Functional Dissection Of Two MicroRNAs In The Regulation Of Gene Expression In Transcriptional Level In Arabidopsis

MiRNAs are singe-stranded small RNAs with a length of18~27nt, which are encodedby the genome. They usually generate from60~70nt precursors. They do not have any ORF,nor do they encode any protein. In most cases, miRNAs play their biological functions byregulating the expression of their target genes. MiRNAs are highly conserved across variouskinds of species. The expression of most miRNAs are specific in different tissues ordevelopmental stages, indicating that they may be involved in comples gene expressionregulation process and play important roles in plant growth and development, as well asresponse to environmental stresses.MiRNAs can regulate gene expression both in transcriptional and post transcriptionallevels. In transcription levels, miRNA usually regulate gene expression through eithermediating target mRNA cleavage or inhibiting the translation of target miRNAs. However,there are only a few reports that support the role of miRNAs in transcriptional levels. It hasbeen report that, in plants, miRNA may regulate gene expression by affecting the methylationof coding sequences of the target genes. Due to the limitation of the research techniques, themechanism of the miRNA mediated gene expression has not been fully understood, and theremight be other mode of functions remained to be discovered.In this study, we aimed at finding miRNAs that interact with gene promoters, thusregulate gene expression in transcriptional level.(1) By analyzing the complementation and the thermal stability of the duplex betweenmiRNAs and their target genes, we used bioinformatical method to indentifed106miRNAswhich may have their targets in over500promoter sequnces in Arabidopsis.(2) Among the predicted miRNAs, there miRNAs, miR171b, miR396b and miR400were chosen for further experimental validation. Using the transient gene expression systemin Arabidopsis mesophyll protoplasts, we found that overexpression of miR171b and miR396b resulted in the increase of the expression of the reporter gene GUS driven bycorresponding target gene promoters.(3) In the seedlings overexpressing miR171b and miR396b, the expression of the targetgenes were also up-reuglated compared with wild type, suggesting that they had the same roleof regulating gene expression in the seedlings.(4) By referring to the Arabidopsis epigenetic map, we found epigenetic modifications inthe promoter regions of most target genes of miR171b and miR396b, indicating that the twomiRNAs might regulate target gene expression in transcription level by altering theepigenetic modifications on their promoters. We also deteced the expression of target genes insome mutants that were absent of different componets of RNA mediated DNA methylation.The results suggested that, miRNA might regulat gene expression in an epigenetic manner.(5) We used qRT-PCR to detect the expression of predicted miRNAs and their targetgenes under high salinity and drought conditions. The expression of miR396b significantlyincreased, as well as its two target genes, AT1G18870and AT3G47640, which suggested thatthe regulation of gene expression by miRNAs in transcriptional level might play importantroles in plant stress response.