多巴胺调控飞蝗型变过程中嗅觉行为的机理研究

We found dopamine signalling pathway modulates the behavioral phase change of the migratory locust. In this study, we choose quantitative PCR, HPLC, in situ hybridization and immunostainning techniques to clarify the expression pattern of dopamine and its receptor in isolation and crowding process and the spatial distribution of this chemical and its receptor in brain. In combination with behavioral assay, we use RNA interference and pharmacological method to confirm the function of dopamine receptor in regulating phase change of this insect. After the injection of dopamine receptor dsRNA or its agonists and antagonists,the fluorescent molecular tracer and inner cell recording techniques are applied to analyze the reaction of the dopaminergic neurocircuit under the stimuli of volatiles from gregarious nymphs. We further use the Y-tube olfactometer to explore the function of dopamine and its receptor in regulating attractive and repulsive behavioral responses to the volatiles from gregarious nymphs in the phase change. After injecting gregarious and solitary nymphs with dopamine receptor dsRNA, we choose the RNA_seq strategy to analyze gene expression profiles of the injected nymphs in isolation and crowding process. The differentially expressed genes are enriched by GO and KEGG enrichment to identify the signalling pathways and gene classes potentially regulating the olfactory stimuli of the migratory locust. The computational analysis of genes differentially expressed revealed by transcriptome data in isolation and crowding will clarify the molecular mechanism of dopamine receptor in regulating olfactory responses in the phase change. This study will provide the basis for the management of locust plague and the research of the behavioral and genetic mechanism in the phase change of the migratory locust.

依据多巴胺信号途径调节飞蝗群居型和散居型行为转变的研究基础，通过定量PCR、HPLC、荧光原位杂交和免疫组化技术阐明多巴胺及其受体在飞蝗群居化和散居化过程中的表达模式以及在脑部的组织分布。并结合行为学分析，通过RNA干扰和药理学方法研究多巴胺受体调节飞蝗型变的功能，确立多巴胺受体与飞蝗型变的关系。采用荧光探针分子示踪以及细胞内记录技术分析群居型和散居型飞蝗在RNA干扰或药理学处理后，多巴胺能相关神经环路对群居型气味的反应。采用Y型嗅觉仪进一步证明飞蝗型变过程中多巴胺对飞蝗吸引和排斥行为的功能调节。同时，利用转录组测序技术研究多巴胺受体干扰的群居型和散居型飞蝗分别在散居化和群居化过程中发生变化的基因表达谱。利用计算生物学的方法富集处理前后差异表达的基因家族类型和信号途径，并通过定量PCR验证，初步阐明多巴胺受体调节型变中嗅觉行为的分子机理，为飞蝗型变行为遗传机理研究以及蝗灾治理奠定理论基础。

本项目的主要研究内容是依据多巴胺信号途径调节飞蝗群居型和散居型行为转变的研究基础，通过定量PCR，HPLC、荧光原位杂交和免疫组化技术以及高通量测序技术阐明多巴胺及其受体调控飞蝗嗅觉行为的分子机制。通过高效液相色谱分析发现多巴胺在群居化过程中显著升高，在散居化过程中降低。通过克隆多巴胺两个受体Dop1和Dop2，发现Dop1在群居化过程中升高，并调节飞蝗的群居化，而Dop2在散居化过程中升高，并调节飞蝗散居化过程。同时发现Dop1调节飞蝗群居过程中同种之间的吸引作用。原位杂交和免疫组化显示Dop1主要分布在飞蝗前脑蘑菇体的肯尼恩细胞中。进一步通过RNA干扰和药理学技术发现Dop1通过调节群居型飞蝗自身产生的化学气味嗅觉反应介导飞蝗表现出同种间的吸引作用。为了进一步研究Dop1调节飞蝗嗅觉行为的分子机制，我们通过注射激动剂激活Dop1，通过RNA干扰阻断Dop1的作用后，通过高通量测序分析Dop1信号影响的下游基因表达谱。结果发现在激动剂注射组里，我们发现731个上调而975个下调的差异基因，而在Dop1干扰后的组里面，我们发现380个上调而359个下调的差异基因。在注释的差异基因中，84个基因同时受到Dop1激活和Dop1抑制之后的影响。将84个基因进行聚类之后发现，腺苷酸环化酶2可能是Dop1下游的靶基因。q-PCR和免疫印迹分析发现AC2 mRNA和蛋白水平在SKF38393注射之后显著升高，而Dop1干扰后显著下降。功能实验发现Dop1下游的AC2能够调节飞蝗对自身气味的选择。因而，AC2是Dop1下游的重要的靶基因，并且介导了Dop1的信号途径。本项目初步探索了多巴胺受体调节型变中嗅觉行为的分子机理，为飞蝗型变行为遗传机理研究以及蝗灾治理奠定理论基础。