鳞翅目昆虫性别决定关键基因鉴定与选择性剪接调控分子机制研究

Sex determination pathway in insects is composed of a cascade of sex-specific alternative splicing of several key regulators.Lepidoptera is the second largest order in insects, including thousands of agricultural pests and the important economic insect silkworm. However, key regulators for sex determination pathway and their functional mechanism in Lepidoptera remain unclear except that homologous genes of downstream doublesex were found. Based on our previous studies, here several methods were proposed to screen for key regulators in the sex determination pathway of Lepidoptera. We will perform: i) RNA pulldown assay using identified sex-specific exonic splicing enhancers in doublesex gene and identify the co-purified proteins by mass spectrum; ii) Yeast three hybrid assay to get positive clones and sequences; iii) Chemical compound screenings that would switch the fluorescence representing female to male transition. These obtained candidate genes will be confirmed as key regulators in the sex determination pathway by RNAi in sex-specific cell lines and transgenic insects. Furthermore, regulatory mechanism will be addressed by mutagenesis studies at levels of the protein functional domains and pre-mRNA splicing elements. These studies will elucidate a mechanism of the long time puzzle of sex determination pathway in Lepidoptera, and would provide a useful method for pest control of Lepidoptera.

多个关键基因的选择性剪接级联反应是昆虫性别决定的一个重要阶段，但鳞翅目昆虫性别决定中除了最下游的doublesex基因，其它关键基因长期以来未能鉴定。鳞翅目昆虫包括经济昆虫家蚕和数千种农业害虫，其性别决定途径与调控机制研究具有重要的理论意义与应用价值。本项目计划在申请人已有工作基础上，通过RNA pulldown，酵母三杂交，转录组测序和小分子化合物筛选等技术寻找鳞翅目昆虫性别决定通路中关键候选基因。此后通过性特异性细胞株进行RNAi沉默、超表达性特异性剪接产物和转基因昆虫等实验确认候选基因是否是真正的关键调控因子。最后利用分子生物学方法进行突变体研究，进一步揭示调控鳞翅目昆虫性别决定的RNA作用元件与选择性剪接调控的分子机制。本项目研究将系统地阐明家蚕性别决定途径级联通路，获得高效的鳞翅目性别特异性剪接信号，并以此为基础探索防治玉米螟与棉铃虫等鳞翅目害虫的防治策略与方法。

多个基因的选择性剪接级联反应是昆虫性别决定的重要调控方式。鳞翅目昆虫包括经济昆虫家蚕和数千种农业害虫，其性别决定途径与调控机制研究具有重要的理论意义与应用价值。但是与模式生物双翅目昆虫果蝇不同，鳞翅目昆虫的性别决定途径中除了最下游的doublesex基因，其它关键基因长期以来未能鉴定。本项目通过酵母三杂交，转录组测序和转基因等技术寻找鳞翅目昆虫性别决定通路中的关键候选基因，获得了结合家蚕doublesex 性别特异性外显子的3个RNA结合蛋白（RBP）。此后通过雌雄细胞株进行了机制研究，揭示出其中的BxRBP1，BxRBP3分别结合外显子3和4；两者协同调控鳞翅目昆虫doublesex的性别特异性选择性剪接。进一步研究发现上游关键基因Masc调控BxRBP3的雄性转录本，从而阐明了一种新的选择性剪接调控机制和具体RNA作用元件。项目执行过程中发表标注SCI论文4篇，分别在RNA Biology, Genes & Development等重要国际期刊。同时，这些成果对于进一步开展鳞翅目经济昆虫利用与农业害虫防治等应用研究具有重要参考价值。