Expression Patterns Of MiR393 And Its Effect On The Plant Growth And Development In Rice

Plant microRNA-target interactions are involved in complex networks of auxin signaling pathways, which affect plant growth and development in numerous biological processes. The target genes of miR393 belong to a gene family of TIR1 auxin receptor, which directly regulate auxin perception. So far, the function of TIR1 auxin receptor in monocots remains unknown. In the present study, we analyzed the expression patterns of OsmiR393 family members in rice (Oryza sativa), and showed that altered expression levels of miR393 affected the growth and development of transgenic lines. In addition, two predicted target genes were identified by using RNAi. Further, interaction of miR393 and its targets was tested in response to exogenous auxin. Our results are as follows:1. Expression analysis of miR393 gene family in rice:a) Total RNA extraction from various tissues of rice at different periods; b) Design primers respectively, according to the sequence of endogenous pre-miR393a and pre-miR393b, then tested relative accumulation level of pre-miRNA; c) Design specific probes according to mature miR393 sequence, then detect mature miR393 in various tissues by Northern blot. The results showed that miR393a mainly expressed in the adventitious roots from underground parts, however, miR393b mainly expressed in the shoot apex meristem, flag leaves and young panicles from above ground parts. This indicates members of rice miR393 gene family have different expression patterns.2. Promoter activity analysis of the rice miR393 gene family:2-2.5kb sequence upstream the stem-loop structure of miR393a and miR393b precursor were isolated and fused to GUS reporter gene to generate pmiR393a::GUS and pmiR393b::GUS construct, and then introduced into rice by Agrobacterium-mediated transformation. The GUS staining results showed that miR393a primarily expressed in adventitious roots, lateral roots primordial, shoot apex meristem and anthers, but miR393b only expressed in the shoot apex meristem. It indicates that the rice miR393a and miR393b genes have obviously specificity of tissues in transcriptional activity of rice plants.3. Overexpression of miR393 on the plant growth and development in Rice:a) Clone the endogenous miR393a and miR393b of rice, over-expression vector 35S::miR393a and 35S::miR393b were constructed and introduced respectively into rice; b) The miR393 over-expression single-copy lines were identified. The phenotypes about the transgenic plants (T3 generation) over-expression of miR393a and miR393b were analyzed, the results showed that over-expression of miR393 dramatically influences the growth and development of rice, mainly in dwarf plant, droopy flag leaf, more tillers, cracked glume, elongate tap roots and reduced adventitious roots.4. The MIM393 transgenic plants were obtained by mimicking target sites for suppressing miR393:Design primers for cloning Arabidopsis IPS1 gene and modifying the miR399-complementary motif of IPS1 into mimic target sites for miR393 ('MIM393'), then generate an over-expression construct 35S::MIM393 to transform rice.4 strains of overexpression of MIM393 were selected by the semi-quantitative and quantitative RT-PCR.5. Identification of rice miR393 target genes:Predict miR393 target genes by bioinformatics approach; initially speculate Os04g32460.1 and Os05g05800.1 the two target genes of rice miR393, respectively named OsAFB2 and OsTIRl according to the sequence clustering analysis. Quantitative RT-PCR analysis revealed that OsAFB2 and OsTIRl were significantly downregulated in the transgenic lines overexpressing miR393 while OsTIRl significantly increased in the flag leaf of 35S::MIM393 transgenic plants, indicating that miR393 negatively regulates the expression of OsAFB2 and OsTIRl at the transcriptional level.6. The transgenic rice plants suppressing expression of OsAFB2 and OsTIRl by RNAi were obtained:Create three RNAi constructs about target genes p35S::RNAi-OsAFB2, p35S::RNAi-OsTIRl and p35S::RNAi-OsAFB2-OsTIRl, and then transform rice by Agrobacterium-mediated transformation, subsequently 22,18 and 13 transgenic lines was obtained respectively for further identification of the effects of suppression of OsAFB2 and OsTIRl expression on the plant growth and development.7. Overexpression miR393 increased resistance to auxin response:a) Calli were induced by culturing wild type seeds on N6 medium (supplemented with 2.5mg/l 2,4-D) after three weeks, but 35S::miR393a and 35S::miR393a transgenic seeds still failed to induced, indicating that overexpressing miR393 enhances resistance to exogenous auxin; b) The 2,4-D treatment of rice seedlings shown that expression of miR393b was significantly up-regulated in both of roots and shoots with 2,4-D while miR393a was down-regulated, but expression of target genes OsAFB2 and OsTIRl did not alert obviously.