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Vector Construction with Bt Gene for Sugar Beet Chloroplast Transformation and Expression Toxin-protein
Jie Cui1,2, Qian Yang2*, De-Chang Xu1
1Faculty of Sugar Beet and Sugar Industry, Harbin Institute of Technology, Harbin 150086, China; 2Department of Life Science and Technology, Harbin Institute of Technology, Harbin 150001, China
Abstract: Based on the high conservation of plant chloroplast genomes in evolution， the PCR primers were designed and synthesized according to the corresponding sequences in the chloroplast genomes of Nicotiana tobacum， Spinacia and Oryza sativa for amplifying and cloning two important chloroplast genes from sugar beet， rbcL gene and atpB gene (with accessory No. as DQ067450 and DQ067451 in GenBank respectively) as homologous recombination fragments to direct targeted foreign genes into the chloroplast genome. Constructed vector of Bt gene CryIAc for sugar beet chloroplast transformation. The constructed vector contain homologous recombination fragments， Bt gene CryIAc， aadA gene， strong chloroplast promoter Prrn and TpsbA. The results of restriction enzyme analysis were in accord with the desired. Bioassays using crude expressed products from host strain of the vector showed that Bt CryIAc gene was expressed and its products were strongly toxic to larvae of Barathra brassicae. The chloroplast transformation vector will be useful value for sugar beet chloroplast genetic transformation and the insect resistant crop improvement. Transformation and consequent works are in progress.
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