MiRNA Identification And Function Analysis Of Rice Against Black - Streaked Dwarf

Rice black streaked dwarf virus (RBSDV) has become the most serious disease in the producing rice area in East China in recent years, and seriously affected the rice yields. It is a fundamental strategy to use of the resistant of plants to cultive disease-resistant corn. miRNA is also as the negativing regulator to regulate gene expression and plants growth and responsing to adverse environmental invarious biological pathways. In this study, constructing small RNA library is based on filtering disease-resistant and susceptible corns from rice breed, combineing with high-throughput sequencing and bioinformatics analysis is to carry on breeding for disease resistance, through the target gene predicting and functional analysis describing is to elaborate the development of plant and the way of disease-related miRNA after the inoculated on plant growthing.Using artificial inoculation method, we selected anti-virus resistance rice variety 9311 and high susceptible variety Nipponbare. We construct eight small RNA libraries containing RBSDV infected and virus-free infected stems and leaves of each variety.Through high-throughput sequencing and bioinformatics analysis,we detected a group of rice miRNAs expressed under RBSDV stress.We also use Northern blot, and Q-PCR to anlyse the expression level of the candidate miRNAs and their target genes. The main results are as follows:1.4795833(?)-5697879 high quality small RNA sequences were obtained from eight small RNA libraries,and 30.48%-45.97% of them matched rice genome. In respond to RBSDVstress,38 miRNA were down-regulated,12 miRNA were up-regulated.31 miRNA members were down-regulated in 9311 and up-regulated in Nipponbare;In contrast, there is only 1 miRNA up-regulated in 9311 and down-regulated in Nipponbare.More over,9 miRNA members were down-regulated in rice stem and up-regulated in leaves; 18 miRNA members were up-regulated in stems and down-regulated in leaves.2. Through Northern blot verification experiment, we detected the abundance of miR156 significantly increased in leaves of 9311 and mildly increased in the stem after infection. In contrast, the expression level of miR156 significantly increased in leaves of Nipponbare and mildly increased in the stem. miR168 were also detected but there wasn’t any significant variation.3. Through qRT-PCR we analysed 18 target genes of 13 highly expressed miRNAs.9 target genes had a negative correlation with their corresponding miRNA a.After analyzing, we found that miR166d and miR444 participated in the modification,degradation and accumulation process of proteins by ubiquitin approach; miR164 invoved to the auxin’s gradient and the original roots and the numbers of petal by regulateing target genes;miR396d and miR160 participated in plant growthing and development by auxin responsing routes; miR171 and miR172 effected to the modification and degradation and the accumulation of proteins by regulating target genes.4. we obtained the cleavage sites of the target genes of miR396d and the target genes of miR444b through 5’RACE.Our study obtained a group of rice miRNA which expressed discrepantly in resistant and susceptible rice through miRNA separation, anthentication and expression analysis after RBSDV stress. We also found these miRNAs participated in the process of rice tissue growth anddevempment. It is the first report about the miRNA related to anti-RBSDV. The study also set basis for ecognization of plant disease-resistance mechanism and geneticl improvement of disease-resistent varieties subsequently.