用。 上述三种质粒分别获得约 30个转化事件。
3. 抗草甘膦功能鉴定
将 6 7叶期的非转基因烟草再生幼苗和 TO代转基因烟草幼苗随机排列， 喷施 2000 ppm草甘膦 (商品名： 农达， 购自深圳诗佳贸易公司）至饱和（每平米喷施 50ml) 7天后观察试验结果发现， 非转基因对照植株严重萎蔫， 转基因烟草结果如下： 其中 28株转^ 7?- 基因的烟草均表现不同 程度萎蔫， 未发现抗性植株； 29株转 CTP-G2基因的烟草中只有 1株抗性植株； 30職 PTP-G2基 因的烟草中有 5株抗性植株。 后续研究中， 均以 PTP作为^ S¾基因的导肽序列。
实施例 4 1^2-£^¾¾基因植物表达载体构建
选择植物双元表达载体 PCAMBIA2300 (购自北京鼎国昌盛生物技术有限责任公司） 作为植物表 达载体， 用 Pnos启动子替换 ΝΡΤΠ基因含双增强子的 35S启动子， 以降低 ΝΡΤΠ蛋白在植物中的表 达。 选择含双增强子的 35S启动子及 Tnos终止子分别作为 MC2-£¾¾基因的启动子和终止子。
用引物 SEQIDNO:25和 SEQIDNO: 26以植物表达载体 pBI121 (购自北京华夏远洋科技有限公 司）为模板扩增 Pnos启动子，使其两端分别带 coRL g/II酶切位点。 50 μΐ PCR反应体系： 10 μΐ 5xPS Buffer, 3 μΐ 2.5 mM的 dNTP 1.0 μΐ ρΒΙ121质粒， 1.0 μΐ PrimeSTAR HS DNA聚合酶（购自 TAKARA 10 μΜ的引物 86(3101\[0:25和86(310]\[0: 26各2.(^1， 以及 31 μΐ的双蒸水。 PCR反应条件： 94 °C 预变性 5 min, 33个循环 (94°C变性 30 s 56°C退火 30 s 72°C延伸 30 s 72°C延伸 10 min„ 通过 EcoR I Bgl II 酶切将所得 PCR产物插入到 pCAMBIA2300(购自 Promega, T4 连接酶盒)获得 pCAMBIA2300-l„
使用引物 SEQ ID NO: 27和 SEQ ID NO: 28， 以 pBI121质粒为模板扩增 Tnos终止子， 使其两端 分别带 cl coRI酶切位点。 50μ1ΡΟ 反应体系： 10 μΐ 5xPS Buffer, 3 μΐ 2.5 mM的 dNTP 1.0 μΐ ρΒΙ121质粒， 1.0 μΐ PrimeSTAR HS DNA聚合酶 （购自 TAKARA 10 μΜ的引物 SEQ ID NO: 27和 SEQ ID NO: 28各 2.0 μ1， 以及 31 μΐ的双蒸水。 PCR反应条件： 94°C预变性 5 min, 33个循环 （94 °C 变性 30s 58°C退火 30s 72°C延伸 30s 72°C延伸 10min。 通过 cl £coR I酶切将所得 PCR产 物插入到 pCAMBIA2300-l获得 pCAMBIA2300-2， 具体构建流程如图 8a所示。
使用引物 SEQ ID NO:29和 SEQ ID NO:30, 以载体 pCAMBIA2300为模板扩增含双增强子的 35S 启动子。 所述两条引物的 5'端分别带 H«d III和 BamH I PCR产物经酶切后插入到 pUC18载体 （购 自 TAKARA) 中， 得到重组质粒 pUC18-35S。 根据公布的 OK (Omega & Kozak) 序列及 PS序列 (Processing & Splicing sequence) (两者已在 ZL 95 119563.8中充分公开） 合成 ¾»wH I-OK-i¾/-J¾o /-PS-S«cI片段， 其序列为 SEQIDNO:31。其中， 该片段的 5'端和 3'端分别带 β H I和 S«c I酶切位 点， 在 OK与 PS之间以 Pst I Xho I两酶切位点相连， 两个酶切位点间插入保护碱基便于后续的酶切 构建。 利用 BamH I和 Sac I酶切， 将 OK-i¾ l-Xho I-PS插入到所述重组质粒 pUC18-35S中， 获得重 组质粒 pUC18-35S-OK-PS
