Differential Expression Of MicroRNAs Under Drought Stress In Maize

Plants growth and development were affected by drought, which is the most important abiotic stress factors restricting agricultural production. Maize is the dryland crop of largest water requirement, more sensitive to drought stress. With global warming and increasing scarcity of water resources, the yield of maize often seriously losses because of the drought. So drought tolerance improvement of maize breeding is particularly important.Drought tolerance in maize is a complex quantitative trait controlled by multiple genes, which needs to be identified in a specific environment, using the conventional method in screening drought tolerant varieties is so slow and less efficient. With the development of molecular biological technology, we have provided the powerful technical support for the research of drought tolerance in maize. Recent studies show that, microRNAs is closely related to the plant drought tolerance, to affect affect plant phenotypes of drought-tolerance by regulating the expression of their target genes under adversity stress. Detecting the differencial expression of microRNAs and their target genes in maize under drought stress, which can provide reference for the molecular genetic research on plant drought tolerance.According to a previous evaluation for drought tolerance, taking maize drought-tolerant inbred line of "81565","87-1" and drought-sensitive inbred line of "21ES","Dan340" as the material in this study, extracting and isolating small RNAs after treating the seedlings with polyethylene glycol6000(PEG-6000) simulative drought treatment, through the microRNA microarry hybridization technology to analyse differential expression of microRNAs in4different drought-tolerance inbred lines under drought stress. Predicting their target genes with WMD3software, and analysing their possible roles in drought response by corn functional annotation database. Analysing mRNA of4maize inbred lines through same treatment by digital expression profiling, compared to target genes regulated by differentially expressed microRNAs, and analysing the regulation modes under drought stress. Detecting differential expression of the miR159and miR168 in4maize inbred lines with LNA-Northern Blotting technology, which play key roles at seedling stage drought stress response pathways upstream.The main results are as follows:1. Analysis of small RNA under drought stress in4different drought-tolerance inbred lines through the microRNA microarry hybridization showed that303microRNAs were identified to be differentially expressed at the1%significant level.185out of which belong to20maize microRNA families (including2microRNAs from custom21new microRNAs),118out of which can be classified into43microRNA families from other11species. But among different maize inbred lines, even different time points from the same inbred line, the expression amount of the microRNAs were not alike. According to the results of cluster analysis, the expression patterns can be divided into8modes: up-regulated or down-regulated expression in the whole process under drought stress; induction or inhibition of expression in the early stage of drought stress, and then returned to normal levels in the later stage; up-regulated or down-regulated expression has been delayed in the late stage of drought stress; up-regulated or down-regulated expression in the early stage of drought stress, in the later stage, but on the contrary in the later stage.2. Taking WMD3software to predict the target genes regulated by microRNAs of drought stress response,128differentially expressed microRNA predicted390potential target genes.46microRNA families were predicted the target genes,21out of which were homologous with maize, and25out of which were nonhomologous with maize.3. Comparing digital expression profiling to target genes regulated by differentially expressed microRNAs show that in the process of each treatment of each inbred line, there were differential expression of predicted target genes. Making function analysis of GO annotation to target genes regulated by differentially expressed microRNAs, they can be divided into several categories according to the function:the genes related metabolism and growth, the enzymes involved in protein modification, the proteins involved in the transcription and translation, transmembrane transport proteins, transcription regulation factors, the signal transduction proteins, the proteins related stress resistant response, and some unknown function proteins.4. Conducting the the comprehensive analysis of the differential expression of microRNA and the differential expression of the target gene, found that in the same family microRNA can simultaneously control serveral multiple target genes, which also participates in the regulation of multiple physiological and biochemical pathways; and more than one family can also be combined with microRNA regulation of a target gene. And the study also disscussed and analysed the potential regulation mechanisms of miRl59, miR168, miRl64, miR165, and miR166under drought stress.5. Using the LNA-Northern blotting to confirm the differential expression of miRl59and miR168in4maize inbred lines. Test results are basically the same with microRNA microarray hybridization results, thus proving the reliability of the microarray data.