向日葵中锈病抗性相关microRNA的挖掘

Sunflower rust（Puccinia helianthi Schw.）is prevalent in sunflower growing regions in China and many other countries. In practice, breeding and the rational utilization of disease-resistant varieties has been shown to be the most effective, most economical and most environmentally sound method for controlling sunflower rust. Exploration of different kinds of resistance genes is a premise for resistance breeding. MicroRNAs (miRNAs) are a type of small non-coding RNA in eukaryotic organisms which play very important roles in plant defense responses. In this study, miRNAs will be surveyed by deep sequencing technique and the significantly different miRNAs will be identified by comparison of miRNA expression differences between resistant and susceptible materials using stem-loop RT-PCR and Northern blotting. The targets of miRNAs will be predicted and identified by bioinformatics tools and degradome sequencing. The target genes of miRNAs and regulation method between miRNAs and target genes will be analyzed by quantitative PCR, 5’RLM-RACE technique, and constructing the in vivo transient validation system of tabacco. Study on biological function of genes will help understand the role of miRNA in plant resistance pathways. The molecular regulation mechanism of miRNA in plant disease resistance will be comprehensively analyzed. Exploration of microRNAs and their targets engaging in the resistance interaction between sunflower and rust has important theoretical and practical values to lead to sunflower genetic improvement.

向日葵锈病（Puccinia helianthi Schw.）是国内外向日葵栽培地区普遍发生的病害，抗病品种的选育和推广是目前防控该病害最为安全、经济有效的手段。寻找各类抗病基因是抗病育种的前提。MicroRNA是真核细胞中一类非编码小RNA，在植物防御反应中发挥着重要作用。本研究通过高通量测序研究 miRNA 在抗、感病材料之间的表达差异，并结合stem-loop RT-PCR和Northern blot技术鉴定出显著性差异的miRNA；采用生物信息学及降解组测序对miRNA靶基因进行预测及鉴定，利用荧光定量PCR、5’RLM-RACE技术及构建烟草的瞬时表达体系验证miRNA下游靶基因和对其的调控方式；通过生物学功能研究明确 miRNA 在植物抗病途径中的角色。综合分析出基于miRNA的抗锈分子调控机理，挖掘抗锈miRNA资源，对指导向日葵遗传改良具有重要的理论意义和实践价值。

向日葵锈病（Puccinia helianthi Schw.）是国内外向日葵栽培地区普遍发生的病害，抗病品种的选育和推广是目前防控该病害最为安全、经济有效的手段。寻找各类抗病基因是抗病育种的前提。MicroRNA是真核细胞中一类非编码小RNA，在植物防御反应中发挥着重要作用。本研究通过高通量测序研究miRNA在抗、感病材料之间的表达差异，并结合stem-loop RT-PCR和Northern blot技术鉴定出显著性差异的miRNA；采用生物信息学及降解组测序对miRNA靶基因进行预测及鉴定，利用荧光定量PCR、5’RLM-RACE技术及构建烟草的瞬时表达体系验证miRNA下游靶基因和对其的调控方式；通过生物学功能研究明确miRNA在植物抗病途径中的角色。综合分析出基于miRNA的抗锈分子调控机理，挖掘抗锈miRNA资源，对指导向日葵遗传改良具有重要的理论意义和实践价值。.通过高通量测序技术及生物信息学分析方法，在向日葵上预测筛选到10个差异表达的新microRNA（miRNA）可能与锈病抗性相关。qRT-PCR的检测结果与前期高通量测序结果80%相一致。利用miRanda软件预测靶基因，10个miRNA共预测到117个靶基因，其中86个靶基因获得其生物信息学功能注释。运用miRBase数据库，对10个新预测miRNA进行同源性分析发现，新预测的10个miRNA与拟南芥、烟草、水稻等作物中参与病害逆境胁迫响应的miRNA有较高相似性（62%～100%）。定量检测和验证miRNA作用的靶基因时发现，抗病组表达量上调的Han-miR21对应的靶基因有1个下调、表达量下调的Han-miR43对应的靶基因有3个上调，符合miRNA与其靶基因互作的规律。5’RLM-RACE方法验证了Han-miR21的一个靶基因可能为抗性相关的蛋白（LOC110894040）。选取miR21、miR25、miR43、miR45通过花絮浸染法分别转化拟南芥，T3代转基因拟南芥接种白粉病菌，与野生型相比均表现出一定抗性变化；有些转基因植株叶片颜色表现为深紫色，可能影响了叶片花青素的积累。