杨树感染溃疡病菌过程中的miRNA效应研究

MicroRNA (miRNA) plays an important role through down-regulating the expression of target genes in plant stress responses. Botryosphaeria dothidea causes stem canker on Populus resulting in significant production losses. Based on the identification of miRNA in Populus inoculated with Botryosphaeria dothidea in our previous research, three kinds of Populus clones with different resistance will be inoculated to further study the regulation roles of miRNA in forest disease resistance. QRT-PCR, Northern blotting and miRNA microarray will be used to analyze the differential expression of miRNA in inoculated Populus clones. Consequently, it will be further elucidated whether the differential expression of miRNA is universal in Populus clones with different resistance. The characteristics of the spacial and temporal expression of miRNA will be identified. MiRNA expressions in different Populus clones and inoculation stages and locations with different distances from inoculaton spots will be analyzed. Types, families and quantities of universal and specific miRNAs in Populus clones will be analyzed to identify the relationship between different resistance of Populus and the differential expression of miRNA. In Situ Hybridization, qRT-PCR and Northern blotting will be used to further analyze the differential expression and positioning of Botryosphaeria dothidea-responsive miRNAs in tissues of inoculated Populus in order to subtly elucidate the effect of miRNA in the infected Populus. Combined with these results, this research might provide some new theory clues for forest pathology and important candidate genes for genetic engineering breeding of plant disease resistance.

miRNA在植物应对逆境胁迫的反应中起着重要的调控作用，杨树溃疡病是严重制约林业生产的一大枝干病害。为了深入研究miRNA在林木抗病中的调控作用，本项目在前期开展杨树溃疡病相关miRNA鉴定的基础上，对3类不同抗性的杨树无性系接种溃疡病菌，用定量PCR、Northern杂交和miRNA芯片的方法深入研究miRNA的表达变化，分析miRNA的差异表达在不同抗性杨树中存在的普遍性及miRNA表达的时空变化特征，鉴定miRNA在不同无性系间、接种不同时间段及侵染点不同距离的杨树中的表达模式，分析不同抗性杨树中表现共性和特性的miRNA种类、家族和数量，揭示杨树的不同抗性与miRNA差异表达的关系。研究响应溃疡病菌侵染的关键miRNA在杨树组织中的表达和定位，以精细揭示溃疡病菌在侵染杨树中miRNA的调控效应，为林木病理学研究和基因工程育种提供新的研究思路和重要的候选基因。

miRNA是植物中存在的一类重要的非编码小分子RNA，长度约为21nt ，在植物应对逆境胁迫的反应中起着重要调控作用，而杨树溃疡病是严重制约林业生产的一大枝干病害。为了深入研究miRNA在林木抗病中的调控作用，本项目用抗性强、中、弱差异的3类杨树无性系为材料，进行了每个无性系125个接种点共计375个接种点的溃疡病菌接种，对照为等同数目的未接种的杨树无性系材料。分别采集发病和未发病的杨树树皮，提取发病和未发病材料的RNA，并提出了一种改良的RNA提取方法。分析了杨树不同无性系感染溃疡病菌过程中miRNA表达变化，比较了溃疡病菌侵染3类不同抗性无性系间的miRNA表达变化差异，鉴定出杨树的不同miRNA种类或家族对溃疡病菌侵染的反应特点，初步建立了不同miRNA种类或家族与无性系对溃疡病抗性的关联。发现16个miRNA的靶基因均涉及植物-病原物互作，因而这些miRNA可能参与杨树对溃疡病的响应。系统鉴定了杨树miRNA的候选启动子，揭示了TATA box和TSS的分布模式，并鉴定了主要的顺式作用元件。该研究结果进一步加深了对杨树miRNA的表达和分子功能的认识。本研究揭示的杨树感染溃疡病菌过程中存在miRNA的调控效应，有助于揭示杨树受溃疡病菌侵染过程中miRNA的调控作用，为林木病理学研究和miRNA在杨树抗溃疡病分子育种中的利用提供了一定的研究思路和调控元件。