烟草中识别极细链格孢菌蛋白激发子Hrip1的受体及其功能研究

The initiation of elicitor-induced plant immunity is a pivotal step in plant immune signal transduction.Protein elicitor Hirip1 (Hypersensitive response-inducing protein 1) was isolated from Alternaria tenuissima, which had been applied for a national invention patent (registration No. CN201110149203.3). Hrip1 can induce the system defense reaction in tobacco and enhance the ability to resist against TMV (tobacco mosaic virus). So far, the work of recongnition between the elicitor Hipr1 and receptor protein in tobacco had not been reported publicly. To uncover the receptor protein of Hirp1 in tobacco, Hirp1 protein was used as a bait to screen the cDNA library of tobacco. Our preliminary work shows that 25 candidate proteins can interact with Hrip1 by yeast two-hybrid system. In this project, bimolecular fluorescence complementation (BiFC) ,GST pull-down and Isothermal titration calorimetry (ITC) were applied to test the interaction with Hrip1 in vitro and in vivo. We would use the technology of virus-induced gene silencing (VIGS) and tobacco genetic transformation respectively to generate transgenic lines with silencing or over-expresson of receptor proteins in tobacco. Subsequently, these transgenic lines of tobacco were used for functional analysis of receptor proteins. Agilent tobacco oligo microarray would be used to analyze differential expression profiling for transgenic lines above. This research will provide theoretic foundation to understand the molecular mechanism of Hirp1-induced defense responses in plants.

植物受体识别蛋白激发子，是植物启动免疫信号转导过程中非常关键的步骤。蛋白激发子Hrip1是本实验室从极细链格孢菌（Alternaria tenuissima）中分离获得，具有自主知识产权(国家发明专利CN201110149203.3）。Hrip1能诱导烟草产生系统防御反应，增强烟草对烟草花叶病毒（TMV）的抗性。但是，烟草中的识别Hrip1的受体工作未见报道。前期工作中，以Hrip1为诱饵蛋白通过酵母双杂交系统筛选烟草cDNA文库，获得了25个互作蛋白。本项目拟采用双分子荧光互补（BiFC）、GST pull-down和ITC方法对互作蛋白进行体内和体外结合验证；运用病毒诱导基因沉默（VIGS）和转基因技术，研究互作蛋白在Hrip1诱导烟草抗病反应中的生物学功能；对VIGS沉默株和超表达植株采用烟草全基因组表达芯片进行表达谱差异分析，为深入揭示Hrip1诱导植物抗病的分子机制提供理论依。

真菌蛋白激发子Hrip1（hypersensitive response inducing protein 1），是病原真菌极细链格孢菌的培养基上清中纯化出的新型超敏反应诱导蛋白。Hrip1能诱导烟草产生了类似于超敏反应的坏死斑，增强烟草对TMV的系统抗性并且提高植物体内相关防卫基因的表达。.通过酵母双杂交实验，成功的在拟南芥中找到1个Hrip1阳性互作蛋白SGT1，用BiFC的方法，确定了Hrip1与SGT1在烟草细胞中特异性结合，与酵母双杂交结果一致，排除了假阳性可能。 SGT1 ( suppressor of the G2 allele of SKP1 ) 是植物R基因抗病信号途径中的重要调控基因。VIGS沉默了烟草SGT1后，植株明显矮化，株型紧凑，节间缩短。在VIGS烟草植株上注射TMV-GFP，通过观察UV光下荧光强度测定植株的抗病性，结果表明VIGS烟草植株对TMV的抗病性低于负对照植株。.为深入了解激发子Hrip1的生物学功能，我们将Hrip1克隆到35S驱动的双元载体pCAMBI1301中，转化农杆菌GV3101后通过浸泡法转化野生型拟南芥Col-0。通过分析3株单插入位点的Hrip1超表达转基因株系，发现Hrip1超表达转基因株系在长日照生长条件下开花时间相对于对照野生型Col-0提前7天，摘取叶片喂食甜菜夜蛾6天其体重相对于对照组减轻25%以上。为了揭示Hrip1提早植物开花时间和抑制甜菜夜蛾生长的作用机制，我们采用RNA-Seq技术比较Hrip1超表达转基因株系和对照组表达谱的差异情况。分析结果显示，Hrip1超表达转基因株系中有40个基因的表达量显著上调，有3个基因的表达量显著下调。通过Real-time PCR验证候选的差异基因表达水平结果与RNA-Seq的结果一致。对差异基因进行KEGG分析，发现差异基因主要参与RNA转运和降解、植物激素信号转导、植物的生理节律等通路。通过进一步分析差异基因，发现Hrip1调控植物开花时间提前，是由于直接或间接作用于成花因子FT而实现植物开花时间提早。Hrip1调控转基因植株抑制甜菜夜蛾幼虫的生长，是由于转基因株系中thionin家族基因和PR-6基因的表达量上调。