黑纹粉蝶夏季滞育/冬季滞育和非滞育的转录组比较与关键基因功能分析

Diapause is one of the basical modes for insect to escape from unfavourable environment, however, there are few genes which were demonstrated to involved in the regulation of insect diapause. In the present project, the cabbage butterfly Pieris melete, a serious multivoltine pest of crucifers, undergoing both summer and winter diapause as pupa was selected as experimental model. Based on the previous studies on photoperiod and temperature regulation mechanism on summer and winter diapause, differential expression of heat shock protein related to summer and winter diapause, sugar content in summer and winter diapausing puape, the present project keep more attention on the juvenile hormone, ecdysone, respiratory metabolism and energy metabolism, which were determined by HPLC-MS. Massively parallel RNA-seq combined with bioinformatics analysis was used to analyses the transcriptome and identify the differential expression genes among the different physiological phase of summer diapause, winter diapause versus nondiapause individuals. Quantitative RT-PCR and RNA inferences will be employed to identify transcriptional events underpinning to diapause response in the related key metabolic of juvenile hormone, ecdysone, respiratory metabolism, and energy metabolism and key signal pathway by comparing gene expression between summer diapause, winter diapause and non-diapause population. RNAi were be selected to inferences the expression of related key genes. Our goal was to explore the dynamics of transcriptional variation by performing a genome-wide RNA-Seq analysis of gene expression during the entire period of summer and winter diapause. Studies on metabolism and signal pathway in summer and winter diapause should be helpful for further understanding the tolerance machanisms of summer and winter diapuase to stress conditions and molecular regulation mechanism of diapause, which may provide new ecological approch in pest management.

滞育是昆虫逃避不利环境条件的基本方式之一，但目前仅极少数基因被证明直接参与了昆虫滞育的调控。项目选择兼具夏季滞育和冬季滞育的十字花科作物害虫黑纹粉蝶为对象，基于前期在滞育光温调控、滞育关联热休克蛋白差别表达和糖代谢生理的研究基础，拟通过测定滞育期间的激素、呼吸和能量物质代谢水平，进一步采用RNA-Seq转录组学和生物信息学分析，比较黑纹粉蝶夏季滞育/冬季滞育和非滞育及其相应各滞育生理阶段的转录组差异，鉴定特异表达的基因。利用qRT-PCR对与滞育特性关联的激素、呼吸和能量生理代谢、信号途径中特异表达的关键基因进行表达量验证。通过RNAi干扰关键基因的正常表达，鉴定其在滞育调控中的作用。项目研究可明确黑纹粉蝶不同滞育类型的转录组差异，对滞育代谢和信号途径关键基因研究可进一步了解昆虫滞育进程中对逆境条件耐受的机制，为探索害虫生态调控的新途径提供参考。

昆虫滞育是逃避不利环境的一种方式，目前证明极少数基因直接参与了昆虫滞育的调控。本项目选择黑纹粉蝶，同时具备夏季滞育和冬季滞育特性。项目采用RNA-Seq转录组学和生物信息学分析，研究比较黑纹粉蝶夏季滞育/冬季滞育和非滞育及其相应各滞育生理阶段的转录组差异夏季滞育、冬季滞育和非滞育之间的RNA-Seq转录组分析；鉴定部分特异表达的基因。从转录组文库中，分别克隆3个Hsp90基因，研究明确低温同时诱导夏季滞育和冬季滞育蛹中Hsp90上调表达；但对非滞育蛹无显著作用；高温刺激逐渐诱导夏季滞育蛹Hsp90上调表达，而在冬季滞育蛹中则快速上调。在不同滞育过程中，31度高温诱导夏季滞育蛹Hsp90表达水平上调，4度低温处理则亦显著诱导Hsp90在冬季滞育蛹中上调表达。针对滞育期间水份和糖类生理代谢分析进行系统检测，分别检测了黑纹粉蝶冬季滞育不同虫态、滞育过程中，分别在自然条件和恒温5℃和20℃下处理不同天数，检测了水分代谢动态变化和能量中总糖、海藻糖和糖原的代谢变化与季节动态。在本项目的资助下，目前已发表SCI论文有4篇，其中发表在一区的SCI论文有1篇，二区的SCI论文有2篇，发表中文刊物论文4篇，会议论文（摘要）3篇，另外还有2篇SCI论文正在修改或准备投稿中。项目研究可明确黑纹粉蝶不同滞育类型的转录组差异，对滞育代谢和信号途径关键基因研究可进一步了解昆虫滞育进程中对逆境条件耐受的机制，为探索害虫生态调控的新途径提供参考。