Hsp基因在麦红吸浆虫滞育中的功能研究

Wheat blossom midge, Sitodiplosi smosellana (Gehin), is a devastating wheat pest in China. The specific diapause and diapause polymorphism is one of key factors in relation to the intermittent serious damage of the pest. Heat shock protein (Hsp), a multigene family, plays very important role in insect diapause. However, members of Hsp family that play concrete function during diapause are different in different insect species. In this study, S. mosellana larvaes of nondiapause, over-summering diapause, over-wintering diapause and postdiapause will be used to screen Hsp genes in relation to diapause of wheat blossom midge that will be characterized by Transcriptome sequencing and northern blotting. Then the entire cDNA sequence will be cloned by RACE based on the gained fragements.The mRNA expression levels of the cloned Hsp genes in various stages of natural and controlled diapause and stress condition including high temperature, low temperature and drought will be detected using real-time quantitative PCR. The regulation functions in diapause of the relevant Hsp genes will be confirmed by RNAi technology. Also, relationship between titre of juvenile hormone and moulting hormone as well as expression level of receptor gene of moulting hormone during insect diapause and expression level of Hsp genes will be analyzed. This study may provide a theory foundation not only for exploring molecular mechanisms of insect diapause polymorphism, but also be important for understanding disaster mechanism and controlling of wheat blossom midge.

麦红吸浆虫 Sitodiplosis mosellana是小麦上的重要害虫，其特有的滞育及滞育多态性是引起其间歇性猖獗成灾的主要原因。热休克蛋白（Hsps）基因家族在昆虫滞育中起着非常重要的作用。本研究以麦红吸浆虫滞育前、越夏、越冬滞育与解除滞育幼虫为材料，采用转录组分析、Northern杂交和RACE技术，筛选并获得其滞育相关Hsp基因；并利用qPCR技术对相关基因在自然与人工控制滞育进程及极端环境胁迫下mRNA表达量进行分析，结合RNAi技术阐明Hsp基因在麦红吸浆虫滞育中的作用；同时研究滞育过程中保幼激素和蜕皮激素滴度、以及蜕皮激素受体基因表达量，分析Hsp基因表达与激素的关系。本项目研究结果不仅为探索昆虫滞育多态的分子机制奠定基础，而且对深入阐明麦红吸浆虫成灾机理、制定合理的综合治理策略具有重要意义。

麦红吸浆虫Sitodiplosis mosellana是一种典型的具有专性滞育的小麦害虫，滞育过程中经历酷暑和严寒。本项目以与昆虫滞育和抗逆性密切相关的热休克蛋白（Hsps）基因为切入点进行研究，不仅有助于深入了解麦红吸浆虫滞育的分子调控机理，而且为进一步探明其滞育过程中对逆境的耐受机制开拓思路。主要研究结果如下:.1. 通过对滞育不同阶段幼虫进行转录组测序分析，鉴定到13种Hsps基因，其中Hsp90/Hsp70/Hsc70/Hsp60/Hsp40/Hsp23/Hsp20.8/Hsp16.4/Hsp11.4与滞育发育密切相关。.2. 采用RACE技术，克隆获得了滞育相关Hsps全长cDNA序列，明确了编码蛋白的基本特性。.3. 通过分析Hsps在自然滞育、人工打破滞育和短期极端高低温环境下的表达模式，明确了与滞育发育的相关性。Hsp90/Hsp70/Hsp40不仅与滞育启动和维持，而且与滞育期间的耐热和耐寒性密切相关；Hsc70/Hsp60/Hsp20.8的表达不受滞育的诱导，但滞育终止后表达水平显著提高，可以作为预测滞育终止的指标；Hsp23/Hsp16.4滞育启动后下调，但滞育终止后显著上调；Hsp11.4随着滞育终止率的提高表达量逐渐提高。短期（15-120 min）极端高（30-50℃）、低温（0--20℃）胁迫可迅速提高越夏和越冬幼虫Hsp基因的表达，但每个基因胁迫诱导的温度和时间范围不同。.4. RNAi实验表明Hsp90/Hsp70/Hsp60/Hsp23沉默后对越冬幼虫发育无影响，但存活率降低。同时成功表达并纯化了麦红吸浆虫Hsp70/Hsp60/Hsp40/Hsp23/Hsp20.8/Hsp16.4重组蛋白，体外分子伴侣活性分析表明，各纯化蛋白均能有效保护苹果酸脱氢酶(MDH)免遭热变性。.5. 通过分析滞育进程中Hsps表达模式与蜕皮激素（20-E）滴度变化的关系及20-E处理（0.1-0.4 pg/nL）后表达水平的变化，表明Hsc70/Hsp23/Hsp20.8/Hsp16.4的表达受20-E调控，但每个基因受激素调控的剂量和时间反应不同。.6. 明确了保幼激素（JH）和20-E信号传导途径相关基因USP和Met/Kh-r1的分子特征及其与滞育发育的关系，Met/Kr-h1的高表达与USP含量的下降可能与滞育诱导和维持