Functional Analysis Of MiRNAs Closely Associated With The Pathogenicity In Tomato

MicroRNAs (miRNAs) are a class of endogenous, noncoding RNAs of 22 nt and widely exist in organism. The recent studies have shown that, miRNA plays an important role in plant and pathogen interaction. When pathogen infect with tomato will cause devastating disease, which bring great economic losses to the production of tomato. Although, at present the research on tomato miRNA related with disease has exist, but the research on miRNA which closely related with the disease in tomato is not system. Based on the above situation, this study by using bioinformatics methods, mining and identified miRNAs that closely related with the disease in tomato, then, we analysed the function of key miRNAs through biological experiment.Firstly, by collecting the literature and miRBase, we found that 34 miRNAs in tomato are closely associated with pathogenicity at expression levels. Subsequently, we predicted their target genes through the use of bioinformatics approaches and built a disease-related regulatory network of miRNAs using cytoscape program. This has led us to identify 13 miRNAs that are closely associated with pathogenicity in tomato from which we selected miR169, miR482, miR5300, miR6024, miR6026 and miR6027 for further analysis based on the association between miRNAs and the number of target genes. Lastly, we performed the analysis of target gene, promoter and real time quantitative PCR verification for these 6 miRNAs. We hope that our study will lay the foundation for the in-depth study of miRNA mechanism.Then, we choose miR482b and miR6024 the two most critical miRNA as our main research objects in this paper, using real-time quantitative PCR technique to analysis the expression patterns of miRNA and their targets in different tissues, induced with pathogens and the disease resistance related signal molecules. The results showed that, the expression of the two miRNAs in the leaves were higher than in roots and stems, and they are not only able to respond to the stress, but negatively correlated with their target genes. This suggests that the two miRNAs may by regulating the expression of their target genes to participate the resistant response in tomato.Finally, in order to further explore the function and mechanism of miR482b and miR6024, we cloned the MIR482b and MIR6024 gene in tomato, and successfully constructed the overexpression vector and transient transformation system, and obtained the transgenic plants. After analysis, we found that the expression level of the miR482b was up-regulated with their corresponding targets down-regulated in transgenic tomato, and the pathogenic ability of pathogenic bacteria to tomato was improved. These results suggested that miR482b and miR6024 may play a crucial role in the stress response of tomato.In a word, by using the bioinformatics and molecular biology experimental technology, to predict, excavate and identify tomato miRNAs that closely related with pathogenic, and analysis their function. Which will not only help us to understand the interaction mechanism of plant pathogen deeply, but also lay the foundation for the tomato breeding.