章鱼胺响应交配介导cAMP-PKA信号通路调控褐飞虱生殖的机制研究

Previous studies have demonstrated that insecticide-induced male reproduction effects can be transferred into females during mating, following a significant post-mating effect that stimulates female egg-laying. However, the molecular mechanism underlying possible pathways that modulate female post-mating effect is not well understood. Our latest results showed that the expression of NlAC9 gene was be correlated with the fecundity of female adult, and octopamine (OA) content of ovary in mated females was significantly increased compared to unmated females. Associating our findings with the current knowledge on physiological functions and signal transduction cascade of OA, a hypothesis that OA response to mating mediates cAMP-PKA signaling pathway modulating the fecundity of N. lugens was posed. Based on previously accumulated achievements, this program aims to confirm this hypothesis from the following aspects. Firstly, we intends to explore the mating-responsive mechanisms of OA, illuminate regulatory roles played by juvenile hormone and OA as well as its Octβ2R receptor in female fecundity, and unravel the function by which CREB1 implicates in N. lugens female fecundity and population growth using RNAi interference, bioassay, western blot and immunofluorescence. By comprehensive analysis of results derived from this program, the functional mechanism of mating-responsive OA that mediates cAMP-PKA-CREB1 signaling pathway modulating the fecundity of N. lugens will be illustrated. This study will elucidate pivotal modulating pathways of the post-mating effects in Hemipteran females and reveal physiological ecology and molecular mechanisms of the pest population outbreak from new insights.

以前研究表明药剂诱导褐飞虱雄虫的生殖效应经交配使雌虫产生显著的交配后效应。但调控雌虫交配后效应的途径及其作用机理不清楚。申请者前期研究发现NlAC9基因表达与雌虫产卵有关，交配的雌虫卵巢内章鱼胺（OA）含量显著增加。基于昆虫OA生理功能及其信号传导的研究进展，我们提出了“OA响应交配介导cAMP/PKA信号通路调控褐飞虱生殖”的假设。本项目拟在前期研究基础上采用RNAi干扰、生物测定、Western blot和免疫荧光等方法，解析褐飞虱雌虫体内OA信号对交配的响应机理，探明OA及Octβ2R受体对雌虫生殖及行为的调控，明确CREB1在雌虫生殖和种群增长中的功能，最终揭示OA响应交配介导cAMP-PKA-CREB1信号通路调控褐飞虱雌虫生殖的作用机制。本研究将从新的角度揭示该害虫种群繁殖、暴发成灾的分子机制。

褐飞虱（Nilaparvata lugens）是中国长江流域及华南和西南广大稻区的重要害虫，也是一种典型的药剂诱导再猖獗。以前研究表明药剂诱导雄虫的生殖效应经交配传导刺激雌虫生殖，但其作用机制不清楚。我们研究结果表明交配后的雌虫卵巢OA含量显著增加，TβH、AC和PKA酶活性、OA和cAMP含量显著增加，TβH和Octβ2R表达水平上调，cAMP/PKA通路关键基因AC9、PKAR、CREB1和CBP表达水平也上调。通过蛋白印迹分析发现CREB1的磷酸化水平增加。未交配的褐飞虱处女雌注射OA后，其体内cAMP含量增加，AC和PKA酶活性增加， CREB1磷酸化水平，cAMP/PKA通路关键基因表达水平显著增加。沉默褐飞虱雌虫体内TβH后其体内OA含量下降，cAMP/PKA信号通路主要相关基因表达下降，CREB1磷酸化水平下降，AC和PKA酶活性降低，雌虫脂肪体和卵巢内蛋白质含量下降、雌虫体重下降；JHAMT、Met、Vg和VgR表达水平也下降，JHIII滴度和Vg蛋白含量均显著下降，导致雌虫卵巢量、产巢历期及雌虫寿命缩短。进一步研究表明，外源OA处理未交配的雌虫后与注射PBS对照相比，JHAMT、Met表达水平增加，JHIII滴度增加，表明OA影响JH合成；进一步通过保幼激素类似物methoprene补偿实验，结果表明TβH 表达量上调，OA含量也显著增加，表明JH影响OA合成。沉默OA／cAMP／PKA通路相关基因后显著抑制褐飞虱雌虫卵巢发育及卵巢小管内Vg的吸收和积累，dsTβH, dsOctβ2R和dsCREB1处理后F1代种群数量、孵化率、种群趋势增长指数均显著降低。本研究结果解析了OA响应交配介导cAMP/PKA通路协同JH调节雌虫生殖的机制。该研究结果揭示了雌虫响应雄虫交配产生交配后效应的可能作用机理，为褐飞虱的防治提供新的途径。