Construction Of Vectors For An Optimized Epsps Gene From Abutilon Theophrasit Medic. Promoted By Multiple Promoters And The Preliminary Analysis Of Its Transgenic Cotton

Selection and utilization of the glyphosate-resistant transgenic cotton have greatly promoted thedevelopment of cotton industry. The appearance of different varieties and types of glyphosate-resistantcotton has brought enormous benefits to economy and environment. With the popularization ofglyphosate-resistant cotton, cultivation of glyphosate-resistant transgenic cotton with independentintellectual property which can be commercialised will produce far-reaching impact to our country ’sagricultural production.Genetically modified glyphosate-resistant(GR) crops are widely commercialized across theworld.However,the pollen of cotton is sensitive to the glyphosate treatment,resulting in poor pollinationand boll abortion.To further improve the glyphosate-resistance trait of cotton and reproductiveabnormalities in GR cotton,we employed a strategy in which the epsps gene are driven by differentpromoters.Our laboratory had cloned and optimized the epsps gene from Abutilon theophrastiMedic．,which had showed glyphosate-resistant after been transferred into tobacco.Based on CaMV35Spromoter and our early study on cotton specific promoter:arf1(preferentially expressed in cottonreproductive organ);GhPG2(primarily expressed in cotton’s anther);PsbP(primarily expressed incotton’s greentissus) and ag2(specifically induced by glyphosate),the epsps gene was driven by thesepromoters and the expression cassettes were constructed,respectively.Furthermore,the tandemexpression cassettes were developed by combining P35S:E cassette with another two expressioncassettes in which epsps is driven by cotton specific promoter. We had transformed cotton with thesevectors,and obtained the following main results:1. Four plant expression vectors had been constructed: pGBI-P35S:E-P35S:E-P35S:E carriesthere epsps expression cassettes all droved by CaMV35S promoter; pGBI-P35S:E-Parf1:E-Pag2:Ecarries there epsps expression cassettes droved by CaMV35S promoter, arf1promoter and ag2promoterrespectively; pGBI-P35S:E-PPsbP:E-Pag2:E carries there epsps expression cassettes droved byCaMV35S promoter, PsbP promoter and ag2promoter respectively; pGBI-PPsbP:E-PPG2:E-Pag2:Ecarries there epsps expression cassettes droved by PsbP promoter,GhPG2promoter and ag2promoterrespectively.Five plant expression vectors contain only one expression cassette and four plant expressionvectors contain two expression cassette were also constructed，including changing the restrictionenzyme site in vectors which only have one expression cassette.2. The preliminary laboratory experiments had determined the glyphosate screeningconcentration threshold in the filed,and the development of the cotton damage symptoms. Four plantexpression vectors were transferred into cotton plant via Agrobacterium based flower dipping approachrespectively.15plants were transformed by vector pGBI-P35S:E-P35S:E-P35S:E,8plants by vectorpGBI-P35S:E-Parf1:E-Pag2:E,7plants by vector pGBI-P35S:E-PPsbP:E-Pag2:E and7plants by vectorpGBI-PPsbP:E-PPG2:E-Pag2:E were obtained after three times glyphosate or Kanamycin screeningcombined with PCR screening. RT-PCR results from parts of the T0generation plants showed that the exogenous gene had obtained transcription. Real-time PCR analysis of the relative expression level infour tissues of the T0generation cotton transferred with different vectors, showed that the spatial andtemporal expression models of exogenous gene is basically consistant with promoters’ function in thesevectors.It had laid a preliminary groundwork for such expression patterns applying to other functionalgene in other species.3. We obtained T1generation transgenic cotton through glyphosate and PCR screening. RT-PCRresults from parts of the T1generation plants indicated that exogenous gene had obtained correctlytranscription.4. The5’ terminal signal peptide and part of the EPSPS protein sequence was truncated,and theremaining part of the nucleic acid fragment was used to construct a prokaryotic expression vectorpET-QME. Then transformed this expression vector into E. coli strain BL21(DE3).After the inducingand culture with different concentration of IPTG,SDS-PAGE gel electrophoresis results showed that thetarget protein had expressed in E. coli, and this had laid a foundation for the preparation of antibody forEPSPS protein to detect the target protein’s expression levels in transgenic cotton.In summary, this paper had constructed multiple plant expression vectors including one,two orthree expression cassettes in which the epsps gene were promoted by different promoters from cottonitself for the first time,and we had transferred vecters which containing three expression cassettes intocotton via agrobacterium.Through glyphosate screening in the filed,we had obtained T0and T1generation transgenic cotton which were transferred by different vectors.We had done the preliminarymolecular analysis about these transgenic cotton,and this had provided a theoretical basis for increasingcotton’s resistance to glyphosate through increasing the gene copy number.