设计引物 SEQ ID NO:32和 SEQ ID NO:33, 以质粒 pUC57-PTP为模板扩增 PTP序列， 使其 5'端 添加 Pst I酶切位点，其 3'端添加 Nco I酶切位点，通过酶切以三片段连接的方式将 Pst I-PTP-Nco I (经 Pst I和 Nco I双酶切 )及 Nco I-MC2-EPSPS-Xho I (将上述 pET28b-MC2用 Nco I禾卩^ ¾o I双酶切 )两 个片段同时插入到 pUC18-35S-OK-PS ( Pst I 和 Xho I 双酶切） 中， 获得重组质粒 pUC 18-35 S-OK-PTP-MC2-PS利用 Hind III和 Sac I酶切将所述重组质粒 pUC18-35S-OK-PTP-MC2-PS 35S至 PS的片段 （SEQ ID NO:34) 插入到 pCAMBIA2300-2中， 并进行克隆测序检测 （感受态细胞 转化、 筛选及测序方法同上）， 获得植物表达载体 pCAMBIA-35S-PTP-MC2 (见图 13 )， 具体构建流 程如图 8 (图 8b-8c) 所示。
参照与 pCAMBIA-35S-PTP-MC2载体构建相同的步骤，获得重组质粒 pUC18-35S-OK-PTP-MC-PS 禾口 pUC18-35S-OK-PTP-G2.PS。 分别将两质粒中 35S至 PS的片段 （分别为 SEQ ID NO:35, SEQ ID NO:36 )插入到 pCAMBIA2300-2的 Hz'«d III禾口 Sac I多克隆酶切位点之间并克隆测序检测（感受态细 胞转化、筛选及测序方法同上），获得植物表达载体 pCAMBIA-35 S-PTP-MC和 pCAMBIA-35 S-PTP-G2。 分别用上述获得的阳性植物表达载体 pCAMBIA-35S-PTP-MC2、 pCAMBIA-35S-PTP-MC 或 PCMABIA-35S-PTP-G2质粒通过电击转化农杆菌 LBA4404, 将筛选得到的阳性转化克隆暗培养过夜 至对数期 （OD6QQ值 0.8 1.2 ) 用于植物转化 （方法同实施例 3 )。
实施例 5 利用农杆菌介导法转化烟草
用实施例 4 中所得的处于对数生长期的分别含表达载体 pCAMBIA-35S-PTP-MC2、 pCAMBIA-35S-PTP-MC或 pCAMBIA-35S-PTP-G2质粒的农杆菌 LBA4404浸染烟草叶盘， 转化方法 及阴性对照烟草获得方法如实施例 3。
取获得的转基因烟草叶片， 提取 DNA (方法参照 《分子克隆》 （第三版））， 使用引物 SEQ ID NO: 37和 SEQ ID NO: 38 ( 50 μΙ ΡΟ反应体系： 5 μΐ lOxEx Buffer, 3 μΐ 2.5 mM的 dNTP， 2.0 μΐ DNA, 1.0 μΐ Ex Taq、 10 μΜ的引物 SEQ ID NO: 37和 SEQ ID NO: 38各 2.0 μ1， 以及 35 μΐ的双蒸水。 PCR反应条件： 94°C预变性 5 min, 33个循环 (94°C变性 30 s， 55 °C退火 30 s, 72°C延伸 2 min)， 72°C延伸 10 min)， 并将鉴定结果为阳性的植株保存备用。 所述每种质粒的转基因分别获得约 80个转化事件。
