Identification And Function Analysis Of Anti-stress Effect MicroRNA In Peanut

MicroRNA is a kind of endogenous non-coding small single strand RNA which is widely exist in animal, plant, viruses and other organisms. It only has about21-25nucleotides[1]. MicroRNAs play an important role in the regulation of gene expression in multicellular organisms, which had been reported that could regulate gene expression negatively on the post-transcriptional levels. The identification and function analysis of miRNAs had made great achievements in the past ten years and had become more and more popular in the field of scientific research.Peanut is one of the world four major oil crops. As the main economic and export oil crops, the molecular biology genetic breeding research of peanut has arouse more extensive attention. But there is little report about the combination of endogenous miRNA and resistance of peanut.In this study, we found out60new conservative peanut miRNA using bioinformatics analysis. Because miRNAs sequence were highly conserved in the plant and their precursor had special secondary structure,60miRNA sequences were classified into16families through homology comparison in miRNAs database. And then, Using Target-align software and BLASTx analysis, we could forecast the target genes of60peanut miRNAs. In addition to two miRNA sequences whose target genes were not found, we forecasted392target gene sequences of the other58microRNAs sequences. Through GO function analysis, we could find that the target gene of the microRNAs could involve in the growth, signal transduction and stress resistance of peanut.Using bioinformatics methods to predict miRNAs has the advantages of convenient, fast and economic. But there are some false positive sequences in the process of predicting. So, we need to verify the prediction through the further experiment. Using poly (A) tail experiment method, we finally identified55of the60miRNA sequences by experiment validation and the success rate was as high as 91.67%. The family of miR159、miR319、and miR394each had one miRNA sequences which was not detected, and miR482family had two microRNAs sequence which were not checked out.The key step to study the role of microRNAs is the confirmation about miRNA target genes. Plant miRNA sequences can cut the target genes in a perfect complementary way, and can cause the degradation of target genes. In this study, we use5’-RACE method to validate the prediction the cutting site of peanut target genes and determine the accuracy of the bioinformatics prediction. In this study we choose20peanut target gene sequence as test object. Through gel electrophoresis detection experiments and cloning sequencing result, we identified14(70%) of peanut miRNA-target genes. The major cutting site of target genes was located at9-11nucleotide, and part of the target genes existed multiple cutting sites.We analyzed the function of target gene sequence and retrieved the function of homologous sequences.26target gene sequences were chosen as the initial object of the stress research. And we measured the expression of miRNAs under the condition of yellow aspergillus infection and drought stress. By using real-time quantitative PCR technology, we study the model about miRNA negative regulating on target genes. Through preliminary determination of target genes-biological function of the miRNA, there were five results which were correct.By means of bioinformatics combined with practice, we found out the peanut stress resistance related miRNAs, and predict its target genes and screening of miRNAs function. In this study we hope to lay a foundation on improving the stress resistance of peanut through strengthening or weakening the gene silencing effect of endogenous miRNA to change gene expression level of the target gene.