类产碱假单胞菌核心杀虫蛋白基因的转化及杀虫活性

目的]利用类产碱假单胞菌核心杀虫蛋白基因(Core Pseudomonas pseudoalcaligenes insecticidal protein gene,cppip)构建植物表达载体并转化烟草,以研究cppip在高等植物体内表达产物的活性.方法]cppip在烟草基因组中的整合及转录通过烟草转化系T0代种子发芽的抗生素抗性分离及其T1代株系的分子检测来证明;烟草转化系后代表达产物的杀虫效果通过测定蝗虫死亡率来分析.结果]证明了cppip能以一定拷贝数插入到烟草基因组内,并按照孟德尔遗传方式传递给后代;比较含信号肽(signal peptide sequence,SPS)和不含信号肽的类产碱假单胞菌杀虫蛋白基因(Pseudomonas pseudoalcaligenes insecticidal protein gene,ppip)表达产物的杀虫活性,发现不含SPS的ppip烟草转化系蛋白表达产物对2～3龄蝗虫幼虫的平均致死率为83.37%,并对幼虫的生长发育有明显抑制作用,而含有SPS的ppip烟草转化系蛋白表达产物对2～3龄蝗虫幼虫的平均致死率为15.65%,两者之间有着显著的差异.结论]推测ppip的SPS会影响该基因在高等植物体内表达产物的活性,本研究结果对于高效利用ppip进行植物转化及抗虫具有重要参考价值.

Transformation of core Pseudomonas pseudoalcaligene insecticidal protein gene and its insecticidal expression in tobacco

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.