家蚕类海龟蛋白BmTLP参与微孢子虫侵染及其功能研究

The spore wall protein (SWP26) plays a specific role in spore adherence to host cells during invasion, and participates in the infection of Nosema bombycis to silkworm. In our previous study, it was found that the turtle-like protein of Bombyx mori (BmTLP, cell adhesion molecule) was determined to interact with SWP26 of N. bombycis via the use of co-immunoprecipitation. To further investigate the function of BmTLP involved in the invasion of N. bombycis to the silkworm, rapid amplification of cDNA ends (RACE) was used to obtain the full-length of BmTLP cDNA sequence. Quantitative real-time polymerase chain reaction (qRT-PCR) was employed toanalyze expression charateristics of BmTLP in infection and non-infection silkworm in different tissue at different time. Further, analysis of the host cell adhesion and infectionwas performed todetermine the influence of BmTLP on the spore adherence and invasion of N. bombycis. In addition, screening and identification of the interactive silkworm protein with BmTLP and RNAinterference were performed to resolve its signal pathways in the response of silkworm to N. bombycis, and its biologicalactivity. The results will lay a foundation for further understanding on the molecular mechanism of interaction between N. bombycis and silkworm, enrich the biological function of the insect turtle protein and fill the blank of the research on the silkworm turtle protein.

家蚕微孢子虫孢壁蛋白SWP26参与了家蚕微孢子虫对宿主细胞的粘附过程，并影响孢子对宿主细胞的侵染。我们前期通过免疫共沉淀实验，筛选到一个与SWP26有相互作用的细胞粘附分子-家蚕类海龟蛋白BmTLP。为了进一步研究BmTLP在家蚕微孢子虫侵染宿主过程中所起的作用及其功能，本项目拟采用RACE技术克隆获得BmTLP基因的全长cDNA，通过荧光定量PCR技术分析健康与患微粒子病家蚕在不同组织不同时间该基因的时相表达特征；然后通过宿主细胞粘附与感染分析，明确BmTLP对家蚕微孢子虫粘附与侵染的影响；最后通过筛选鉴定家蚕细胞中BmTLP的互作蛋白与RNA干扰实验，解析其在家蚕微孢子虫感染过程中可能介导的信号途径以及该蛋白的生物学活性。研究结果将为深入了解家蚕微孢子虫与家蚕之间的相互作用分子机制奠定基础，并可丰富昆虫海龟蛋白的生物学功能，填补家蚕海龟蛋白研究领域的空白。

家蚕是鳞翅目昆虫的模式生物，是对人类贡献最大的经济昆虫之一。细胞粘附分子是一类位于细胞膜表面的糖蛋白分子，与细胞发育、炎症反应、免疫应答等密切相关。本项目通过克隆前期筛选到的细胞粘附分子家蚕类海龟蛋白—BmTLP的cDNA，发现BmTLP基因的mRNA前体通过不同的剪接方式可产生3种剪接异构体：Bmtutl-464、Bmtutl-519与Bmtutl-810，这3种剪接体在家蚕不同组织与不同发育时期的表达特征存在较大差异；当家蚕微孢子虫感染时，与其它2种家蚕类海龟蛋白亚型相比，Bmtutl-519基因的相对表达水平变化最为显著，亚细胞定位发现Bmtutl-519在BmN细胞质和细胞膜中均有分布，可以以细胞表面受体或调制因子的方式参与家蚕微孢子虫的感染过程；通过稳定细胞系构建、孢子粘附与家蚕细胞感染分析，证实Bmtutl-519在一定程度影响了家蚕微孢子虫对宿主细胞的感染。在酵母双杂交中项目组筛选到一个与Bmtutl-519具有相互作用的家蚕伸展蛋白BmELP，当家蚕微孢子虫感染时可引起BmELP基因转录水平的上调，尤其在细胞水平更为明显。本项研究揭示了Bmtutl-519在家蚕微孢子虫侵染宿主细胞过程中的生物学功能以及可能参与的调控途径，已在TOP期刊《Developmental and Comparative Immunology》上发表，研究结果对了解家蚕微孢子虫与家蚕之间相互作用分子机制奠定了基础。2018年获得云南省科技进步奖三等奖1项。