基于microRNA的抗玉米弯孢叶斑病分子调控机理研究

MicroRNA (miRNA) are a class of non-coding small RNAs, which play an important role in plant disease resistance. But the molecular regulation mechanism based on the miRNA has not been studied. In this study, the significant different miRNA was identified by comparison of miRNA expression differences between resistant and susceptible variety using miRNA microassay, combined with results of stem-loop RT-PCR and Northern blotting. miRNA cis-elements and target were predicted by bioinformatics. The upstream transcription factors of miRNA was identified by the yeast hybrid and ESMA, and the target genes of miRNA and regulation relationship between miRNA and target genes was analyzed by quantitative PCR technology and the 5 `-RACE techniques and Western blotting. The mechanism and role of miRNAs in plant disease resistance pathways was explored through study of the gene function. The molecular regulation mechanism of miRNA in plant disease resistance was comprehensively analyzed, which will fill miRNA research on maize resistance to Curvularia lunata and provide a theoretical basis and technical support for China's maize breeding for disease resistance.

MicroRNA（miRNA）是一类非编码小RNA，在植物抗病性过程中起重要作用。目前基于miRNA的抗玉米弯孢叶斑病分子调控机理尚无人研究。本研究利用miRNA微阵列研究miRNA在抗、感病材料之间的表达差异，结合stem-loop RT-PCR和Northern blotting技术鉴定出显著性差异的miRNA；采用生物信息学对miRNA顺式元件和靶基因进行预测，通过酵母杂交、凝胶迁移滞后实验鉴定出miRNA上游转录因子，利用荧光定量PCR技术、5`-RACE技术和Western blotting验证miRNA下游靶基因和对其的调控方式，研究miRNA的上下游调控关系元件和调控关系；通过基因功能研究明确miRNA在植物抗病途径中的机制与角色；综合分析出基于miRNA的抗玉米弯孢叶斑病分子调控机理，填补玉米抗弯孢叶斑病miRNA研究的空白，为我国玉米抗病育种提供理论基础和技术支撑。

通过高通量测序技术鉴定了玉米与弯孢亲和和非亲和过程的microRNA，采用miRNA 微阵列和stem-loop RT-PCR技术鉴定出玉米与玉米弯孢叶斑病菌亲和和非亲和互作过程中有显著性差异表达的miRNA；采用降解组和生物信息学技术解晰了miRNA的靶基因，通过比较生物学筛选了抗病相关的microRNA和相应的靶基因，利用荧光定量PCR 技术和5`-RACE 技术验证抗病相关的miRNA－靶基因对之间相互关系和调控方式，其中明确miRNA732在植物抗病途径中的机制与作用，揭示抗玉米弯孢叶斑病的分子调控机理, 为我国快速培育玉米抗病品种提供理论基础和技术支撑。