Preliminary Investigation Of The Transcription Processing And Stress-tolerant Function Of MiR393 In Arabidopsis

An increasing amount of studies demonstrated that microRNAs play critical roles in gene expression of both developmental patterning and physiological processes. The expression of several plant miRNAs was found to be up or down regulated by different environmental stresses and may participate in these stress adaptations. We investigated that how plant miR393 genes were regulated and biogenesis, and its potential roles in the responses to drought and salt stresses in Arabidopsis. Our experimental results were shown as followings:1. Several of plant expression cassettes containing three different kind of stem-loop precursor were constructed: 1) Endogenous pre-miRNA393a and pre-miRNA393b backbones were cloned. And two over- expression cassettes of 35S::miR393a and 35S::miR393b were constructed. 2) 35S::amiR393 was constructed with an at-miR319a stem loop. 3) 35S::mmiR393 was constructed with a mus-miR155 precursor backbone from murine.2. Using Agrobacterium tumefaciens-mediated transformation, five transgenic lines were obtained by hyg-resist screening, GUS, PCR and RT-PCR analysis. No obvious phenotype changes were observed in these lines.3. Realtime RT-PCR and Northern blots analysis showed that levels of both pre-miR393b and mature miR393 were increased significantly in three transgenic lines. Although level of pre-mmiR393 was increased, accumulation of mature miR393 was very limited in 35S::mmiR393 transgene lines. It suggested that miRNA precursor from animals might be processed inefficiently in plant.4. Using PEG-mediated drought treatment and NaCl stress assay, growth of transgenic and several mutants of hyll-1, dcll-1 and ago1, were measured. The results represented that dcll-1 and agol mutants were hypersensitive to drought and NaCl, and hyll-1 mutant was more tolerant to drought comparative to wild type. miR393 overexpression transgenic lines exhibited no obvious tolerance change in response to drought and NaCl treatment.5. 1.5～2kb putative promoter sequences upstream the fold-back structure of MIR393a or MIR393b precursor were isolated and fused to GUS reporter gene to generate pmiR393a:: GUS and pmiR393b::GUS transgenic plants. Under normal cultural condition, GUS expression was detected in veins of seedlings of pmiR393b::GUS transgenic lines but not in pmiR393a::GUS lines. The different expression pattern of MIR393a and MIR393b was suggested to be regulated by different stresses and may participate in these stress adaptations.