芒果果实抗炭疽病相关基因的克隆及功能分析

The plant fungal pathogen Colletotrichum gloeosporioides (Penz) is the causal agent of mango fruit anthracnose during storage and transportation. Domestic and overseas reports have evidenced that there were great differences in disease resistance among different variety of mangos and suggested that disease resistance genes can be isolated from mango fruit. But till now, the molecular mechanism of mango fruit with resistance to anthracnose is still unclear. In our early study, we have accomplished mango fruit transcriptome sequencing of resistant varieties (Zill) and susceptible varieties (Tai Nong 1) when inoculated with C. gloeosporioides conidia. In this study, we will study fruit transcriptomes through RNA sequencing(RNA-seq) and compare the differential genes expression profiling between resistant and susceptible varieties at different time points after inoculation. To identify the differentially expressed genes with potential relevance in disease resistance, subsequent metabolic pathway analysis of the differential expressed genes, combinational analysis of small RNAs and the comparison with known resistance gene of other species are needed. Moreover, to analyses the candidate genes function, the candidate genes functional analysis will be conducted by qRT-PCR technology, or by over expression and gene silencing technology in Arabidopsis. The research result will be advantageous in uncovering mango fruit disease resistant molecular mechanism; It is hopeful to provide disease resistance genes resource and help establish stable basis in mango disease resistance breeding.

炭疽病菌是引起贮运期芒果果实腐烂的主要诱因，国内外报道不同品种的芒果对炭疽病的感病性存在较大差异，表明有抗病基因可以利用。但迄今为止，芒果抗炭疽病的分子机理还不清楚。前期工作我们已完成接种胶孢炭疽病菌后抗病品种'Zill'与感病品种'台农'的果实转录组测序。本项目仍以上述抗/感品种为材料，对接种后不同时间点的抗/感果实进行RNA-seq测序，通过横向比较抗病品种与感病品种基因表达谱、以及纵向比较相同品种接种前后不同时间点的基因表达谱，筛选差异表达的基因, 并对差异表达基因进行后续的代谢通路分析、与小RNA的联合分析、以及与其它物种已知的抗病基因比较等，寻找芒果抗炭疽病相关基因；并通过果实接种后实时荧光定量PCR检测候选基因的表达水平，和构建植物表达载体，对拟南芥同源基因进行抑制或过表达来研究候选基因功能。研究结果有利于揭示芒果抗炭疽病的分子机理，可为培育抗病新品种提供基因资源和奠定基础。

项目的背景.果实采后炭疽病每年对芒果产业造成巨大损失。研究报道表明，芒果树中存在抗病基因，值得开发应用。本课题通过转录组学等技术寻找芒果抗炭疽病相关基因，并和拟南芥同源基因比对，筛选抗病基因并研究其功能。.研究内容.以抗/感病芒果品种果实为材料，对接种后不同时间点的抗/感果实进行RNA-seq测序，通过比较抗病品种与感病品种基因表达谱、以及相同品种接种后不同时间点的基因表达谱，筛选差异表达的基因；再对差异表达基因进行后续的代谢通路分析、以及与其它物种已知的抗病基因比较等，寻找芒果抗炭疽病相关基因，并通过果实接种后实时荧光定量PCR检测候选基因的表达水平，及转化拟南芥，接种炭疽病菌，评价抗病效果。.重要结果和关键数据、科学意义.RNA-seq测序获得了芒果抗/感果实转录组数据，对抗病和感病样品基因表达差异分析发现，共有3671个基因上调或下调，选择了部分基因，进行Real-time PCR验证，发现与转录组表达结果一致。对Unigene所参与的代谢途径进行了具体分析，基本涵盖了植物防卫反应的各个途径。根据RNA-seq 数据库，利用 RT-PCR 和 RACE 技术，克隆和鉴定了芒果果实中 10 个抗病相关基因，包括两个病程相关基因 MiPR1 和 MiPR5，先天免疫途径的关键组分 MiFER1、MiCDPK、MiFRO，MiMPK3家族成员等。通过农杆菌介导的花序浸染法将 MiPR1 和 MiPR5转化野生型拟南芥植株，已经获得了 30 株转基因后代。接种转基因植株的抗病表型发现，与野生型拟南芥抗炭疽病能力相比，转MiPR1 和 MiPR5拟南芥植株似乎有一定抗性，重复实验和抗病机理正在进行中。.另外，我们还筛选自然界低毒抑菌化合物防治芒果采后病害，对外源物质作用机理进行了蛋白质组分析，以此开发了新型杀菌剂一款，在多种热带果蔬病原真菌室内毒性测定上有较好抑菌效果。