Synthesis Of Cry1ca Pesticidal Gene, Expression In E.coli And Transgenic Indica Rice Development

Accoding to plant preferred codons and frequency, optimization and modification on cry1Ca gene did not alter the amino acid sequence of wild cry1Ca protein. We began to improving GC concent, regulating and removing instability sequence element for RNA, addding plant expression leader sequence and terminators sequence, adding restriction endonuclease BamHI to its two ends. The whole length of 1912 base squence is completed and synthetized finally.Prokaryotic fusion gene expression vector, PET-28bca, was constructed. Expression of fusion protein in E.coli was carried out by IPTG induction. Inclusion body was purified according to its six His-Tag which might chelated with Ni ions by affinity chromatography, and gained about 67kDa size protein. After renaturation, it was as antigen to immune rabbits. ELISA assay showed that the titer of the prepared antiserum against cry1Ca protein was about 1:10000, and it maintained highly specific.Double T-DNA plant expression vector, pCDMARCA, was constructed. Recombinant vector was transferred into indica rice of Minghui 86 through Agrobacterium tumefaciens-mediated transformation. It showed that cry1Ca gene had integrated into rice chromosome genome through southern blot analysis. ELISA assay suggested that cry1Ca gene had expressed and its protein reached to 0.01%-0.28% of whole soluble proteins in leaf. During nature conditions, resistant-insect was carried out, we gained thirty-three transgenic resistant insect To lines.