实施例 6 过表达 EPSPS转基因烟草 T0代植株抗草甘膦功能鉴定
将 6〜7叶期的非转基因烟草再生幼苗和 T0代转基因烟草幼苗随机排列， 喷施 2000 ppm草甘膦
(商品名： 农达， 购自深圳诗佳贸易公司）至饱和（每平米喷施 50 ml) 。 7天后观察试验结果发现， 非转基因对照 （CK1 ) 植株严重萎蔫， 而分别转 P P-MC2-£ ¾ ¾、 PTP-MC-EPSPS PTP-G2基 因的烟草均有部分植株表现出草甘膦抗性， 其中转 PTP-MC2-EPSPS基因烟草的抗性植株比率为 33.7%, 显著高于转 P7P-MC- PSPS基因烟草的抗性植株比率 （17.3%) 和转 Ρ7?-<¾基因烟草抗性 植株比率（13.9%) (统计结果见表 2 ) 。 CK1为用草甘膦处理的非转基因对照， CK2 为未处理的 非转基因对照。
Figure imgf000012_0001
喷施 2000 ppm草甘膦 15天后，对上述抗性转基因烟草再次喷施 3000 ppm草甘膦，喷施方法同上。 7天后观察结果（见图 9a-9d)发现，转 ^?-^^-£^»¾基因的 28株烟草中除 5株叶片轻度发黄、 萎蔫外， 其余 23株均生长正常 （图 9a为两个转 7?- 1^2-£^»¾基因烟草植株， 其余抗性植株 与其类似， 在此未示出） ； 而 14株转 P P-MC-£ ¾ ¾基因烟草 （图 9b为两个转 PTP-MC-EPSPS 基因烟草植株， 其余植株与其类似， 在此未示出） 和 11株转 (^基因烟草 （图 9c为两个转 Ρ7?- (^基因烟草植株， 其余植株与其类似， 在此未示出） 均表现不同程度叶片发黄、 萎蔫， 甚至 死亡， 在所述草甘膦浓度处理下未筛选到抗性植株； 非转基因对照植株 （图 9d) 死亡。 所述结 果表明， 转 PTP-MC2-EPSPS 基因烟草植株的草甘膦抗性显著高于转 PTP-MC-EPSPS 基因或 (^基因烟草植株的草甘膦抗性。
根据喷施 3000 ppm草甘膦的鉴定结果，随机选取其中转 ？？-^^ ^^^基因植株中的抗草 甘膦和不抗草甘膦转基因烟草植株各 3棵、 转 PTP-MC-EPSPS和转 PTP-G2不抗草甘膦转基因烟 草植株各 3棵、 及 1棵非转基因烟草植株， 用植物 RNA提取试剂盒 （mv rogen) 分别提取叶片 总 RNA。 紫外分光光度测定总 R A在 260 nm和 280 nm的吸光度值， 计算各 R A浓度。 依照 invitrogen反转录试剂盒 ( Superscript III Reverse Transcriptase) 所提供的方法进行反转录 (1 总 RNA作为模板， 反转录引物分别为 SEQ ID NO: 38) 。使用引物 SEQ ID NO: 37和 SEQ ID NO: 38 扩增 PTP， 检测该融合蛋白的相对表达情况 与 £^»¾基因融合表达， 检测 的转录 水平即代表 基因的转录水平） 。
50 μΐ PCR反应体系： 50 μΐ PCR反应体系： 5 μΐ ΙΟχΕχ Buffer, 3 μΐ 2.5 mM的 dNTP， 2.0 μΐ cDNA, 1.0 μΐ Ex Taq^ 10 μΜ的引物 SEQ ID NO: 37禾卩 SEQ ID NO: 38各 2.0 μ1， 以及 35 μΐ的 双蒸水。 PCR反应条件： 94°C预变性 5 min， 33个循环 （94°C变性 30 s， 55°C退火 30 s， 72 °C 延 伸 2 min ) ， 72 °C 延 伸 10 min 。 