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

项目名称： 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

项目编号： No.31472045

项目类型： 面上项目

立项/批准年度： 2015

项目学科： 动物学

项目作者： 徐永镇

作者单位： 中国科学院上海生命科学研究院

项目金额： 87万元

中文摘要： 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。

中文关键词： 昆虫性别决定；鳞翅目；选择性剪接；关键基因；分子机制

英文摘要： 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.

英文关键词： Insect sex determination;Lepidoptera;alternative splicing;Key regulator;molecular mechanism