Construction Of Small RNA Library Of Tomato Induced By Phytophthora Infestans And Functional Analysis Of Sly-miR396

MicroRNAs (miRNAs) are a class of endogenous, non-coding RNAs of19-23nt and widely exist in eukaryocyte. Previous studies have demonstrated that miRNA are involved in either plant development or biotic and abiotic stresses. Recently, miRNA play vital roles in plant-pathogen interactions have drawn great attentions. Tomato is a common economical crop and cultivated throughout the word. Late blight caused by P. infestans could lead gravely damage to tomato production. However, there is no report of miRNAs associated with P. infestans inoculation in tomato. For the reasons above, we constructed two small RNA libraries and analyzed the function of miR396in tomato.Firstly, using high-throughput sequencing, we sequenced two small RNA libraries from normal and P.infestans-inoculated tomato (Solanum pimpinellifolium) leaves. Then, we identified230known and67novel miRNAs; then,118miRNA differentially expressed after Phytophthora infestans inoculation. In addition, eleven miRNAs were validated by qRT-PCR. Target prediction showed P.infestans-responsive miRNAs had important roles in plant-pathogen interaction.MiR396was a disease-related miRNA in our libraries.The characterization of miR396was analyzed by bioinformatics methods. QRT-PCR analysis demonstrated that miR396response to different stresses, including pathogen inoculation and abiotic stresses.To further elucidate the function of miR396, the gene of miR396was cloned. Over expression vector used for transformation was also constructed. Transient expression in tomato and transformation in tobacco suggested that overexpression miR396inhibited the level of their targets in both tomato and tobacco. Also, higher level of miR396had resistance to late blight. These results suggested that miR396were involved in the regulation of stress-related target genes. MiR396might play a crucial role in disease resistance of tomato.In a word, this study investigated the solution of late blight from the sight of miRNA for the first time. In additon, it also analyzed the function and characterization of miR396systematically. This study has important implications for further identification of miRNAs under pathogen stress.