尖孢镰刀菌浸染西瓜过程中效应蛋白FonSIX6与非编码RNA互作机制研究

The defense mechanism of RNA interference was used in Arabidopsis and other plants to resist Fusarium oxysporum. Pathogens often interact with non-coding RNAs to inhibit host RNA silencing to accelerate the infection process. This shows that the interaction between the effector protein and RNAs is very important for the regulation of immune system..The effector Fonsix6 is a avirulence factor of Fusarium oxysporum f.sp.niveum, and it is a key factor affecting pathogenicity. Bioinformatics analysis shows that it has RNA binding motif. The study of the interaction between the effector FonSIX6 and non-coding RNA is very important for the analysis of the regulatory network and the disease resistance mechanism of watermelon..This project will carry out the following research: .1, It will be determined whether the defense mechanism of RNA interference was used in watermelon to resist Fusarium oxysporum f.sp.niveum, and whether the effector protein FonSIX6 has the ability to inhibit RNA silencing in host watermelon. .2, RNAs in combination with FonSIX6 protein and its sequence information were obtained by High-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP) method; These RNA target sequencing information will be analysised using GO metherd. After Confirmed by RNA co immunoprecipitation method, the non-encoding RNA that can bind FonSIX6 through will be Conducted functional verification by over expression, RNA interference, subcellular localization and other methods to understand its function in watermelon resistant response, this will be the basis and help for disease resistant breeding and disease prevention.

拟南芥等植物在抵抗镰刀菌危害时使用RNA干涉防御机制, 有些病原菌通过效应蛋白与非编码RNAs的互作抑制寄主RNA干涉来加速侵染过程，这说明效应蛋白与RNAs的互作对抗病免疫系统调控十分重要。效应蛋白Fonsix6是尖孢镰刀菌的无毒因子，是影响致病性的关键因子，生物信息学分析表明，它具有RNA结合的基序。研究它与非编码RNA的互作关系对解析西瓜抗病反应的调控机理至关重要。本项目将进行：1, 探明西瓜对镰刀菌的防御机制是否采用RNA干涉,效应蛋白FonSIX6是否具有抑制寄主RNA干涉的能力。 2，采用HITS-CLIP方法筛选获得与FonSIX6蛋白结合的RNAs及其序列信息，对这些RNA靶点测序信息进行GO分析，利用RNA免疫共沉淀等方法验证后，对确实能够与FonSIX6结合的非编码RNA通过超表达、RNA干涉、亚细胞定位等方法了解其在西瓜抗病反应中的功能，为抗病育种和病害防治提供依据。

①本项目对GoldCLIP方法进行了优化，用GoldCLIP技术分别在西瓜和烟草植株中筛选到了与FonSIX6蛋白结合的西瓜和烟草中的RNAs序列8条，其中包括PHD结构域有关序列。在NCBI基因组数据库中进行同源比对，其分别与野生稻、菠萝、甜瓜、烟草、玉米基因组序列高度同源。对这些RNA靶点测序信息进行GO分析，其序列对应蛋白分别与PHD结构域E3连接酶、RNA聚合酶sigma因子、吩嗪合成调控蛋白(PBR)高度同源。.② 采用酵母双杂交的方法筛选到与FonSIX6互作的西瓜PHD结构域蛋白ClPHD23。综上两方面的结果，与FonSIX6蛋白互作的非编码RNA，以及与FonSIX6蛋白互作的蛋白都与PHD结构域蛋白有关，选择西瓜PHD结构域蛋白ClPHD23开展功能研究很有必要。.对ClPHD23基因进行瞬时超表达，并采用病毒诱导的基因沉默（VIGS）的方法以及采用创建ClPHD23基因过表达和敲除材料，研究西瓜ClPHD23在枯萎病抗病响应中的功能，证实西瓜ClPHD23正调控西瓜枯萎病的抗性。.开展了ClPHD23基因的亚细胞定位、时空表达等基本特征研究，ClPHD23基因被定位在细胞核上。采用VIGS的方法对ClPHD23基因在西瓜抗旱中的作用也进行了研究，研究发现ClPHD23基因被干涉，植株抗旱能力下降。.③开展了西瓜抗逆相关基因SOD和NCED对枯萎病菌侵染的响应机制研究.采用生物信息学手段分别对ClSOD和ClNCED基因进行分析，对它们基因结构、进化关系、蛋白结构、亚细胞定位、启动子顺式作用原件及表达模式等进行分析。并利用荧光定量 PCR分别分析了西瓜ClSOD和ClNCED基因的时空表达模式及其在抗感材料中的表达模式。