Computational Prediction And Functional Analysis Of Maize MiRNAs Under The Infection Of Corn Sheath Blight

microRNAs(miRNAs) is widely present in eukaryotic cells of a large class of endogenous,18-25 bp length of the non-coding small RNA(non-coding RNA,ncRNA).It plays an important role in multicellular organisms to regulate and control gene expression,and work in response to the outside plant environmental stress and disease stress defense.Sheath blight and other fungal diseases of the hazard are one of the major diseases of the main maize growing areas southwest of the production.It is an important study of the meaning of theory and practice for sheath blight of maize resistance varieties to cultivate by researching miRNAs in the role of sheath blight resistance and mechanisms.Downloaded from the miRBase database of Arabidopsis,rice and other plants have been discovered miRNAs,miRNAs were predicted in the maize by bioinformatics homologous blast Methods,their target genes were predicted using miRU online prediction software.In conditions of artificial inoculation of inbred lines R15(R) and Ye 478(S),using semi-quantitative RT-PCR and real-time fluorescence quantitative PCR(QPCR) method,it is analysised the expression of miRNA under the conditions of resistant to sheath blight.Excavation and identification of anti-fungal diseases of maize related to miRNAs regulation and control strategies and methods,access to a number of maize candidate miRNAs.The main findings are as follows:1.Downloaded from the miRBase database of Arabidopsis,rice and other plants have been discovered miRNAs,a total of 31 miRNAs were predicted in the maize by bioinformatics homologous blast Methods,their target genes were predicted using miRU online prediction software.2.Through semi-quantitative RT-PCR method to detect the expression of precursor of 31 of miRNAs and the miR168 in R15(resistant disease) and Ye 478(susceptible) under bacteria stress:the 10 miRNA precursors can be obtained RT-PCR-positive bands in Ye 478 and R15,through the recovery of PCR products,cloning and sequencing to prove the authenticity of sequence;the expression of the 5 miRNAs precursor sequences(miR168-1, miR168-2,miR165,miR173 and miR845) is a certain difference between the two tissue materials,it may be related to maize self-expression and regulation under sheath blight bacteria stress.3.By improving the tail-primer-extension real-time fluorescence quantitative PCR method,Level of precursor materials in two significant differences in the expression of miRNA molecule(miR165,miR168b,miR168b(~*),miR173),and has been found associated with maize miRNAs resistance elements(miR171 and miR319),Designed and synthesized the relevant primers for PCR of mature miRNAs molecular amplification and expression of authentication.The expression of 6 mature miRNAs and quantitative:In disease resistant maize material R15 and susceptible materials Ye 478,miR165,miR168b,miR168b(~*) and miR173 were an Up-regulated expression,miR171 and miR319 were down-regulated expression.The expression of all 6 miRNAs was a significant difference in 12h tissue sheath treatment between esistant/susceptible materials,although the leaves have a similar difference, but the difference was smaller.4.Through the plant miRNA target gene prediction software miRU,it is found that 6 miRNAs' targets are related to the resistance of the plant:Multi-pathogen-associated polygalacturonase enzyme(PG);plant cell wall-related cellulose synthase,hydroxyproline rich glycoprotein(HRGP);plant disease resistance signal transduction related ubiquitin-enzymes,Auxin effiux carrier protein,serine/threonine phosphatase,serine/ threonine kinase,PP-2C-like protein phosphatase;and glutathione-S-transferase,Stress inducible protein and Wound-induced basic protein.5.According to a large number of our group research and the results of this study,that miRNAs in maize may control the following resistance mechanisms:(1) When the maize by fungal pathogens invasion,Up-regulated expression of miR168b~*,miR173 may be involved in the degradation of pathogenic bacteria secrete toxins and enzymes to inhibite against pathogens;(2) up-regulated expression of miR165,miR168,miR173 through negative regulation of target gene serine/threonine phosphatase,PP-2C-type phosphatase enzymes reduced the expression of protein phosphatase;miR171 and miR319 were down-regulated expression so that serine/threonine protein kinase phosphatase enzymes to be released, breaking the protein kinase/phosphatase balance,making a large number of protein kinase expression,leading to protein phosphorylation cascade,start with the increase of defense gene expression to inhibit the growth of pathogenic bacteria;(3) Down-regulated expression of miR171 and miR319 may be induce the expression of glutathione-S-transferase,Stress inducible protein and Wound-induced basic protein,so that the whole plant defense against pathogen invasion.