结膜吸吮线虫不同发育阶段差异表达基因及其功能分析

Thelazia callipaeda (T.cp) is frequently reported as being responsible for thelaziosis of humans, carnivores(dogs and cats) ,whose geographical distribution in Asian countries. Although T.cp is an underestimate parasitic nematode, and may seem to be scattered distribution, human thelaziosis is highly endemic in some areas in Asia, particularly in China. However, a lack of genomic information has restricted studies on the T.cp’s genes and its related function. Now, the research of T.cp is mainly focused on the morphology , life cycle and epidemiology. In this program, we will try to establish the animal model of T.cp based on its local epidemic in ZunYi city. Then, we utilized the Digital gene expression profiling (DGE) approach to examine the differenence expression genes (DEGs) and expression abundance in the dynamic process of developmental stages. These data will provide a strong foundation for gene function annotation and biological pathway enrichment analysis, which will helpful to excavate the significant difference expression genes and biological pathway that is critically important for key parasite functions such as establishment and maintenance of infection, development, reproduction, and survival in the host. A regulatory network will be established based on above results，and expression of candidate gene is validated by qPCR and RT-PCR methods. The purpose of this study is to explore the unique and common genes and their corresponding biological mechanisms that enable T.cp to grow and develop, to maintain their symbiotic association with the host, as well as the host strategies to counter parasitic infections.The results can not only enrich Thelazia callipaeda genomics information, but also offer support to deeply study.For example, some of these may be useful targets for vaccines or new drug treatments, and RNAi research for T.cp.

结膜吸吮线虫（Thelazia callipaeda，T.cp）是一种多分布于亚洲，寄生于人、犬、猫等宿主眼部的人兽共患寄生虫。T.cp作为一种较少见的寄生线虫，多散在分布，也存在局部流行。目前由于T.cp基因组信息缺乏，对其基因与功能的相关研究甚少，对T.cp的研究主要集中在生活史、形态学及流行病学。本研究利用结膜吸吮线虫病在贵州遵义市正安、绥阳等地区局部流行的特点，拟建立T.cp动物模型，利用数字基因表达谱分析技术检测T.cp不同发育阶段虫体的差异表达基因及其表达丰度，进行基因功能注释和生物通路富集分析，挖掘表达呈显著差异的基因和生长发育相关的重要生物通路，初步建立调控网络，并对候选基因进行表达验证。本研究旨在解析T.cp生长、发育及侵入不同宿主过程中的重要基因及功能模块，由此不仅可丰富T.cp基因组学信息，也为进一步筛选重要功能基因进行靶基因研究或RNA干扰等深入研究提供支持。

结膜吸吮线虫（T.cp）是一种人兽共患的眼部寄生线虫，多散在分布，也存在局部流行。由于T.cp基因组信息缺乏，对其基因与功能的相关研究甚少。本研究首先对已完成测序的结膜吸吮线虫基因组数据完成组装和注释，得到6333个基因，结合转录组denovo文库进行长转录组预测。公共数据库注释包括KOG、KEGG、GO等富集分析，专有数据库注释包括TCDB、PHI、CAZyme，其中PHI注释到基因数最多（1498）；蛋白序列注释到259个分泌蛋白，与此前认为寄生线虫基因中完全或较少存在CYPP450基因家族不同，注释到分属于19个基因家族的238个CYP450基因，主要集中在CYP5家族。进一步体外培养收集新生幼虫，喂饲法感染实验室自繁冈田绕眼果蝇（Amiota okadai），收集感染后24h后果蝇进行差异表达谱分析。通过对T.cp雌、雄虫和幼虫的差异基因表达谱分析显示，与生长发育、异源化合物代谢途径相关基因，以及与宿主互作、蜕皮及移动功能相关基因有显著差异。因此，我们重点研究高差异表达的细胞色素P450基因-CYP2B11基因，进行真核表达和功能验证。通过果蝇感染前后的差异表达谱筛选其影响感染的基因模块发现，许多基因都富集到免疫相关的Toll和IMD通路，初步绘制信号通路。分泌蛋白组中鉴定到一些具有抗原性分泌蛋白，其中有6个分泌型虫源MIF（Tcp-MIF），经差异表达谱筛选出一个高差异表达的虫源Tcp-MIF，其与人源MIF具有较高同源性。Tcp-MIF表达后功能验证发现，对小鼠和人巨噬细胞有免疫刺激效应,推测其可能通过细胞因子模拟作用调节宿主免疫反应，机制有待进一步研究。本项目通过解析T.cp生长、发育及侵入不同宿主过程中的重要基因及功能模块，不仅可丰富T.cp基因组学信息，也为进一步筛选出的重要功能基因进行寄生虫与宿主免疫互作或RNA干扰等深入研究提供支持。