南方根结线虫寄生致病新基因MiMsp40的功能研究

South root knot nematode (Meloidogyne incognita) is an obligate, sedentary, endoparasitic plant nematode. Its host range was more than two thousand plants. It was estimated annual lose causing by root knot nematodes will cost over 50 billion US dollar in the world. This plant parasitic nematode is called as one of the most destructive plant pathogen. Unfortunately, the environmental-friendly approach for management of this nematode is still lacking. Understanding of the function of parasitic genes will help to establish an important foundation for control of this nematode. In this research, we will using a series of techniques and methods such as in situ hybridization and immunohistochemistry technique, subcellar location in onion epidermis, real time PCR, yeast two hybridization, and Gst pull down and BiFC, plant transgenic technique, DNA microarray, in vitro and in vivo RNAi and rescue of mutants of interacting proteins of Arabidopsis thaliana to explore the function of pioneer parasitic gene designed MiMsp40 isolated from Meloidogyne incognita, we expect to obtain the gene expression organ, expressing differential of developmental stages of nematode, Screening and validation of the protein of Arabidopsis thaliana interacting with MiMsp40, investigation of the morphological changes and sensitivity of the plant of Arabidopsis thaliana over-expressed MiMsp40 to nematode infection, and gene expression differential of the transgenic Arabidopsis thaliana, and survey of plant pathogenesis after RNAi MiMsp40. We expect all our results will ascertain if the MiMsp40 of south root knot nematode has a role to regulate host plant metabolism or suppress immune reaction of plant,and then helping root knot nematode to establish parasitic relationship with its plant host. Our results will provide some basic information for further elucidating the molecular mechanism of parasitism and pathogenesis of root knot nematode. And the results will also pave some theoretical basis for screening new RANi target gene of nematode and working out a new strategy for control of root knot nematodes.

南方根结线虫是重要的专性内寄生植物病原线虫，可寄生二千多种植物，据估计，全世界因其危害造成的损失高达数百亿美元，被称作最具破坏性的植物病原物，目前仍缺乏安全有效防治措施，揭示线虫寄生致病基因的功能是防治线虫的重要基础。本研究拟利用原位杂交、发育表达模式分析、免疫组织定位和植物细胞瞬时表达、酵母双杂交、Gst pull down和BiFC、植物转基因和基因芯片及RNAi等技术明确该线虫新基因MiMsp40产物的表达部位及在不同虫龄的表达量差异，获得MiMsp40与拟南芥的互作蛋白，分析转MiMsp40基因的拟南芥其形态、线虫敏感性和基因表达变化，利用RNAi和拟南芥缺失突变体研究其功能，明确该新基因是否具有调控植物代谢或抑制其免疫反应、促进线虫与植物建立寄生关系的功能；研究结果将为进一步阐明和完善根结线虫致病分子机理提供重要数据，为寻找防治线虫新靶标及制定防治新策略提供依据。

南方根结线虫是专性内寄生植物病原线虫，可寄生近2000种植物，据估计，全世界因根结线虫危害造成的损失高达500亿美元，被称作最具破坏性的植物病原物，目前仍缺乏安全有效防治措施，揭示线虫寄生致病分子机制是防治线虫的重要基础。本研究的主要研究内容包括：1）MiMsp40基因的遗传进化和表达规律研究；2）MiMsp40基因与寄主植物的相互作用研究；3）利用RNAi和拟南芥互作基因突变体，研究MiMsp40基因的功能。经过几年的研究，取得了明显进展，利用原位杂交技术明确了MiMsp40基因在南方根结线虫腹食道腺中表达、定量PCR分析表明其在寄生后的2龄幼虫中表达量较高；植物体内RNAi干扰该基因后线虫的致病能力明显下降，而在拟南芥中超表达该基因后导致植物根变长且转基因植物对线虫更敏感，应用烟草瞬时表达系统和胼胝质沉积技术研究表明该蛋白可抑制寄主的PTI免疫反应；应用酵母双杂交、免疫共沉淀和双分子荧光互补技术发现并验证MiMsp40蛋白与拟南芥的Hspro1和STZ蛋白存在互作并确定了互作关键区域，利用转录组学分析过表达MiMsp40拟南芥植株发现其在茉莉酸类（JAs）合成及信号传导通路上的重要基因明显下调，代谢组学分析发现转基因植物中茉莉酸甲酯的含量明显下降，而回补该激素后过表达植物对线虫的敏感性下降。烟草表达系统显示STZ负调控JA合成关键基因LOX3。综合上述结果表明该基因具有抑制寄主植物免疫反应、通过与Hspro1和STZ蛋白互作、干扰植物茉莉酸合成从而促进寄生关系建立，结果为阐明根结线虫调控植物的代谢过程和抑制植物免疫反应提供了重要数据，为寻找防治根结线虫新靶标并制定防治新策略提供了新途径。在项目执行期间，获得了北京市农业推广技术一等奖（第2名）和国家发明专利各1项、申请了发明专利1项，发表了SCI论文2篇，培养了博士后和博士研究生4名。承办或协办了第12、13届全国植物线虫学术讨论会，并应邀在会上作大会报告，扩大了本研究的学术影响力。