MicroRNA介导的Wolbachia调控赤眼蜂产雌孤雌生殖的分子机理研究

Wolbachia is widely distributed in animal known arthropod, much attention is given for which altering the host's reproduction. There were a few studies that declared Wolbachia modifying the miRNA in Cytoplasmic Incompatibility hosts for colonization. Up to present, there is not reported that function of miRNA in regulating the mechanism of Parthenogenesis-Inducing (PI) hosts. Based on the systemic analysis of Trichogramma biological characteristics of reproduction and control application in the fields, the materials, including adults and ovums, will be investigated that performed key function in sex determination during Trichogramma development. Variance miRNAs between the insects infected and non-infected (antibiotics removing) by Wolbachia will be isolated by miRNA array and transcriptome sequencing technology in adults and ovums. The true miRNAs will be checked by Northern blotting and qPCR. The target sites of miRNAs involved in reproduction regulation will be identified on the basis of total mRNA degradation sequencing and transcriptome sequencing technologies, and uncovered further the miRNA regulating objective genes or sequence through qPCR and Western blotting.To clarify the molecular mechanism resulted in PI reproduction regulated by miRNA, the relationship between the expression of miRNAs and their target genes and the concentration of Wolbachia will be analyzed under difference phenotype of reproduction and development stage. The project will be useful to enrich the theory of Wolbachia regulating host reproductive mechanism, and application widely of symbiosis and host in the field of agriculture and health.

Wolbachia（Wol）在节肢动物中广泛分布，因其可以改变宿主生殖方式所以倍受关注。研究表明Wol可改变胞质不亲和宿主miRNA来完成其定殖，然而，miRNA在Wol调控产雌孤雌生殖（PI）机制中的作用至今未见报道。项目在系统研究Wol对赤眼蜂PI调控生物学及生防应用的基础上，以赤眼蜂性别决定关键虫态：成虫和卵为材料，采用miRNA阵列结合转录组测序筛选两虫态下感染和未感染（去除）Wol赤眼蜂中差异miRNA，通过Northern和qPCR进一步验证。基于降解组和转录组测序，鉴定miRNA的靶位点；并通过qPCR和Western明确miRNA对靶基因的调控。通过分析不同生殖表型、发育阶段赤眼蜂miRNA及其靶基因表达与共生菌滴度的关系，明确miRNA介导调控PI的分子机理。研究有助于丰富共生菌对宿主生殖调控的理论，对于共生菌及其宿主在农业和卫生安全领域的应用具有重要的科学意义。

通过22 ℃、25 ℃、28 ℃和31 ℃四个温度连续5代对食胚赤眼蜂进行处理，分析其寄生数、羽化率和雌雄性别等生物学指标。结果表明，食胚赤眼蜂单头雌蜂的寄生卵粒数、羽化率和雌雄性后代比例在不同温度处理下都存在显著差异，而在不同世代之间并未有显著差异，温度和世代对食胚赤眼蜂的生殖力和雌雄性后代比例存在显著的交互作用。重要的是，以25 ℃为对照对不同虫态的赤眼蜂进行30℃和35℃短暂高温处理，分析其寄生数、羽化率和雌雄性别等生物学指标。在短期高温处理后，卵态和幼虫态经历高温后性比变化较大，卵态和幼虫态对高温比较敏感。.高温（≥25℃）及继代处理可改变营孤雌生殖赤眼蜂的生殖方式及其体内Wolbachia 含量，且随着温度的升高以及处理时间的延长作用显著。即高温对营孤雌生殖赤眼蜂生殖方式改变程度与其体内Wolbachia 含量负相关，而Wolbachia 含量与宿主生殖方式正相关。且只有Wolbachia 滴度达到一定阀值，高温才能改变宿主生殖方式。在28 ℃处理下F1~F5代食胚赤眼蜂体内Wolbachia 的wsp基因核苷酸序列发生变化，主要是核苷酸的突变，表明温度可能影响共生菌表面识别蛋白来改变其与宿主互作。.首次报道豆芫菁可以被Wolbachia感染。通过MLST测序方法检测食胚赤眼蜂-米蛾，捕食螨-米蛾和赤拟谷盗-米蛾三个寄生、取食关系中的Wolbachia的遗传多态性，与wsp基因分析结果基本一致，表明Wolbachia 在宿主间未发生水平传播。. 采用生物信息学方法在全基因组水平鉴定短管赤眼蜂Argonaute蛋白基因家族，鉴定并命名6个TpAGO蛋白。EST表达分析发现TpAGO基因在昆虫的卵期、幼虫期、蛹期和成虫期均有不同程度的表达，而且靶向EST在同一物种中具有单一性别（雌性或雄性）表达特性，表明短管赤眼蜂TpAGO蛋白基因参与赤眼蜂性别及发育调控。. 研究内容为目前研究Wolbachia与宿主昆虫相互作用研究提供了理论参考，对揭示共生菌操控宿主生殖等热点理论具有重要指导意义。.