菲利普孢囊线虫HfSvG434效应蛋白功能研究

The wheat production was seriously threatened by cereal cyst nematodes (CCNs) in China. Of all the CCNs, Heterodera filipjevi virulence was much stronger and the yield loss was much higher than the Heterodera avenae. Our previous study showed the H. filipjevi novel effector HfSvG434 play a key role during the feeding site development and maintenance. The present research planning to use the in situ hybridization to determine where HfSvG434 gene are expressed, with the genome southern blot to detect HfSvG434 gene copy number variation and with the real time PCR to detect HfSvG434 gene expressed difference at different stage. Then cloned HfSvG434 effector genes and transiently expressed them in Nicotiana benthamiana, to detect HfSvG434 effector could whether induce cell death in N. benthamiana, with the subcellular localization assays to determine where HfSvG434 gene could be localized in the tobacco cell; and with the immunohistochemistry technique to detect where HfSvG434 gene could be localized in the syncytium cell. Moreover, combined the virus induced gene silencing (VIGS) with the dsRNA soaking interference biological technique, to determine the influence on the syncytium development and pathogenicity in the different resistant host after HfSvG434 gene expression inhibited. Thereafter, with the HfSvG434 gene overexpression and RNAi in the Barley, to determine the influence on the syncytium development and maintains. This study will promote our understanding of the process of wheat-H. filipjevi interactions, and the research achievement will help us to establish a sound foundation to the cyst nematode feeding cite establishment, and possess important theoretical for the wheat-H. filipjevi interaction. The research achievement will help us to establish the H. filipjevi study technology research platform.

禾谷类作物孢囊线虫（CCN）是我国小麦上重要的致灾病害。在CCN中，菲利普孢囊线虫比小麦禾谷孢囊线虫毒性更强，危害更大。前期研究发现菲利普孢囊线虫新效应蛋白HfSvG434与合胞体的形成和维持有关。本项目拟采用原位杂交，Southern杂交和荧光定量PCR检测技术，明确HfSvG434在线虫的组织定位，基因拷贝数和基因表达量差异分析；采用免疫组化分析和亚细胞定位技术，揭示HfSvG434在寄主合胞体内互作位点和植物细胞中的表达定位；通过VIGS和dsRNA干扰技术，解析HfSvG434对不同抗、感寄主致病力和合胞体发育的差异影响；采用HfSvG434基因过表达和植物介导的RNAi基因沉默技术，明确HfSvG434对合胞体形成和维持的影响。探索菲利普孢囊线虫与小麦互作的分子机理，将为解析孢囊线虫的合胞体形成机制研究奠定坚实的基础，为菲利普孢囊线虫致病分子机理提供理论依据和技术研究平台。

禾谷类作物孢囊线虫（CCN）是我国小麦上重要的致灾病害。在CCN中，菲利普孢囊线虫比小麦禾谷孢囊线虫毒性更强，危害更大。本项目采用原位杂交，Southern杂交和荧光定量PCR检测技术，明确了HfSvG434在线虫的组织定位主要位于亚腹食道腺；该基因在菲利普孢囊线虫中为高度保守的多拷贝基因，拷贝数为2；通过基因表达量差异分析，发现该基因在线虫侵染的四龄时期表达量最高，为合胞体形成和发育维持基因。通过烟草瞬时表达和小麦原生质体的亚细胞定位技术，揭示了HfSvG434在寄主合胞体内互作位点和植物细胞中的表达定位主要位于细胞核和细胞膜上。通过RNAi 浸泡、VIGS和dsRNA干扰技术，明确了HfSvG434基因沉默可以有效降低二龄幼虫的侵染数量，同时对抗、感寄主致病力和侵染后幼虫的发育均有促进作用，表明了该基因可以促进线虫侵染和合胞体的形成发育；采用HfSvG434基因过表达和植物介导的RNAi基因沉默技术，明确了HfSvG434转基因拟南芥对植株的形态发育具有抑制作用，对根系生长同样具有抑制效果。最后，我们利用酵母双杂交膜系统文库，共筛选到46个互作蛋白，经PCR检测，28个能够被检测到含有目的基因条带，测序及比对后，注释结果主要与解毒，蛋白质水解，氧化还原，控制细胞分裂等基因相关。研究结果为解析HfSvG434效应蛋白对孢囊线虫的合胞体形成机制和孢囊线虫致病分子机理的阐明提供了理论依据，为分子育种和抗性标记提供了数据支撑。截止目前，项目累计发表文章11篇（SCI论文4篇，北大核心3篇，国际会议论文2篇，中文会议论文1篇）；授权专利7项（发明专利2项，实用新型专利5项），参加大会报告1项。

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