基于组织学和转录组学对甘蔗宿根矮化病的致病机理解析

Sugarcane ratoon stunting disease is the worldwide disease. In this research, tissue culture sugarcane without lxx was as study object. GFP-Lxx mutant was inoculated in sugarcane, and the infecting process was surveyed under fluorescence microscope. In effect interaction between the Lxx and sugarcane, differential gene expression profile of Lxx were acquired using the second-generation high throughput sequencing method. The sequencing results were aligned with KEGG and COG protein database, and noted the protein function and the pathway, also known differential expression gene of lxx were participated in which metabolic pathway and signal transduction pathway. The key pathogenic genes were cloned using race and genome walking method and analyzed with bioinformatics software. Function of pathogenic genes were study using the southern blot, northern blot and real-time PCR. Lxx pathogenicity were studied in this project not only in histology, but also in transcriptome. And the main metabolic pathway and signal transduction pathway were identified in connection with pathogenicity, and gained the proprietary intellectual property rights of pathogenic genes. It not only laid the foundation for clarifying the pathogenic mechanism of sugarcane ratoon stunting disease, but also provide the technical support for selecting the resistant avrieties of sugarcane. This project has important significance in theory and in practice.

甘蔗宿根矮化病是世界性的甘蔗细菌病害。本项目以不带Lxx甘蔗组培苗为材料，利用表达GFP基因的Lxx突变菌株接种研究Lxx侵染的组织学过程；通过转录组测序法高通量分析与甘蔗互作过程中Lxx基因差异表达谱，通过KEGG和COG等蛋白质数据库进行比对，获得蛋白质和pathway注解；确定差异表达基因参与主要代谢途径和信号转导途径；筛选Lxx致病关键基因，通过RACE和Genome walking技术获得致病关键基因,通过生物、信息学和Southern分析基因结构，通过定量PCR和Northern杂交，对致病关键基因功能进行解析。以上研究不仅从组织学方面解析Lxx致病性，更直接深入到转录组水平，确定Lxx致病的信号转导途径和代谢途径，并获得具有自主知识产权的致病基因。这不仅为阐明甘蔗宿根矮化病的致病机理奠定基础，也为今后甘蔗宿根矮化病的防治和抗病品种选育提供技术支撑，具有重要的理论和实践意义。

甘蔗宿根矮化病（Ratoon stunting disease,RSD）是世界性的细菌性病害。本项目以Badila为材料，人工接种病原菌研究Lxx侵染的组织学过程；通过转录组测序法高通量分析与LXX互作过程中甘蔗获得基因差异表达谱，确定差异表达基因参与主要代谢途径和信号转导途径，并通过q-PCR进行验证，具体结果如下：.1、.从染病的Badila中成功分离、培养LXX病原菌；.2、.世界上首次利用自主研发标记LXX抗体的免疫磁珠富集LXX病原菌,使得从侵染的甘蔗汁中直接分离LXX用于转录组测序成为可能；.3、.接种LXX的Badila茎和叶片组织冰冻切片原位PCR结果显示lxx主要分布在蔗茎的木质部、韧皮部，主叶脉中的木质部、近上表皮处维管束鞘细胞，叶肉组织的维管束鞘细胞壁以及周围的叶肉细胞；病理切片观察到叶片维管束鞘外侧“花环”叶肉细胞明显壁薄且皱缩不规则,蔗茎维管束中木质部、韧皮部细胞散裂明显，这两个结果相互佐证从组织学方面解析了Lxx致病性；.4、.染病蔗茎LXX定量分析与病理切片分析结果表明蔗茎维管组织中的韧皮部和木质部的损坏程度与Lxx菌定植密度呈正相关，茎节组织结构与茎间的差异推测是阻碍lxx菌由基层向上慢慢迁移富集的原因；此外，染病的蔗茎芽轴、芽原基染色较深，推测其作为甘蔗无性繁殖传播LXX的原因之一；.5、.对LXX胁迫条件下甘蔗不同时期转录组开展对比研究结果显示差异表达基因按功能主要参与植物激素信号转导、苯丙氨酸代谢、苯丙素生物合成、碳代谢、光合作用、光合生物固氮、植物与病原菌互作等途径；SA信号转导启动激发PR基因表达使甘蔗产生系统获得性抗性；苯丙氨酸代谢途径的激活响应lxx胁迫，启动防卫系统；光合生物固碳途径的关键基因表达普遍下调，结合之前的LXX定位和病理切片结果，从组织学和转录组学推测Lxx破坏了甘蔗光合作用的正常运作，使蔗茎表现矮化病症。