化学感受蛋白CSPs在荔枝蒂蛀虫产卵行为中的功能研究

The litchi fruit borer, Conopomorpha sinensis Bradle, is the most destructive pest of lithci and longan fruits. Due to its special life habits and occurrence, it is very difficult to control this borer pest. Pest control via interference of feeding or oviposition behaviors are effective strategies to break this bottleneck problem. Insect oviposition is a complex process that depends on a wide range of chemoreception and behavioral responses. Chemosensory proteins (CSPs) are believed to play a crucial role in both chemoreception and physiological processes of insects due to their widely distribution and highly conservation. Nevertheless, the potential physiological function of CSPs in insect oviposition behavior remains unclear. Based on our previous study on olfactory mechanism of C. sinensis and results of preliminary experiments, we postulated the hypothesis that CSPs could influence the oviposition behavior of C. sinensis via a wide range of effects on localization and recognition of host plants, and egg-laying of the insect. Firstly, C. sinensis will be treated with different oviposition stimulates. The differential expression CSPs of C. sinensis will be discovered by using whole transcriptome sequencing (RNA-seq). Then, by expression profile analysis, molecular docking and fluorescence competitive binding assay, the function of CSPs in insect oviposition behavior will be predicted, and the oviposition-related CSPs will be further selected. Finally, the solid evidence for distinct role and regulation mechanism of CSPs in C. sinensis oviposition behavior in vivo will be obtained via RNA interference (RNAi). This project can not only reveal the potential CSPs functions in insect oviposition behavior, but also provide a solid foundation for establishment of the blockade technique of pest oviposition behavior by using CSPs genes as effective new targets.

荔枝蒂蛀虫是荔枝龙眼生产中的首要害虫及防控难点，通过干扰其取食产卵等行为的防控方法是打破该虫防控难题的突破口。昆虫产卵是由多种因子参与调控的对寄主信息感知反应过程。化学结合蛋白CSPs以其广泛分布及高度保守性被认为在昆虫对化学刺激的行为反应及生长发育中扮演重要角色，但是有关CSPs在昆虫产卵行为中的具体生理功能却知之甚少。本项目基于前期对荔枝蒂蛀虫嗅觉识别机制的研究，提出假设：CSPs通过影响昆虫对寄主植物定位、识别和排卵三个环节，参与荔枝蒂蛀虫产卵行为调控。拟利用RNA-seq筛选不同产卵刺激后荔枝蒂蛀虫差异表达CSPs；通过表达谱分析、分子虚拟对接和荧光竞争结合进一步筛选该虫产卵相关CSPs；利用RNAi技术在活体水平研究关键CSPs在昆虫产卵行为中的生理功能。该研究有助于阐明CSPs对昆虫产卵行为的调控作用及相关机制这一科学问题，对探索利用CSPs靶向控制害虫产卵行为也有重要意义。

寄主化学信息刺激对昆虫产卵行为起着决定性作用，气味结合蛋白OBPs和化学感受蛋白CSPs是昆虫化学信息感知的关键载体蛋白。前期研究发现荔枝蒂蛀虫GOBPs与荔枝果实挥发性物质结合特性与行为学实验结果并不完全相符，推测有其他化学感知相关蛋白参与荔枝蒂蛀虫对寄主植物识别及产卵选择过程中。在此基础上，本项目深入研究CSPs对荔枝蒂蛀虫产卵行为的调控作用及相关机制，阐明CSPs在蛾类昆虫产卵行为中的生理功能。主要研究内容包括：不同产卵刺激后荔枝蒂蛀虫差异表达CSPs的鉴定；荔枝蒂蛀虫产卵行为关键CSPs的筛选；活体水平研究关键CSPs对荔枝蒂蛀虫产卵行为的调控作用及相关机制。.利用转录组测序技术获得不同产卵刺激后荔枝蒂蛀虫后的853个DEGs，其中包括274个表达上调基因和579个表达下调基因，我们对DEGs进行了GO功能富集和KEGG通路聚类分析后发现：DEGs并未如我们预期的显著富集于运载或化学感知相关生物学过程或通路，我们最终仅从不同产卵刺激处理后荔枝蒂蛀虫转录组数据中筛选到差异表达的4个CSPs基因。根据4个CSPs基因在不同时期和不同组织表达量均有不同，根据其结果选定CSP6和CSP10为后续研究基因。CSP6和CSP10分别由6个和5个α-螺旋形成一个疏水性的结合口袋用于结合各类化合物，但是与荔枝蒂蛀虫寄主化学信息识别关系并不紧密。结合注射法RNAi基因沉默效率检测结果，我们重点对CSP6在产卵行为相关生理功能进行了研究。CSP6表达量下调并不会影响荔枝蒂蛀虫产卵行为中寄主定位和识别环节，但是CSP6可能通过保幼激素通路调控卵黄原蛋白合成，影响荔枝蒂蛀虫卵巢发育，进而影响荔枝蒂蛀虫排卵环节。.本研究确定了CSP6参与荔枝蒂蛀虫产卵行为调控的作用环节，并初步探索相关作用机制，该基因可作为荔枝抗虫育种的新靶标，对探索利用CSPs靶向控制害虫产卵行为也有重要意义。