褐飞虱雄虫生殖相关基因SPATA5和PHF7的功能研究及对雌虫生殖的调控

The brown planthopper, Nilaparvata lugens , is a classic example of an insecticide-induced resurgence of rice pest. In the past, studies on resurgence mechanisms have focused on the physiological and ecological aspects of insecticide-induced stimulation of fecundity in adult females, while the role of N. lugens male in the resurgence is ignored. We have found that protein expressional levels of two genes (SPATA5, Spermatogenesis - associated protein 5 and PHF7,testis development protein NYD-SP6)associated with reproduction of N. lugens male treated with triazophos was significantly higher than the control using proteomics approach, however, their function is unclear. Therefore, this project will investigate temporal and spatial expression patterns of the two genes (SPATA5 and PHF7) of the N. lugens male from the protein and mRNA levels, using immunohistochemical staining and situ hybridization, respectively, to confirm specificity of SPATA5 and PHF7 genes expression and the role in the regulation of spermatogenesis and testis development in N.lugens. In aadition, we will examine their regulatory function in female reproduction via mating using RNAi feeding, analyse effects of different insecticides on sperm formation of N.lugens male and expression level of the two reproductive genes, and clarify the role of insecticide-induced stimulation of reproduction of adult males in N. lugens resurgence. The present findings will provide valuable information for the mechanisms of N. lugens resurgence and enrich the theory of pest resurgence, moreover, may discover a approach for development of new insecticides or control of N. lugens.

褐飞虱是一种典型的再猖獗水稻害虫。过去研究药剂诱导害虫再猖獗仅局限于对雌虫生殖的刺激，忽视了雄虫在再猖獗中的作用。申请者利用蛋白质组学方法发现三唑磷处理诱导褐飞虱雄虫生殖基因SPATA5和PHF7及蛋白质表达量显著高于对照，但其功能不清楚。本项目在此基础上，采用免疫组织化学染色法和原位杂交法分别从蛋白质和基因mRNA水平分析褐飞虱雄虫生殖基因SPATA5和PHF7的时空表达模式，明确两基因的表达特异性及在褐飞虱雄虫中调控精子形成和精巢发育中的作用；同时采用RNAi饲喂法分析褐飞虱雄虫生殖基因SPATA5和PHF7 的功能，揭示它们通过交配对雌虫生殖的调控；分析不同药剂处理对褐飞虱雄虫精子形成及生殖基因表达的影响，明确褐飞虱雄虫生殖基因在药剂诱导褐飞虱再猖獗中的作用。本研究将揭示药剂刺激害虫再猖獗的另一新机制，丰富害虫再猖獗理论，而且可能为新农药的研制提供有价值的信息或害虫防治提供一条途径。

褐飞虱是一种典型的农药诱导的再猖獗型害虫。我们以前研究表明三唑磷40ppm浓度处理的褐飞虱雄虫体内SPATA5（spermatogensesis-associated protein 5-like） 和PHF7（PHD finger protein 7）基因的表达水平与对照相比显著上调。药剂处理的雄虫与雌虫交配产卵量显著高于对照组。也有研究报道SPATA5和PHF7基因在精巢特异性高表达。因此，我们推测褐飞虱雄虫NlSPATA5和NlPHF7基因的表达水平的变化可能与雄虫、雌虫的生长和生殖有关。研究结果表明，分别沉默雄虫NlSPATA5和NlPHF7基因对雄虫和雌虫的的生长和生殖产生了显著的影响。沉默NlSPATA5与对照相比显著延长了产卵前期，产卵量和雄虫附腺蛋白含量显著减少，雄虫生殖系统发育畸形。敲除NlSPATA5后与对照相比雌雄虫体重和寿命、产卵期无显著性差异；但减少了脂肪体和卵巢内蛋白质含量、类共生菌数量、影响了卵巢发育和Nlvg基因表达。同样沉默NlPHF7基因与对照相比显著缩短了雌虫的产卵期、产卵量和雄虫附腺蛋白含量显著减少，雄虫生殖系统发育畸形。敲除NlPHF7后与对照相比显著影响了雌雄虫的体重，显著减少脂肪体和卵巢内的蛋白质含量、类共生菌数量、也影响了卵巢发育和Nlvg基因表达。然而，沉默NlPHF7后对雌虫的产卵前期和寿命无显著性影响。我们推断增加NlSPATA5和NlPHF7基因表达是三唑磷农药诱导褐飞虱再猖獗的分子机制之一，也是导致害虫种暴发的原因之一。因此，我们认为NlSPATA5和NlPHF7可以作为控制褐飞虱种群暴发的新靶标基因。我们的研究结果将有助于揭示种群繁殖、暴发成灾的发生机制，为制定科学、合理的害虫防治策略提供新途径，对多种农业害虫的综合治理有重要的指导意义；同时也完善和发展一个系统和完整的雌雄虫生殖刺激共同导致害虫再猖獗的理论。