以 烟 草 内 源 Actin 基 因 (http://www.ncbi.nlm.nih.gOv/nuccore/AB158612.l) 作为内参， 对上述转基因样品进行 RT-PCR 检测。 反转录引物为 SEQ ID NO: 40; PCR引物为 SEQ ID NO: 39和 SEQ ID NO: 40。 反转录及 PCR体系和条件同上。 PCR产物电泳结果如图 10所示： 1-3为转 基因的抗 3000 ppm草甘膦 T0代转基因烟草植株， 4-6为转 P7P-MC- PSPS基因的不抗 3000 ppm草甘膦 T0代 转基因烟草植株， 7-9为转 (^基因的不抗 3000 ppm草甘膦草甘膦 T0代转基因烟草植株， 10-12为转 基因不抗 3000 ppm草甘膦的 T0代转基因烟草植株， 13为非转基因 对照烟草。
上述检测结果表明,对照烟草植株中没有 基因的转录； 不抗草甘膦的转 7?-^2-£^»¾ 基因烟草植株中目的基因转录较弱或没有转录； 抗 3000 ppm草甘膦 T0代转 ？？- ^ ^^^基 因的烟草植株与不抗草甘膦的转 ^7?-1^-£^»¾基因和转 PTP-G2基因的烟草中目的基因转录程 度基本一致 （第 9泳道转 基因植株转录较弱除外） 。 以上结果表明， 在所转基因的转录 基本水平一致的情况下， 转 PTP-MC2-EPSPS 基因烟草植株的草甘膦抗性显著高于转 Wy-MC- rara基因或 PTP-G2基因烟草植株的草甘膦抗性。
实施例 7 利用农杆菌介导法转化棉花
使用农杆菌介导法进行棉花的遗传转化。 具体操作步骤如下：
1. 无菌苗制备
(1) 棉花种子用浓硫酸 (H2S04)脱去短绒， 自来水洗掉种子表面的硫酸， 晾干后用 70%乙醇 浸泡对种子进行表面消毒 1 mm， 再用 10% 15%过氧化氢 (H202)处理 2 4 h， 用无菌水冲洗 2~3 次；
(2) 在无菌水中浸泡 18~24h,待种子露白，再在无菌条件下剥去种皮，种入种苗培养基 (1/2MS (含 9.39 mM KN03， 0.625 mM KH2P04， 10.3 mM NH4N03, 0.75 mM MgS04， 1.5 mM CaCl2， 50μΜΚΙ， 100μΜΗ3ΒΟ3， ΙΟΟμΜ MnS04， 30μΜ ZnS04， 1μΜΝα2Μο04, O.^MCoCl2, ΙΟΟμΜ NaEDTA, 10(^MFeSO4) +琼脂 6 g/L， pH 6.8)中； (3) 25 °C~28°C光培养 3~5天备用。
2. 棉花外植体与农杆菌的共培养
取无菌苗的下胚轴，用解剖刀切成 0.5-0.6 cm小段，浸入含表达载体 pCAMBIA-35S-PTP-MC2 质粒的农杆菌菌液 （OD6。。为 0.8 1.2 ) 中 5~10 mm， 然后取出下胚轴段， 用灭菌滤纸吸干多余的 菌液， 放在共培养培养基上 (MS + 2， 4-D 0.1 mg/L + KT 0.1 mg/L + 葡萄糖 30 g/L + 乙酰丁香酮 200 mg/L + 琼脂 6 g/L， pH 5.0， 培养基表面铺一层灭菌滤纸)， 用封口膜封口。 于 22°C 25 °C在 暗处共培养 2天。
3. 愈伤组织诱导及抗性愈伤组织的筛选
(1) 愈伤组织的诱导
将所述共培养后的下胚轴段放入愈伤组织诱导培养基中 （MS + 2,4-D 0.1 mg/L + KT 0.1 mg/L
+ MgCl2 0.91 g/L + 脱乙酰吉兰糖胶 2.0 g/L + 卡那霉素 50 100 mg/L +头孢霉素 500 mg/L + 葡 萄糖 30 g/L， pH 5.8)， 在 25 °C培养 2个月（一个月换一次相同的培养基)。
4. 愈伤组织的增殖继代
