棉铃虫糖受体的配体鉴定及表达特性分析

Sugars serve as not only some of the simplest, most easily metabolized forms of energy available to insects life but also the key signals for host recognitions and stimulating feeding. Different sugars are recognized by sugars receptors (SRs) in neurons distributed in different gustatory sensilla styloconicam. Studies in SR were less than those of insects odor receptors. Firstly, most ligands of SRs are unknown though 3 ligands of corresponding SRs were indentified. Secondly, it is unknown that different SRs function by forming heterodimers or polymorphism. The present applying project aim to indentify the corresponding ligands of our previously gained putative SRs in Helicoverpa armigera by ca2+ imaging in HEK293T cells, firstly. Then the expressions of heterodimers or polymorphism of different SRs will be characterized by RNAi and single sensillum recording technique（SSR）. Furthermore, the differences of spatial expressions of differently funtional SRs between peripheral sensilla and central nervous systerms will be compared using FISH. This project may have better understanding the sugar sensory mechanism and enrich the hypothesis of "labelled lines" of gustatory neurons coding mechanism of Helicoverpa armigera, namely each neuron in one sensillum styloconicam responding to one kind of stimulars.

糖是昆虫生命活动中最简单和最易获得的能量物质，也是昆虫识别寄主和刺激取食的重要信号物质之一。昆虫依靠外周味觉感受器内神经元上的糖受体识别配体糖。但是相对于昆虫嗅觉受体，昆虫味觉受体包括糖受体的研究极为不足，主要表现在：（1）目前仅鉴定出昆虫4个糖受体的对应配体；（2）糖受体是否与其他受体形成二聚体或多聚体的形式才能表现其应答活性？本申请项目拟在前期获得的多个棉铃虫糖受体基因的基础上，（1）利用细胞钙成像技术分析棉铃虫不同糖受体在体外表达细胞中对不同测试糖的反应及应答专一性；（2）利用RNAi技术结合单感受器记录技术测定和分析不同糖受体可能的异二聚体或多聚体表达特性；（3）进一步，利用mFISH技术分析不同糖受体在外周神经系统和中枢神经系统中的空间表达差异，以期深入阐述棉铃虫的糖化学感受机理和我们提出的棉铃虫幼虫味觉神经元编码的"单线标记"假说，即一个神经元应答一类刺激物质的假说。

利用单感受器记录技术分析了棉铃虫Helicoverpa armigera幼虫下颚外颚叶上中栓锥味觉感器和侧栓锥味觉感器内不同糖受体细胞对不同糖的反应及应答专一性；利用RNAi 技术结合单感受器记录技术测定和分析了棉铃虫糖受体对棉铃虫幼虫取食选择行为和味觉电生理的反应的影响；利用免疫荧光技术分析了棉铃虫幼虫中栓锥感器和侧栓锥感器内味觉受体细胞在中枢神经系统中的空间投射位置异同。结果表明中栓锥感器和侧栓锥感器内糖受体细胞对不同糖的反应谱存在显著差异；幼虫糖受体HaLgr1的表达具有组织和性别特异性; HaLgr1 RNA干扰后能够显著影响幼虫对海藻糖、果糖、葡萄糖、麦芽糖和蔗糖的电生理和行为反应。因此，HaLgr1可能与棉铃虫幼虫识别多个糖受体相关；而棉铃虫幼虫中栓锥感器和侧栓锥感器内味觉受体细胞在中枢神经系统中的空间投射位置没有发现显著差异。本项目的结果对进一步了解昆虫味觉编码机制及利用昆虫味觉控制害虫提供了理论基础。