镉污染对泽兰实蝇雄性附腺蛋白及功能基因的胁迫研究

Procecidochares utilis，as an introduced host specific stem gall fly of a invasive weed -- Eupatorium adenophorum，which was safe to other plants, its control to E. adenophorum has showed the obvious effect. However, preliminary research suggested that P. utilis was threatened by contaminants in mining area. Preliminary research suggests that Cadmium(Cd) stress can influence fecundity, mating effect, and cell structure of reproductive organs of P. utilis, accordingly reduced the effect of P. utilis for controlling E. adenophorum. This project intends to start from the result of proteome analysis and In silico expression profile, selects the differentially expressed genes which up-regulated and down-regulated and associated with reproduction as the object of this study, and carries on gene cloning and expression. And we also plan to analysis the expression differences of gene in the reproductive organs by real-time fluorescent quantitative detection, locate its transmission path in the mating process by Western blot, and use RNA interference to make clear the biological function of gene. The results of this project will lay a foundation for elucidate the reproductive injury mechanism of P. utilis caused by cadmium stress, and will provide experimental and theoretical basis for research biological mechanism of heavy metal detoxification; and also and will provide theoretical guidance for laying feasibility method to increase biocontrol efficiency for control harmful species.

泽兰实蝇专性寄生入侵性杂草紫茎泽兰，对其他植物安全，且对紫茎泽兰的控制作用明显。但是泽兰实蝇在矿域生境中经常受到重金属的威胁，前期的研究工作表明，镉胁迫影响到了泽兰实蝇的生殖力、交配效应以及生殖器官的细胞结构，从而降低了泽兰实蝇对紫茎泽兰的防治效果。本项目拟结合蛋白质组和数字基因表达谱分析结果，选定对镉反应为上下调的与生殖相关的差异表达基因作为研究对象，对其进行基因克隆与表达，采用实时荧光定量分析方法检测其在各生殖器官中的表达差异，采用免疫印迹法探明其在交配过程中的传递路径，并采用RNA干涉的方法明确基因的生物学功能。本项目的研究结果将为阐释重金属镉胁迫对泽兰实蝇的生殖毒性损伤机理奠定基础，为生物对重金属解毒相关机制研究提供重要的实验和理论依据；也为提高重金属污染区外来有害物种生物防治效率可行性方法的制定提供理论指导。

泽兰实蝇是入侵性杂草紫茎泽兰的专性天敌，但其在生境中经常受到重金属的威胁，造成其寄生率和生殖能力的下降，从而降低了泽兰实蝇对紫茎泽兰的防治效果。本项目利用高通量测序技术平台Illumina对泽兰实蝇受镉胁迫和非胁迫条件下的雄性附腺样品进行转录组测序。对雄性附腺的Unigene进行功能注释、同源性和代谢通路分析，包括与NR、Swiss-Prot、KEGG、COG、KOG、GO等数据库的比对，共获得52743条Unigene的注释结果。进行基于Unigene库的基因结构分析，获得了SSR标记并进行了CDS预测和SNP分析。受镉胁迫后有150个基因的表达水平发生改变，其中80个上调，70个下调，并进行了模式聚类、功能注释以及富集性分析；对3个差异基因进行了定量分析,金属硫蛋白Mnt1基因在成虫中表达量高，在镉胁迫下有上调的趋势，特别是在消化道和雄性附腺中；细胞色素P450基因在幼虫中表达量高，在镉胁迫下有下调趋势，在消化道、马氏管、雄性附腺中的趋势更明显；热激蛋白Hsps基因在成虫期表达量最高，低剂量镉处理后表达量下调，高剂量处理反而有上升趋势。另外，为了更好的了解雄性附腺受镉胁迫后的响应机制，我们还进行了精巢和消化道的转录组测序作为对照研究。本项目已发表文章5篇，其中SCI1篇，授权专利1项，培养研究生2人。