大豆孢囊线虫取食管形成相关基因(Hg-FT)的功能及作用机制研究

The soybean cyst nematode (SCN, Heterodera glycines) is a worldwide distributed pathogen of soybean, it caused significant economic yield losses and potential threaten to soybean production and food security in China. There have been several investigations characterizing the size limitation of molecules taken up through the feeding tube，unfortunately, the size cut-off for molecular uptake into SCN, has never been determined. In previous studies，we found that 28kDa GFP was observed to be taken up and a putative feeding-structure genes Hg-FT were identified from transcriptomic data of soybean cyst nematode. In this study, we plan to study the uptake of variously sized fluorescent proteins expressed in transgenic roots into H. glycines at different developmental stages, we investigate the biological function of Hg-FT gene family using HIGS and over-expression. The action site of Hg-FT in plant cell will be tested by immunodetection and subcellular localization, identify the physically interact proteins of Hg-FT by yeast two-hybrid system and confirm the interaction between host interact proteins and Hg-FT by BIFC and GST Pull Down, determine the molecular mechanism of host interact proteins via CRISPA and over-expression. The results will supply evidences to understand the interaction molecular mechanism between SCN and host, supply the new evidences for the structure of feeding tube and it is very important beneficial to make decision control strategies.

大豆孢囊线虫病是世界范围内的大豆毁灭性病害，严重威胁大豆的生产安全。植物线虫取食管对吸取的食物分子量有严格的限制，但SCN所能取食的分子量特征及取食管形成机制尚未见报道。在前期研究中，初步明确了SCN能够取食大于28kDa的GFP，并从中筛选出与取食管形成密切相关的Hg-FT基因。本研究通过发根系统表达一系列的荧光蛋白，明确SCN在不同发育阶段所能取食的最大分子量；采用HIGS和过表达技术明确Hg-FT家族的生物学功能，采用免疫组织定位和亚细胞定位技术明确Hg-FT的作用位点，采用酵母双杂筛选Hg-FT的互作蛋白，采用BiFC和GST Pull Down技术进行互作验证，采用CRISPA和过表达技术明确互作蛋白的生物学功能，从而解析SCN取食管的分子筛功能及Hg-FT效应蛋白的生物学功能。其研究结果为明确SCN的取食管形成提供直接证据，为利用生物技术控制大豆孢囊线虫奠定了坚实的基础。

大豆孢囊线虫病是世界范围内的大豆毁灭性病害，严重威胁大豆的生产安全。植物线虫取食管对吸取的食物分子量有严格的限制，但SCN所能取食的分子量特征及取食管形成机制尚未见报道。在本项目的资助下，我们确定大豆孢囊线虫能够取食27kDa mCHERRY和26.7kDa GFP、38kDa 和53kDa荧光蛋白，接种后12天，对于38kDa的荧光蛋白的取食率为18%，对于53kDa的荧光蛋白的取食率为9%，从而确定大豆孢囊线虫最大能够取食53的荧光蛋白。在此基础上，我们从大豆孢囊线虫中分离克隆出2个Hg-FT的候选基因33E05和34B08。亚细胞定位表明，33E05和34B08定位于细胞膜，qPCR表明，2个基因在大豆孢囊线虫寄生阶段大量表达。采用体外和体内RNAi分析结果表明：33E05和34B08转录水平在三个时间处理中下降明显，但接种后第8天和30天线虫侵染情况无显著性差异。采用异源表达系统,33E05和34B08过表达后，线虫侵染率无显著变化，但线虫发育明显加快。33E05和34B08体内RNAi介导基因沉默后，线虫发育明显延迟。采用酵母互作验证、荧光双分子互补和GST-pull down验证，明确了34B08-SP和ATD2-03互作可与互作，加速线虫的发育。发表研究论文6篇，其中SCI论文3篇，英文会议摘要1篇，获得黑龙江省科技进步二等奖1项。