北非蝎昆虫毒素AaHIT1基因的原核表达和转基因分析

将北非蝎昆虫毒素 (AndroctonusaustralisHectorinsecttoxin ,AaHIT1)基因克隆入温度诱导表达载体pBV2 2 0 ,并在宿主菌E .coliDH5α中进行诱导表达 .表达定位分析发现 ,AaHIT1大部分存在于包涵体中 .蛋白质N 端测序证实了体外表达的AaHIT1蛋白的正确性 .将AaHIT1基因转化入烟草中并获得了转基因植株 ,基因组PCR、RT PCR及Northern印迹分别证实AaHIT1基因已正确地插入到烟草基因组中并已得到表达 .抗虫实验表明 ,该转基因烟草对白粉虱有明显的抗性 .该研究为蝎毒AaHIT1的生物学毒性实验以及获得抗虫转基因作物奠定了基础

Prokaryotic Expression and Transgenic Analysis of a Scorpion Toxin AaHIT1 Gene

The coding sequence of AaHIT1, an insect toxin purified from the venom of North African scorpion Androctonus australis Hector, was cloned into the temperature-induced prokaryotic expression vector pBV220 and expressed in E.coli DH5α host cells. Localization assay using AaHIT1 produced from E.coli showed that most of the toxin protein was found in inclusion bodies. The authenticity of in vitro expressed protein was confirmed by N-terminal peptide sequencing. AaHIT1 gene was also used to transform tobacco leaf discs. Genomic PCR, RT-PCR and Northern Blot analyses of putative transgenic plants confirmed the integration of the AaHIT1 coding sequence into tobacco genome and the successful expression of AaHIT1 in transgenic plants, respectively. In summery, an insect toxin gene AaHIT1 was expressed in E.coli cells and the protein was found largely localized in inclusion bodies. When the AaHIT1 gene was transformed into tobacco genome, it was found that the transgenic plants were able to deter aleyrodids from feeding on the leaves.