Transformation of core Pseudomonas pseudoalcaligene insecticidal protein gene and its insecticidal expression in tobacco
类产碱假单胞菌核心杀虫蛋白基因的转化及杀虫活性

Objective] We studied the effect of the signal peptide sequence (SPS) on the expression of Pseudomonas pseudoalcaligenes insecticidal protein gene (ppip). Methods] We obtained the core pseudomonas pseudoalcaligenes insecticidal protein gene (cppip, ppip without the UTR and SPS) by PCR and ligated it into pCAMBIA2301 to generate plant express vector pCPPIP, which was then transformed into tobacco to investigate the insecticidal activity of cppip expression products by locust bioassays. The Kanamycin resistance segregation ratio was determined by the germination rate of T0-generation seeds of the transgenic tobacco. Integration of ppip into genomic DNA was detected by PCR and confirmed by Southern blotting. Results] The bioassay with the 2nd and 3rd instar larvae of Locusta orthoptera showed that the crude proteins extracted from cppip transformed plants caused an average mortality of 83.37%. In contrast, the protein extracts from ppip transformed plants caused a much lower mortality (15.65%). The growth of locust was highly inhibited by the expression products of cppip when compared with the locusts fed with the protein extracts from wild type tobacco or tobacco transformed with intact ppip gene. Conclusion] The results indicated that the SPS might affect the insecticidal activity of ppip expressed in plants. The data of this study are helpful for cost-effective genetic engi-neering of plants with ppip gene.