西瓜枯萎病菌效应蛋白FonSIX6及其西瓜互作蛋白功能研究

Fusarium wilt in watermelon caused by Fusarium oxysporum f.sp.niveum is one of the most serious diseases in watermelon production. Wild germplasm PI296341 is an excellent gene resource for Fusarium wilt resistance. It is especially important to obtain genes of resistance diseases and to understand mechanism of resistance diseases..Several effector proteins to inhibit or promote plant resistance responses were secreted into xylem while interaction between Fusarium oxysporum f. sp. lycopersici and tomato plant, including SIX6(Secreted In Xylem) which was proved to be an effector protein existing in the watermelon specific Fusarium strain (Fon). In order to determine function of effector protein FonSIX6 in Fusarium oxysporum f.sp.niveum and its interacting proteins in watermelon we have cloned the gene of FonSIX6 and have constructed its deletion mutants. A yeast expression library have been constructed using root tissue of watermelon germplasm PI296341 induced by Fusarium oxysporum f.sp.niveum. 14 target interacting proteins have been obtained. On this basis, in this research item we prepare for:.1..Based of response on resistant and susceptible watermelon germplasm will be inoculated with deletion mutant strain, function of effector protein FonSIX6 to inhibit or promote plant resistance responses will be defined.. 2. Obtained 14 target interacting proteins during plant resistance responses will be analyzed and investigated, function of 1-2 genes about resistance disease will be verified. Location of interacting proteins in cell, and expression level of the gene induced by Fon will be researched. Target gene silencing will be down by virus-induced gene silencing. After watermelon germplasm was gene silencing, seedling were inoculated with deletion mutant strain and wild type strain, according to their phenotype, function of the gene during resistance disease will be comprehended. This study will provide theory foundations for Fusarium wilt control, advancing selection and breeding of new varieties.

枯萎病是西瓜最主要的病害之一。利用优异抗病种质资源获得抗病基因并明确其抗病机理显得尤为重要。镰刀菌和番茄相互作用时分泌几个效应蛋白进入木质部启动或抑制抗病反应，其中仅SIX6 (Secreted In Xylem)效应蛋白存在于西瓜专化型尖孢镰刀菌（Fon）中。为了明确FonSIX6蛋白及其与西瓜互作蛋白的功能，我们已克隆获得了FonSIX6基因并构建了其缺失突变体；构建了西瓜专化型尖孢镰刀菌诱导野生西瓜种质PI296341根部组织后的酵母表达文库并初步筛选获得14个相互作用靶蛋白。据此，本研究计划：1、根据抗、感枯萎病种质材料接种真菌缺失突变体后的抗病反应，明确FonSIX6蛋白是启动还是抑制抗病反应；2、对获得的14个靶蛋白进行分析，选择其中的1-2个抗病相关基因进行功能研究。 通过了解其在植物中的作用部位、是否相应病原菌诱导，以及病毒介导基因沉默后的抗病反应等，明确其在抗病中的作用

摘要：枯萎病是由西瓜专化型尖孢镰刀菌（Fon）引起的，是西瓜生产中最主要的病害之一。明确Fon-西瓜相互作用过程对理解其致病机理及防治枯萎病具有重要的意义。.目前，Fon存在三个生理小种0、1、2，PI296341-FR是抗三个生理小种的优异种质。根据报道，镰刀菌和番茄相互作用时分泌效应蛋白进入木质部启动或抑制抗病反应，这类蛋白统称为SIX（Secreted In Xylem）蛋白。通过对已知的SIX在Fon中筛选后发现，仅SIX6效应蛋白存在于Fon中（命名为FonSIX6）。为了明确FonSix6蛋白及其与西瓜互作蛋白的功能，进行了如下研究：.西瓜专化型尖孢镰刀菌存在三个生理小种0、1、2，本研究发现，FonSIX6存在生理小种0和1中，生理小种2缺失该基因。采用染色体步移的方法，获得了FonSIX6的侧翼序列。构建了Fon1的缺失突变体、互补菌株、Fon2互补菌株，通过培养发现，该基因不影响真菌的生长发育。RT-PCR监测发现，在真菌侵染三天后FonSIX6表达。通过接种Fon1野生型、缺失突变体、互补菌株、Fon2野生型、互补菌株后发现，缺失突变体的致病力明显增加，将FonSIX6基因导入到生理小种2后接种发现致病力下降，这些结果说明FonSIX6在镰刀菌-西瓜互作过程中起无毒因子的作用。以FonSIX6为诱饵，采用酵母双杂交的方法，在西瓜抗枯萎病材料PI-296341-FR中筛选获得了一个含有C4HC3结构域的相互作用的PHD类型蛋白，命名为ClC4HC3。.为了对该基因的功能进行分析，建立了西瓜的再生及筛选体系。构建了ClC4HC3的植物表达载体，通过农杆菌介导的方法转化到西瓜材料Crimson Sweet中。目前，已获得转化该基因的西瓜转基因植株，接下来将进行进一步抗病性分析。