木薯叶片应答细菌性枯萎病病原菌的基因表达谱构建及抗病相关基因鉴定

Cassava (Manihot esculenta) is a starchy root and a major tropical food crop in Africa, Latin America and Asia. Cassava, as important as it is however is susceptible to many pathogens that decrease yields. More than thirty bacterial, fungal, viral, virus-like, mycoplasma and nematode agents had been reported to attack cassava. Xanthomonas axonopodis pv manihotis (Xam) is a causative agent of cassava bacterial blight (CBB) which is considered as a major biotic constraint for cassava production in all cassava-cultivating regions of the world. The most appropriate approach to control bacterial blight is by growing resistant cultivars, as element of an integrated control system Genotypes with different levels of resistance were reported. This resistance is thought to be polygenic and additively inherited. Recently, the cassava-Xam interaction has been described as pathotype cultivar specific.The defense genes are expressed during different genotypes. Changes in gene expression are important for activation of defense mechanisms and transcriptional activation or repression of genes has been reported in several plant-pathogen interaction systems. Genomic-scale methods are revealing a complex web of signaling cascades involved in plant defense response. Transcript profiling plays an important role in annotating and determining gene functions, and different methods have been developed to study plant-pathogen interactions such as differential display, serial analysis of gene expression (SAGE). The recent development of RNA-Seq is a recently high-throughput sequencing method that uses deep-sequencing technologies to produce millions of short cDNA reads. It has long been recognized as an efficient method for gene discovery and remains the gold standard for annotation of both coding and non-coding genes. .In this study, screening cassava genotypes for resistance to cassava bacterial blight was performed by observing symptom development in our field under strong disease pressure. The resistant and susceptible cultivars were screened. The transcriptome and digital expression profile would be carried out using Illumina RNA-Seq technology. The differentially expressed genes of cassava leaf response to Xam would be identified through comparing the difference between the resistant and susceptible cultivars. The full-length cDNA would be isolated by RACE and other technology. The function of the candidate genes will be verified through biochemical analysis, transgene and other means. The project will be implemented to obtain the cassava disease resistance gene expression profiles, a number of cassava disease resistance gene would be identified from the genomic level. This work begins to reveal potential molecular metabolism for cassava disease resistance, the novel genes and functional verification will provide gene resources for cassava resistance breeding.

木薯是热带地区重要的粮食作物和能源作物，随着国际油价高涨，国内外木薯市场供不应求。生产上细菌性枯萎病是限制木薯产量的重要生物制约因素之一。但至今为止，木薯抗细菌性枯萎病的分子机理还不清楚，抗病相关基因资源的报道缺乏，防控技术研究基础薄弱，这已成为木薯产业持续健康发展亟待解决的现实问题。.课题组前期已从100份木薯种质资源中筛选出高抗和高感细菌性枯萎病的品系。本项目拟采用第二代高通量测序技术对抗、感木薯品系受病原菌浸染后的转录组和基因表达谱进行分析，通过两次差异对比，筛选木薯应答病原菌相关基因，构建木薯抗病差异基因表达谱。经RACE等技术分离全长基因，并通过生化分析、转基因等手段进行候选基因的功能验证。项目的实施将获得木薯抗病差异基因表达谱，从基因组水平识别一批抗病相关基因，为木薯抗病分子机理的解析提供基因资源和理论基础；新基因的获得及功能验证将为木薯抗病育种提供基因资源。

木薯是热带地区重要的粮食作物和能源作物，随着国际油价高涨，国内外木薯市场供不应求。生产上细菌性枯萎病是限制木薯产量的重要生物制约因素之一。但至今为止，木薯抗细菌性枯萎病的分子机理还不清楚，抗病相关基因资源的报道缺乏，防控技术研究基础薄弱，这已成为木薯产业持续健康发展亟待解决的现实问题。植物先天免疫反应是近年来植物抗病分子生物学研究的前沿领域，调控先天免疫信号传导通路的关键基因表达有望获得具有广谱抗性的新种质。.项目组根据模式植物先天免疫研究结果结合木薯基因组数据，从木薯基因组中分别候选了植物先天免疫反应关键节点的调控基因，包括4个类受体蛋白激酶（FLS2、BIK1、CERK1、EFR）、MAPK级联反应基因家族、ERF基因家族、RBOH基因家族以及下游mark基因等，利用生物信息学手段对这些基因的表达谱、基因结构、系统进化、染色体复制与扩张等进行了分析，明确了这些基因的基本生物学特征；采用实时定量PCR对这些基因应答病原物刺激的表达模式进行了分析，根据分析结果候选了MeBIK1、MeMAPK1、MeRbohA基因。开展了亚细胞定位、拟南芥中的功能验证以及转基因木薯功能鉴定等工作，明确了调控这2个基因的表达能显著提高转基因植株的抗病性。酵母双杂交结果表明，木薯PTI反应信号通路可能与拟南芥是保守的。为进一步研究木薯与CBB互作的分子机制，项目组对受到Xam浸染前后的差异基因表达谱进行了测序，获得了一批差异表达基因。.通过项目的实施，项目组建立了较系统的木薯抗病分子研究体系。原生质体瞬时表达体系、木薯遗传转化体系的构建为木薯功能基因组学的开展、转基因抗病育种工作提供了技术平台。另外，通过病原菌资源的收集、种质资源的抗谱鉴定等工作，课题组对生产上木薯病害的发生情况、基本规律有了更深入的认识，明确了木薯抗病种质的早期筛选指标，为木薯抗病分子育种提供了可用的基因资源和种质资源。另外，项目实施过程中凝聚了团队，为木薯研究储备了人才。