将诱导出的抗性愈伤组织接入增殖培养基 （MS培养基 + MgCl2 0.91 g/L + 脱乙酰吉兰糖胶 2.0 g/L + 葡萄糖 30 g/L, pH 5.8)中， 25 °C培养， 每隔一个月继代一次， 直到愈伤组织分化。 在第 一次和第二次转入增殖培养基后有部分愈伤组织褐化死亡， 正常愈伤组织增殖也不快， 第二次继 代后， 愈伤组织增殖速度才加快。
5. 愈伤组织的分化及转基因苗移栽
愈伤组织经几次继代后， 有的愈伤组织转成米粒状颗粒， 将其转入分化培养基中 (无 NH4 +、 且 KN03加倍的 MS +谷氨酰胺 1.0 g/L +天门冬酰胺 0.5 g/L + MgCl2 0.91-1.35 g/L + 脱乙酰吉兰 糖胶 2.0 3.0 g/L + 葡萄糖 20 30 g/L, pH 5.8)， 进一步分化成胚状体， 胚状体长成为幼苗后再转 入大的三角瓶中， 待根长好后练苗移栽。 洗去再生棉株根部的培养基， 栽到灭菌蛭石中， 浇足 1/2MS (含 9.39 mM KN03， 0.625 mM KH2P04， 10.3 mM NH4N03, 0.75mM MgS04， 1.5 mM CaCl2， 50 μΜ KI, 100 μΜ Η3ΒΟ3， 100 μΜ MnS04， 30 μΜ ZnS04， 1 μΜ Na2Mo04, 0.1 μΜ CoCl2, 100 μΜ Na2EDTA, 100 μΜ FeS04, 蔗糖 30 g/L；)。 栽好的再生棉苗放入控温 22°C、 控湿 80〜85%的人工培 养箱中 5〜7 d， 再在温室中培养 10〜20 d后移栽到土盆或大田中。
取获得的转基因棉花叶片，提取 DNA (方法参照《分子克隆》（第三版）），如实施例 4，利用 PCR 方法鉴定转基因苗， 保留经鉴定为阳性的植株。
使用与上述相同的农杆菌介导的方法， 分别使用含植物表达载体 pCAMBIA-35 S-PTP-MC或 pCAMBIA-35S-PTP-G2的农杆菌 LBA4404转化棉花， 并通过 PCR进行转基因幼苗检测。 所述每种质粒 分别获得约 30个转化事件。 实施例 8 过表达 Ρ7?-Μ^£Λ»¾转基因棉花 TO代植株抗草甘膦功能鉴定
将 4〜5叶期的非转基因棉花幼苗和 TO代转基因棉花幼苗随机排列， 喷施 3000 ppm草甘膦 （商 品名： 农达， 购自深圳诗佳贸易公司） 至饱和 （每平米喷施 50 ml) 喷施 10天后观察试验结果 （见 图 l la-l ld) 发现， 非转基因对照植株都严重萎蔫甚至死亡 （图 l id) ， 转 P7?-MC2-£ ¾ ¾基因 的 31 株棉花中除 8 株叶片轻度发黄、 萎蔫外其余 23 株均生长正常 （图 11a 为两个转 ^P-MC2-£ ¾ ¾ 基因抗性棉花植株， 其余抗性植株与其类似， 在此未示出） ； 而 27 株转 ^^-^^^^^^基因棉花 （图 l ib为两个转^^-^^- ^^^基因抗性棉花植株， 其余抗性植株与 其类似，在此未示出）和 29株转 基因的棉花（图 11c为两个转 ^7?-(^基因抗性棉花植株， 其余抗性植株与其类似， 在此未示出） 均表现不同程度叶片发黄、 萎蔫， 在所述草甘膦浓度处理 下未筛选到抗性植株。 所述结果表明， 转^7?- 1^2-£^»¾基因棉花植株的草甘膦抗性显著高于 转 Ρ7?-Μ(^-£Λ»¾基因或 PTP-G2基因棉花植株的草甘膦抗性。
