大猿叶虫滞育准备期滞育相关基因的鉴定及其功能分析

Insect diapause is a fascinating and important scientific problem and requires more efforts of study. Identification and functional analysis of diapause-associated genes is esential to understand the molecular mechanisms of insect diapause. The cabbage beetle Colaphellus bowringi is a serious pest of crucifers which has facultative reproductive diapause. Diapause preparatory phase is short and clear. All diapausing adults exhibit digging behavior and burrow into the soil after diapause preparatory phase. Therefore, the cabbage beetle is an ideal animal model for exploring the molecular regulation mechanism of insect diapause. In current project, basing on the transcriptomics data of the cabbage beetle, we plan to screen the differential expressed gene/protein using the methods of digital gene expression profile and comparative proteomics during the diapause preparatory phase of female adults bewteen normal strain and the selected non-diapause strain against photoperiod in this species. Then,we plan to identify the candidate diapause- associated genes, analyze its expression patterns, and clone the full-length cDNA sequences of main candidate diapause-associated genes using RACE, qRT-PCR and other approaches. Finally, a set of RNA interference system will be established to analysis the function of the main candidate diapause-associated genes. Our results will be helpful for understanding the diapause regulation mechanism at the molecular level during diapause preparatory phase in the cabbage beetle, getting more intuition about insect diapause, and providing a new concept for insect pest management.

昆虫滞育是非常奇妙而尚需认识的重要科学问题，寻找昆虫滞育相关基因并分析其功能，是昆虫滞育调控分子机制研究的核心工作。大猿叶虫Colaphellus bowringi是一种具兼性生殖滞育的十字花科蔬菜害虫，滞育准备期明确且持续时间短、易于从钻土行为判断成虫是否滞育，是探索昆虫滞育调控分子机制的理想材料。本项目依托大猿叶虫转录组数据，以大猿叶虫正常品系和针对光周期滞育诱导筛选的同源非滞育品系为材料，采用数字基因表达谱和比较蛋白质组学技术，分析滞育准备期雌成虫的基因/蛋白差异表达，筛选滞育相关基因。同时，采用RACE、qRT-PCR等技术，鉴定候选基因并分析表达模式、克隆主要候选基因cDNA全长序列，并建立RNA干扰体系分析主要候选基因功能。研究结果不仅可探明大猿叶虫滞育准备期的滞育调控分子机理，为解析昆虫滞育调控分子机制提供新线索，也可为创新害虫防治技术提供新思路。

昆虫滞育是昆虫生长、发育、繁殖的停顿状态，是大多数温带昆虫避开不利物候而采取的季节性适应策略。寻找昆虫滞育相关基因并分析其功能，是昆虫滞育调控分子机制研究的核心工作。本项目依托大猿叶虫转录组数据，采用RNA-Seq和iTRAQ技术鉴定出大量参与生殖滞育准备调控的候选基因和蛋白，首次在分子水平上明确了生殖滞育昆虫在滞育准备期脂类和糖类代谢途经被激活，而蛋白质代谢途经被抑制。结合RNA干扰技术分析，发现保幼激素以Met依赖的形式诱导卵巢发育和卵黄原蛋白基因转录，同时抑制脂肪累积和虫体抗逆基因表达等滞育表型，最终促进生殖发育而抑制滞育的发生，证明了长日照和短日照型昆虫均通过JH-Met这一保守的信号途径调控生殖滞育。明确了JH-Met信号的缺乏是诱导大猿叶虫滞育准备期营养代谢相关基因典型表达模式产生的关键，发现脂质积累对滞育个体含水量和抗逆基因表达的调控作用。此外，高通量测序发现大猿叶虫产卵前期和滞育准备期肠道微生物群落组成表现出明显差异，其中Bacteroidetes，Firmicutes和Proteobacteria等与人类肥胖相关的肠道微生物与滞育呈现较好的相关性，初步建立了昆虫滞育与肠道微生物的相关性，为滞育研究提供了新思路。研究结果部分揭示了大猿叶虫滞育准备的调控机理，为解析昆虫滞育的分子机制提供了新线索，也为创新害虫防治技术提供新思路。