气味结合蛋白在茶翅蝽越冬聚集行为中的功能研究

The brown-marmorated stink bug, Halyomorpha halys (Stål), has obvious overwintering aggregation behavior. It is reported that aggregation related semiochemicals can mediate insect overwintering aggregation behavior, and odorant binding proteins (OBPs) play important roles in perception of these compounds. In the present work, the body volatiles will be collected from overwintering adults of H. halys using solid phase microextraction. Semiochemicals in body volatiles can evoke electrophysiological and aggregation behavioral responses from overwintering adults of H. halys will be identified by coupled GC-MS, GC-EAD and insect olfactometer. Based on the OBP genes identified from H. halys genome, antennal-specific and differentially expressed OBP genes in H. halys before and after overwintering aggregation will be identified by RT-PCR and real-time quantitative PCR technique. By recombinant protein expression and fluorescence binding assays, the relationships between OBPs and aggregation related semiochemicals will be investigated. The effect of specifically knocked out the OBP genes in perception of semiochemicals and overwintering aggregation behavior will be further studied using the CRISPR-Cas9 system. Accomplishment of this project will enhance our knowledge of the physiological roles of OBPs in H. halys overwintering aggregation behavior, and the chemosensory molecular mechanisms of overwintering aggregation in H. halys, which can provide a theory base for designing new lures to regulate the overwintering aggregation behavior of H. halys.

茶翅蝽具有明显的越冬聚集行为。前期研究表明，聚集相关信息化合物能够介导昆虫越冬聚集行为，气味结合蛋白（OBPs）在识别此类化合物中发挥重要功能。本申请以茶翅蝽为研究对象，通过固相微萃取收集越冬成虫的虫体挥发物，结合GC-MS、GC-EAD和昆虫嗅觉仪鉴定对越冬成虫具有电生理及行为活性的聚集相关信息化合物。在茶翅蝽基因组鉴定的OBPs基础上，利用RT-PCR和荧光定量PCR技术筛选越冬成虫触角特异且聚集前后差异表达的候选OBP基因。采用重组蛋白表达和荧光竞争结合试验，解析候选OBP蛋白与聚集相关信息化合物的对应识别关系。利用CRISPR-Cas9系统敲除靶标OBP基因，评价缺失靶标OBPs对茶翅蝽识别信息化合物及越冬聚集行为的影响。本研究有助于明确气味结合蛋白在茶翅蝽越冬聚集中的生理功能，阐明该虫越冬聚集化学通讯分子机制，为开发新型引诱剂调控茶翅蝽越冬聚集行为提供理论依据。

茶翅蝽越冬成虫体表挥发物包括：十三烷、十二烷、十一烷、反-2-葵烯醛、反-2-辛烯醛、4-氧代-反-2-己烯醛、反-2-己烯醛和乙酸-反-2-癸烯酯，其中反-2-葵烯醛能够引起茶翅蝽越冬成虫强烈的电生理及趋避行为活性，未发现具有聚集行为活性的组分。共鉴定茶翅蝽越冬成虫气味结合蛋白OBP基因41个，化感受蛋白CSP基因17个，大多数OBPs在触角中高表达，仅少数CSP在触角中高表达。重组表达HhalOBP14、HhalOBP15、HhalOBP16、HhalOBP20、HhalOBP25和HhalCSP15，荧光竞争结合实验显示，HhalOBP蛋白能与虫体挥发物反-2-辛烯醛、反-2-葵烯醛及其类似物结合，此外与寄主相关挥发物如苯甲酸-3-己烯酯同样具有结合能力；HhalCSP蛋白可有效结合寄主相关挥发物组分，但与虫体挥发物不结合。建立了茶翅蝽CRISPR/Cas9基因编辑方法，成功敲除HhalOBP25基因。综上，茶翅蝽越冬成虫中未发现聚集信息素，气味结合蛋白可能介导报警信息素及寄主相关挥发物的识别；已建立的茶翅蝽基因编辑方法，为茶翅蝽行为靶向调控奠定了重要工作基础。