根据抗性鉴定结果， 随机选取所述转 PTP-MC2-EPSPS基因植株中的抗草甘膦棉花植株 2棵 和不抗草甘膦转基因棉花植株 3棵、 转 PTP-MC-EPSPS基因和转 PTP-G2基因不抗草甘膦转基因 棉花植株各 3棵、 及 1棵非转基因棉花植株提取 R A， 进行 RT-PCR检测， 所用引物及操作步骤 如实施例 6所述。 以陆地棉内源 Actin基因 （http：〃 www.ncbi.nlm.nih.gov/nuccore/32186889 ) 作为 内参，对上述转基因样品进行 RT-PCR检测。反转录引物为 SEQ ID NO:42； PCR引物为 SEQ ID NO: 41： 和 SEQ ID NO: 39。 反转录及 PCR体系和条件同实施例 6中烟草 Actin基因的检测。
扩增产物电泳结果如图 12所示： 1-2为转 ^?- 1^2-£^»¾基因的抗草甘膦 TO代转基因棉 花植株， 3-5为转 PTP-MC-EPSPS基因的不抗草甘膦 TO代转基因棉花植株， 6-8为转 PTP-G2基 因的不抗草甘膦的 TO代棉花， 9- 1 1为不抗草甘膦的转 Ρ7?-Μ^£Λ»¾基因的 TO代棉花， 12为 非转基因对照棉花。
上述检测结果表明,对照棉花植株中没有 PTP的转录；不抗草甘膦的转 ？？- ^-^^^^基因 棉花植株中目的基因转录较弱或没有转录；抗草甘膦 TO代转 Ρ7?-Μί^-£Λ»¾基因棉花植株与不 抗草甘膦的转 PTP-MC- PSPS基因和转 7?- (^基因棉花中目的基因转录程度基本一致 （第 8泳 道转 (^基因棉花转录较弱除外） 。
上述结果表明， 在所转基因的转录水平基本一致的情况下， 转^？？- ^ ^^^基因棉花植 株的草甘膦抗性显著高于转 Ρ7?-Μ^Λ»¾基因或转 PTP-G2基因棉花植株的草甘膦抗性。
从实施例 2、 6禾卩 8的结果可知， MC2-EPSPS融合蛋白的抗草甘膦能力比单独的 MC-EPSPS蛋 白或 G2蛋白的抗草甘膦能力有显著提高，并且所述草甘膦抗性的提高是由于蛋白结构的改变引起的， 而不是由于基因转录或蛋白表达量的增加引起的。 因此， 可在外源 EPSPS基因表达水平相同的情况 下， 获得草甘膦抗性更强的转基因植物， 从而可规避通过增强外源基因表达水平来提高抗性这一技术 方案可能影响受体植物正常生长发育的负面风险。

Claims

权 利 要 求 书
1. 一种融合蛋白， 其序列如 SEQ ID NO:2所示。
2. 编码权利要求 1所述融合蛋白的核苷酸序列。
3. 权利要求 2所述的核苷酸序列， 其具有如 SEQ ID NO: l所示的核苷酸序列。
4. 一种重组表达载体， 其包含权利要求 2或权利要求 3所述的核苷酸序列， 其中所述核苷酸序 列与表达载体的表达控制序列可操作地连接； 所述表达载体优选 pET28b或 pCAMBIA2300。
5. —种重组细胞， 其包含权利要求 2或 3所述的核苷酸序列或者权利要求 4所述的重组表 达载体， 优选为重组大肠杆菌细胞或重组农杆菌细胞。
6. 一种改善植物草甘膦抗性的方法， 包括： 将权利要求 2或 3所述的核苷酸序列或者权利 要求 4 的重组表达载体导入植物细胞、 组织、 器官或植株并使其表达； 优选地， 所述植物为烟 草或棉花。
7. 一种制备融合蛋白的方法， 其包括将权利要求 2或 3所述的核苷酸序列插入表达载体并 使所述核苷酸序列与所述表达载体的表达控制序列可操作地连接， 然后将所得的重组表达载体 导入生物体使所述核苷酸序列表达； 优选地， 所述生物体为大肠杆菌、 烟草或棉花。
8. 权利要求 1所述的蛋白、 权利要求 2或 3所述的核苷酸序列、 权利要求 4所述的重组表 达载体或者权利要求 5 所述的重组细胞用于改善植物草甘膦抗性以及用于植物育种的用途， 优 选地， 所述植物为烟草或棉花。
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