Studies On Molecular Characterization Of Small RNA TaMIRs On Responding To Salt Stress In Wheat (Triticum Aestivum L.)

Using the 32 small RNA molecules (TaMIRs) that are scurrently released in the international microRNA database (http://www.mirbase.org/) as the basis, the molecular characterization of the tested TaMIRs on responding to salt stress were elucidated based on semi-quantitative RT-PCR approach. The main results are as follows:1. Large variations on the expression levels of the 32 tested wheat TaMIRs exist. Under nomal growth condition, four groups could be classified, including high-level group such as TaMIR159b, TaMIR1120, TaMIR1122, TaMIR1129, TaMIR1130, TaMIR1134, TaMIR1136 and TaMIR139;mid-level group such as TaMIR159a, TaMIR160, TaMIR164, TaMIR167, TaMIR399, TaMIR408, TaMIR444, TaMIR1117, TaMIR1118, TaMIR1119, TaMIR1126, TaMIR1130, TaMIR1132, TaMIR1133 and TaMIR1137 ; low-level group such as TaMIR174, TaMIR1121, TaMIR1127, TaMIR1128 and TaMIR1135;silencing group such as TaMIR1123, TaMIR1124, TaMIR1125 and TaMIR1138. These results clearly indicate that the tested wheat TaMIRs play important roles on plant growth and development through distinct expression models.2. Under salt stress condition, there were eight among the tested TaMIRs to be up-regulated, including TaMIR159a, TaMIR160, TaMIR167, TaMIR174, TaMIR399, TaMIR408, TaMIR1124 and TaMIR1133. The putative target genes interacted by the salt induced TaMIRs varied in the numbers. Among them, no target gene was identified in TaMIR399. The target genes identified were 4 for TaMIR159a, 3 for TaMIR160, 2 for TaMIR167, 2 for TaMIR174, 5 for TaMIR408, 7 for TaMIR1124 and 5 for TaMIR1133.3. The biological functions of the target genes interacted by the salt induced TaMIRs are involved in diverse classes, such as signal perception, protein phosphoryzation, transcriptional regulation, primary metabolism, defensive response, phytohormone response, protein synthesis, cell division, nucleic acid metabolism, and apoptosis. Therefore, the salt induced wheat TaMIRs are involved in sigal transduction of multiple pathways associated with salt signaling.4. Using the precusor sequence of TaMIR1124, a wheat miRNA that was responded significantly to salt stress, as the basis, two binary plasmids fused the sense and antisense sequences of TaMIR1124 into vector pCAMBIA3301 were constructed. The two binary plasmids were introduced into Agrobacterium tumefaciens strain EHA105 by heat shock method. Futhermore, the transgenic tobacco lines integrated sense and antisense sequences of TaMIR1124 were generated based on Agrobacterium tumefaciens-mediated trasformation approach.5. Except the control (CK) that was transformed the empty binary vector, the specific segments derived from TaMIR1124 precusor were PCR amplified in the transgenic tobacco lines, including 10 lines integrated the sense sequence of TaMIR1124 and 9 lines integrated the antisense sequence of TaMIR1124. Under salt stress condition, the transgenic lines with sense and antisense integrations of TaMIR1124 diaplayed phenotypic variations, shaowing a pattern that sense expression of TaMIR1124 enlarged the plants whereas antisense expression of TaMIR1124 reduced the plants. These results suggested that the expression levels of TaMIR1124 could regulate the capacities to responding and withstanding salt stress in plants.