表达蝎毒素的重组斜纹夜蛾核型多角体病毒(SpltMNPV)的构建及毒力

Construction and toxicity the recombinant SpltMNPV expressing the scorpion toxin gene

【目的】开发高毒力的重组斜纹夜蛾核型多角体病毒(Spodoptera litura multicapsid nucleopolyhedroviruse,SpltMNPV)杀虫剂。【方法】构建编码蜕皮激素UDP-葡萄糖基转移酶(ecdysterioid UDP-glucosyl transferase gene,egt)基因缺失并插入东亚钳蝎神经毒素(B.martensi Karsch,BmK ITa1)基因的重组转移载体,重组转移载体与SpltMNPVⅡ基因组DNA共转染斜纹夜蛾细胞,通过荧光斑法与有限稀释法相结合筛选重组病毒。【结果】成功筛选出缺失egt基因的、早期启动子(ie-1)启动的、表达BmK ITa1成熟肽的重组病毒SpltMNPV-Δegt-Pph-egfp-ie-1-BmK ITa1。生物测定结果显示,重组病毒的杀虫速度(LT50)较野生病毒提前0.7-0.8 d。【结论】通过在SpltMNPV病毒基因组中插入外源毒素基因可明显增强病毒的杀虫效果,结果说明开发高毒力SpltMNPV生物杀虫剂具有可行性。

[Objective] To develop a high toxic recombinant Spodoptera litura multicapsid nucleopolyhedroviruse（SpltMNPV） insecticide.[Methods] We constructed a recombinant transfer vector that was characterized by disrupting of ecdysterioid UDP-glucosyltransferase（egt） gene and expressing the mature peptide of the Chinese scorpion,B.martensi Karsch（BmK ITa1） gene at the control of ie-1 promoter.The transfer vector and the SpltMNPV II DNA co-transfected the SpLi cells.Recombinant viruses were purified by the end point dilution and fluorescent spot purification.[Results] We successfully screened the recombinant SpltMNPV-Δegt-Pph-egfp-ie-1-BmK ITa1 of which the egt gene was knocked out and expressed the mature peptide of the BmK ITa1 gene at the control of ie-1 promoter.Bioassays showed that,compared to the wide-type SpltMNPV,the speed of the recombinant virus killing the S.litura（LT50） increased by 0.7-0.8 days.[Conclusion] The insecticidal effect of SpltNPV could be increased by inserting the foreign gene,which provided a further opportunity to develop the SpltNPV into commercially viable products to control the S.litura.