EPSPS Gene And Bar Gene Cloning And Functional Verification Of The Castor

Castor(Ricinus communis L.) is one of the world’s top ten oil crops. Currently, there is not have a transgenic herbicide-resistant castor varieties. In this study,we used castor male parent “2129” as the material,using PCR amplification method, cloning EPSPS genes and Bar gene,then construct expression vector pCAMBIA3301-Bar-EPSPS, utilizing Agrobacterium-mediated shall EPSPS and Bar gene go to the castor body at the same time.Lay the foundation for cultivating new castor verieties of herbicide-resistant.The results are as follows:1 According to gene sequence of EPSPS which published in GenBank and the principle of overlap extension PCR designed 19 pairs of primers which length at 60 pb, and adjacent two peimers have 20 pb overlap, in 19 pairs of primers at both ends added digestion sites XhoI. According to gene sequence of Bar and principles of overlap extension PCR designed 11 pairs of primers which length at 60 bp,and adjacent two primers have 20 bp bases overlap, in 11 pairs of primers at both ends added digestion sites NcoI, BstEⅡ. Using PCR amplification,get Bar and EPSPS gene full-length sequence,namely through recycling,recycling,ligation,transformation,identification,sequencing,analysis and comparison, got EPSPS and Bar 1529 bp and 831 bp full-length sequence.2 Make expression plasmid pCAMBIA3301 linearized,with NcoI, BstEⅡcut Bar gene from the cloning vector,and then connected,after transformed, identification,acquire pCAMBIA3301-Bar plasmds. The pCAMBIA3301-Bar lindarized by XhoI,cut the EPSPS gene from the cloning vector,and then connect,after transformed, identification, acquire pCAMBIA3301-Bar-EPSPS expression vector, and to determine the correct connection direction of EPSPS gene.3 The constructed plant expression vector pCAMBIA3301-Bar-EPSPS transformed into Agrobacterium competent EHA105 by freeze-thaw method.4 Screening anti-glufosinate concentration of castor cotyledon node, and ultimately determine in the anti-screening culture medium, adding 0.1mg / L glufosinate is the most suitable concentration.5 Using Agrobacterium-mediated method, pCAMBIA3301-Bar-EPSPS for castor cotyledon node genetic transformation,afte resistance screeing,ultimately got 72 resistant plants.By PCR amplification detection,1 of 72 is transgentic plant.