干涉BmNPV极早期非必需基因ie-2的转基因家蚕系统构建和抗性检测

Construction and Resistance Assay of Transgenic Silkworm Expressing RNAi Targeting Nonessential Immediate Gene ie-2 of BmNPV

家蚕核型多角体病毒(BmNPV)是严重危害家蚕的病原之一。为了探究通过转基因干涉BmNPV非必需基因抑制病毒的增殖,提高家蚕抗性的可行性,选择BmNPV极早期非必需基因ie-2作为靶标,构建转基因干涉载体,通过显微注射获得转基因家蚕系统A4Pie2H-A和A4Pie2H-B。3龄幼虫添食感染BmNPV后,A4Pie2H-A与A4Pie2H-B的幼虫死亡率分别比非转基因对照品种降低19%和24%;定量PCR结果显示A4Pie2H-A与A4Pie2H-B的幼虫体内的ie-2 mRNA表达量分别为非转基因对照品种的50%和20%,病毒DNA含量分别为非转基因对照品种的32%和12%。此外,2个转基因家蚕系统的全茧量和茧层率与正常家蚕品种比较无显著差异。研究结果表明,干涉BmNPV极早期非必需基因ie-2可以显著抑制转基因家蚕体内病毒的复制增殖,使幼虫死亡率降低。

Bombyx mori nucleopolyhedrovirus（BmNPV） is one of the pathogens causing severe damage to silkworm（Bombyx mori）.In an attempt to explore the feasibility of inhibiting BmNPV proliferation and enhancing resistance of silkworm through introducing RNAi targeting nonessential gene of BmNPV,the nonessential gene immediate early-2（ie-2） of BmNPV was chosen to construct transgenic RNAi vector,and to generate transgenic silkworm lines A4Pie2H-A and A4Pie2H-B via microinjection.After oral infection of BmNPV,the mortality rate of the 3rd instar larvae of A4Pie2H-A and A4Pie2H-B decreased by 19% and 24% respectively compared to that of the non-transgenic silkworm.Quantitative PCR revealed that the mRNA level of ie-2 in A4Pie2H-A and A4Pie2H-B was 50% and 20% to that of the non-transgenic silkworm respectively,and the accumulated BmNPV DNA content was 32% and 12% to that of the non-transgenic silkworm respectively.Besides,cocoon weight and cocoon shell rate of the two transgenic silkworm lines had no significant difference with those of the normal silkworm variety.These results showed that RNAi of the nonessential immediate early gene ie-2 could significantly suppress the proliferation of BmNPV and reduce the mortality of transgenic larvae.