复制可控型AcMNPV载体的开发及在鳞翅目昆虫基因功能研究中的应用

缺少高效的研究工具和方法，成为制约鳞翅目昆虫基因功能研究的重要因素之一。苜蓿银纹夜蛾核型多角体病毒（AcMNPV）能经口感染30多种重要的鳞翅目害虫，对其复制必须基因进行改造，使病毒复制受人为调控，从而使AcMNPV成为能携带外源基因进行过表达、基因沉默和蛋白标记等的载体，将为鳞翅目昆虫基因功能研究提供一个高效工具。我们的前期研究证明，将AcMNPV复制必须基因dnapol置于诱导表达系统Tet-on的调控之下，能够在细胞水平实现AcMNPV的可控复制。在此基础上，我们将在甜菜夜蛾或黏虫个体水平验证病毒的可控复制，再通过对几丁质合成酶A的RNAi来检验重组AcMNPV介导基因沉默的效果。该病毒载体的成功开发，与本项目组正在进行的高通量AcMNPV病毒库构建技术相结合，将可在30多种鳞翅目昆虫中实现高通量正向遗传筛选，为这些昆虫的功能基因组研究提供一个好的平台。

One of the most important impeding factors for molecular characterization of Lepidoptera is few high efficient research tools and methods being available. To solve this problem, this project will develop a recombinant AcMNPV vector, which will be able to carrying foreign genes for overexpression, RNAi mediated gene silence and protein epitope tagging, by controlling the expression of a replication essential gene for AcMNPV and making the replication of AcMNPV being controllable. Our previous studies showed that after replacing the original dnapol cassette, a virus DNA replication essential gene, with a Tet-on inducible expression one, replication of the recombinant AcMNPV could be induced by doxycycline in sf9 cells. We will then verify the controllable repliaction of AcMNPVs in Spodoptera exigua Hübner and Pseudaletia separate larvae, and check the RNAi effect on SechsA with the AcMNPV vector. With the success on constructing this recombinant AcMNPV vector, in combination with our on-going development of highthrough-put recombinant AcMNPV library construction, we will be able to make forward genetic screening on more than 30 species from Lepidoptera, thus build a good platform for functional genomics research on these species.