基于比较转录组分析的苹果轮纹病抗性相关基因筛选及其功能研究

Ring rot (Botryosphaeria dothidea) is one of the highly devastating diseases to apple in China. Although biological property of the pathogen, infection pathway, pathogenicity condition have been investigated, the defense mechanism of apple plant against B. dothidea and interaction between host and pathogen still remain unclear. In the present project, comparative transcriptome analysis apporach will be used to analyze the infected tissues of 'Fuji' and '95-76', susceptible and resisitant to ring rot disease,respectively, at the early stage of the disease in order to obtain transcriptome information of both host and pathogen. Differently expressed genes in 'fuji' and '95-76' responding to B. dothidea infection will be screened with non-infected tissues as control for excluding the interference of different host genetic background. The candidate resistant-related genes will be determined according to expression abundance and predicted gene fuction, and transformed into tobacco and apple plant to futher explore their roles playing in the defense responses to B. dothidea. At the same time, refering to the genome data of other pathogenic fungi in Genbank the pathogenic-related candidate genes will be determined from transcriptome information, and their fuction will be investigated using knock-out and genetic complement experiment. The aim of this project is to predict the signal pathway involved in the defense of apple plant against B. dothidea, and determine resistant-related genes of host and pathogenic-related genes of pathogen, which is the base of further work for cluciating the interaction mechanism between host and pathogen.

轮纹病是苹果主要病害之一，在我国危害严重。尽管对轮纹病病原的生物学特性，发病规律及侵染途径有一定的了解，但对苹果本身的防御机制及病原与宿主的互作机制研究相对缺乏。本项目拟利用比较转录组分析途径，对具有明显抗性差异的苹果材料'富士'和'95-76'在轮纹病病原侵染后发病初期的染病组织进行分析，以同时获取宿主和病原的基因转录信息。通过宿主发病组织与非侵染对照的转录组数据比较排除宿主遗传背景差异的干扰，筛选响应病原侵染的宿主差异表达基因，并根据基因表达丰度、预测的基因功能确定抗性相关候选基因，进一步转化模式植物和苹果分析基因功能；同时，参照已有病原真菌的效应子基因信息，确定病原致病相关基因的候选基因，并通过基因敲除和功能互补实验分析其功能。通过本项目的实施，拟初步确定参与苹果对轮纹病抗性的信号转导途径，确定宿主的抗性相关基因及病原的致病相关基因，为进一步深入研究病原与宿主的互作机制奠定基础。

轮纹病是苹果主要病害之一，在我国危害严重。尽管对轮纹病病原的生物学特性、发病规律及侵染途径有一定的了解，但对苹果本身的防御机制及病原与宿主的互作机制研究相对缺乏。本项目在转录组测序的基础上分析了苹果植物参与轮纹病防御基因，并对侵染与模拟侵染样本之间的差异表达基因进行分析，阐明了参与防御轮纹病病原侵染的相关信号转导途径。其中植物与病原互作的相关途径、芪类化合物/二芳基庚烷和姜酚生物合成途径、核糖体生物合成途径、类黄酮生物合成途径参与了苹果植物对轮纹病的防御反应，这些途径的关键基因表达受轮纹病病原菌Botryosphaeria dothidea诱导提高表达，而光合作用、淀粉和蔗糖代谢途径、甘油酯代谢途径在轮纹病侵染过程中受到抑制。对这些途径的重要差异表达基因功能进行分析，揭示其在轮纹病防御过程中的作用机制。证明MdXEGIP1基因参与了苹果植物对轮纹病病原侵染的防御反应，发现MdCNGC1基因是苹果对轮纹病防御反应的负调控子，鉴定了参与苹果对轮纹病病原识别的基因MdCERK1-2。同时利用农杆菌的遗传转化法(ATMT)构建轮纹病病原真菌B. dothidea的T-DNA插入突变体库，获得了960个插入突变体。从这些突变体中筛选46个致病相关突变体并鉴定了两个致病性相关基因。本项目为进一步深入研究轮纹病病原与苹果植物之间的相互作用机制奠定了基础。