家蚕和斜纹夜蛾味觉受体的全基因组鉴定及苦味受体的功能研究

Gustatory receptors play critical roles in the feeding, oviposition selection and courtship behaviors of phytophagous insects, most of which are bitter receptors. However, there are few reports on the function research of bitter receptors. In this proposal, we will perform the genome-wide identification of gustatory receptors in the monophagous silkworm and polyphagous tobacco cutworm, and then study the functions of bitter receptors in these two lepidopteran insects. Bombyx mori is an important economic insect for silk production. Spodoptera litura is one of the most widespread and destructive agricultural pests in Asia, which holds a strong detoxification ability. Based on the genome data, we identified 65 bitter receptors in B. mori, in which BmGr19 was highly expressed in larval thoracic legs and BmGr27–31 was a set of clustered genes expressed in adult legs. The results of RNAi experiments revealed that BmGr19 played a critical function in food selection while BmGr27–31 was involved in the courtship behavior. 223 bitter receptors were identified in S. litura. It is an interesting result that SlituGR177 and SlituGR176 were inserted into detoxification related CYP9a gene cluster in the genome and highly expressed in midgut of S. litura. The expression of CYP9a was decreased after knock down of SlituGR177 and SlituGR176, suggesting that the two SlituGRs may be involved in the detoxification process of S. litura. In this project, we will take a variety of research methods, including biological behavior, transcriptional regulatory region identification, and interaction between receptors and ligands, to reveal and clarify the biological functions of bitter receptors in the food selection, mating and detoxification process of insects in vitro and vivo. The implementation of this project will provide theoretical basis and target genes for genetic improvement of silkworm and pest control.

味觉受体在昆虫取食、产卵位置选择和交配行为中发挥重要作用，大部分味觉受体都是苦味受体。目前关于苦味受体的功能报道很少，本项目对单食性经济昆虫家蚕和多食性农业害虫斜纹夜蛾的味觉受体进行全基因组鉴定，进而研究苦味受体功能。基于基因组数据，鉴定到65个家蚕苦味受体，基因干涉实验发现幼虫胸足高量表达的苦味受体基因BmGr19参与取食行为，成虫腿部特异表达的苦味受体基因簇BmGr27–31参与交配行为。鉴定到223个斜纹夜蛾苦味受体，中肠特异表达的SlituGR177和SlituGR176在基因组中插入到解毒代谢基因簇CYP9a中，干涉这两个苦味受体基因导致CYP9a整簇基因下调表达，暗示它们可能参与斜纹夜蛾的解毒代谢。本项目将从生物行为、调控区域鉴定、受体与配体相互作用等方面，在细胞和个体水平上，研究苦味受体在昆虫食性、交配、解毒中的功能，从而为家蚕遗传改良和农业害虫防治提供理论基础和靶标基因。

味觉受体在昆虫之间及昆虫与宿主植物的相互作用中具有重要作用，参与多种行为。单食性经济昆虫家蚕和多食性农业害虫斜纹夜蛾的比较基因组研究，为找到参与这些行为的候选基因提供良好线索。通过基因鉴定、转录组、进化树、PCR分析发现，BmGr27-31在斜纹夜蛾中没有同源基因，且在家蚕成虫腿部特异表达。siRNA、转基因干涉实验表明干涉BmGr27-31，特别是降低BmGr29-31的表达，家蚕雌雄蛾的交配时间显著延长。覆盖前、中、后腿感受器的雄蛾，和正常雌蛾的交配时间延长，表明去掉腿部感受器，即缺乏跗足感受器中表达的化学感受基因后，蚕蛾的交配质量显著降低。人工去除雌蛾鳞毛，10min内有69%的雄蛾无法完成交配。GC-MS分析了鳞毛上的碳氢化合物成分，并采用顶端记录实验对鉴定到的差异物质进行筛选，结果表明腿部第五跗足上的感器可以响应其中的一些代表性物质。在化学感受进程中，化学感受蛋白（BmOBPs）和气味结合蛋白（BmCSPs）可以结合和运输无法溶于水的亲脂性化学物质，将物质运输到味觉受体上。LC-MS鉴定到17个BmOBPs和16个BmCSPs在鳞毛上存在。竞争性结合实验表明其中一些BmOPBs或BmCSPs对筛选到的代表性鳞毛化合物具有结合能力。因此，本研究认为家蚕雄蛾腿部表达的味觉受体通过对雌蛾鳞毛的表皮碳氢化合物的识别来协调家蚕的交配行为。本项目研究成果在基础研究和应用研究两方面都具有重要参考价值。