Identification And Functional Characterization Of Stress-regulated MicroRNAs And Functional Analysis Of MicroRNAs Promoters In Arabidopsis Thaliana

High salinity, drought and low temperature are three common environmental stress factors that seriously influence plant growth and development worldwide. Recently, microRNAs (miRNAs) have been linked to plant stress responses. MicroRNAs are designated as endogenous small sized RNA with 19-25 nt in length, and were found to be widespread in both prokaryotic and eukaryotic organisms. Their important roles as a class of gene expression regulators have been widely recognized, and were proven to be involved in many processes including leaf development, auxin signaling, phase transition, flowering and genome maintenance. Recently, there has been strong evidence leading to the proposal that miRNAs are hypersensitive to abiotic or biotic stress as well as to diverse physiological processes. Hence, efforts to identify novel stress-regulated miRNAs and determine their expression patterns could improve our understanding of their functions in stress adaptation. The main results were as follows:1. We identified 14 stress-inducible miRNAs using microarray data in which the effects of three abiotic stresses were surveyed in Arabidopsis thaliana. Among them, ten high-salinity-, four drought- and ten cold-regulated miRNAs were detected. miR165, miR319 and miR393 were upregulated by both high salinity and cold, miR167 was induced by both high salinity and drought, and miR168, miR171 and miR396 were observed to respond remarkably to all three stresses.2. To determine which locus is responsive to stress conditions, we performed semiquantitative RT-PCR analysis using specific primers designed to amplify fold-back precursor transcripts in Arabidopsis, and our RT-PCR results were ultimately consistent with the microarray data for all miRNAs tested. Expression profiling by RT-PCR analysis showed great crosstalk among the high-salinity, drought and cold stress signaling pathways.3. To further elucidate the inducibility of these products, we analyzed the 1000 bp upstream promoter sequence of 20 miRNAs by using the PlantCARE database. We identified several known stress responsive elements, such as the ABREs (ABA-response elements), AREs (anaerobic induction elements), MBS (MYB binding site involved in drought-inducibility), HSEs (heat stress responsive elements), LTRs (low-temperature responsive elements) and TC-rich repeats (defense and stress responsive elements)The existence of stress-related elements in miRNA promoter regions provided further evidence supporting our results.4. Analysis of transgenic plants with the gus reporter gene that under the control of different sequences upstream miRNA precursors shows that all these sequences can function as miRNA promoters.5. GUS staining experiment with the transgenic plant on different development stages shows that the promoter of OsmiR159a, OsmiR398a, AtmiR396b, AtmiR397b, AtmiR400 is constitutive promoters. However, AtmiR393a and OsmiR443 are specific expressed miRNA which might be under the control of their promoters due to their different functions.