Characterize The Roles Of MiRNAs In Responding To Short-term Waterlogging In Zea Mays. Root

Maize is the one of the world's most important crop for food, feed and energy. And it is also the largest crop for food in China. In south China, waterlogging has become to one of the most important abiotic stress in restricting the development of maize production. In eukaryotic genome, there are a large number of non-conding RNAs, which directly involved in the regulation of the developmental and stress response gene expression in the transcriptional and post-transcriptional level.In this study,the roots of three inbred line with different sensitivity to waterlogging, which are Hz32(waterlogging tolerant), B73(mid-tolerant) and Mo17(waterlogging sensitive), under short-term waterlogging (1-4h) were used for the miRNA, their promoter and targets analysis. We focused on the characterization the roles of miRNAs in rapidly responding to short-term waterlogging in maize root cell, which will give a new sight of the miRNA regulation mechanism under waterlogging stress and help on the marker-assisted selection of the waterlogging resistance gene in order to breed excellent waterlogging resistance inbred lines.1. To investigate the role of miRNAs under hypoxia stress in Hz32, small RNAs (smRNA) were isolated from waterlogging tolerant line (Hz32) root tissues subjected to4h (signal transduction stage) of waterlogging treatment and control sample (no waterlogging treatment) for Illumina high-throughput sequencing. The total number of sequence reads was9,251,451for the treatment sample and9,541,782for the control sample. Form the libraries, we have found24candidate differential expressed mature miRNA signatures, representing64known miRNA and2novel miRNA loci.2. To characterize the involvement of miRNAs and their targets in response to short term hypoxia conditions, a quantitative real time PCR (qRT-PCR) assay was used to quantify the expression of the24candidate mature miRNA signaturesand their92predicted targets in three inbred Zea mays lines (waterlogging tolerant Hz32, mid-tolerant B73, and sensitive Mo17) under3time point treatment (1h,2h,4h). Based on our studies, miR159, miR164, miR167, miR393, miR408and miR528, which are mainly involved in root development and stress responses, were found to be key regulators in the post-transcriptional regulatory mechanisms under short term waterlogging conditions in three inbred lines.3. RNA-seq analysia of Hz32under4h waterlogging treatment has validated most of the expression of the miRNA targets and the5'-RLM-RACE assay also validated the miRNA cleavage site of6miRNA targets genes. Comparing the exoression pattern of the miRNA targets with their miRNAs shows that around50%of the exoression pattern of miRNA targets shows the nagetive correlated with their miRNAs' expression and most of them transcription factor that are participate in responding to hypoxia stress. It is suggested miRNA play a key role in the stress response of maize seedling underwaterlogging at the post-transcriptional level.4. The promoter regionof30differentially expressed miRNA genes, which have the TSS information from the known database, were used for computational cis-motif predictions using all experimentally derived position weight matrices (PWMs) from JASPAR core Plantae (http://jasper.genereg.net/). We found the occurrence of18stress and development associated transcription factor binding sites (TFBS) in the selected promoter region of miRNAs. Andwe contracted a TF library, formed by712arabidopsis TFs for the Y1H analysis, which indirectly validated the cis-motif of the miRNA regulated by conserved transcription factor.5. Based on all the results, using the Cytoscape software to build a short-term waterlogging regulated miRNAs and their target genes regulatory networks. From the network,we found a possible negative feedback regulatory pathway between MYB and miR159/319and it is confirmed by the Y1H.