The Molecular Characterization And Function Analysis On Tolerance To Low-pi Stress Of Wheat TaMIR1139、TaMIR167 And It’s Target Genes And TaMIR1136

Micro RNAs(mi RNAs) are small non-coding RNA molecules and they regulate expression of the interacting target genes through post-transcriptional level or translation repression mechanism based on their complementary pairings with the interacted targets in plants. Previous studies have shown that micro RNAs are involved in mediating plant growth and development, and in regulating plant responses to biotic and abiotic stresses. In this study, the possible target genes of Ta MIR1139 were predicted by an online tool referred to ps RNARarget. Using a phosphorus-efficient wheat variety(Shixin828) as materials, we investigated the molecular characterization and the expression pattern of the target genes under the low-Pi stress. DNA recombinant technology and gene genetic transformation approach were adopted to generate the transgenic tobacco plant lines with antisense expression of the target genes. The transcription profiles of the low Pi-treated transgenic tobacco plants over-expressing Ta MIR1139 and Ta MIR167 were determined based on the digital gene expression tag profiling(DGE) approach. In addition, as one of important target gene interacted by Ta MIR167, Ta ARF6 was shown to be low Pi responsive and its roles in mediating plant response to Pi deprivation were defined based on transgene analysis. Aside from above two wheat mi RNA members, Ta MIR1136, a wheat mi RNA member that is also response to the low-Pi stress, was cloned and subjected to binary expression vector construction as well as genetically transformed in tobacco. The main results are as follows:1. Prediction analysis revealed that Ta MIR1139 interacts nine target genes, including T1(TC449025), T2 GLYCINE(CJ636238), T3 SCAFFO(TC384616), T4(TC397135), T5 RIBO(TC400227), T6 PEROXI(CB412183), T7 MYB1(TC397585), T8(TC382427) and T9(CD930311). Expression pattern analysis showed that the transcripts of the target genes of T1, T3, T4 and T5 were gradually decreased along with the low-Pi stress progression. Other target genes did not show altered expressions upon treatment of the Pi deprivation.2. Using the transgenic lines over-expressing Ta MIR1139 and the wild type(WT) as materials, the phenotype features, activities of cellular antioxidant enzymes, and the differential transcription profiles were investigated. Under the low-Pi stress, the transgenic plants exhibited much more improved phenotypic feature, higher fresh and dry weights per plant than WT. These results are in consistent with the higher antioxidant enzymatic activities of SOD, CAT, POD and lower content of MDA in the transgenic plants. Differential transcription profile analyses revealed that 3205 genes were differentially expressed in the low Pi-treated transgenic plants, in which 1683 were up-regulated and 1522 were down-regulated. These results indicated that Ta MIR1139 mediates plant response to the low-Pi stress through its transcriptional regulation of large quantity of the downstream genes.3. Using transgenic lines antisense expressing Ta MIR167 and WT as materials, the phenotype fetures, activities of cellular antioxidant enzymes, and the transcription profiles were also investigated. Under the low-Pi stress, the transgenic plants grew weaker and accumulated less fresh and dry weights per plant than WT. These results are closely associated with the lower antioxidant enzymatic activities of SOD, CAT, POD and higher content of MDA in the transgenic plants. Transcription profile analyses of the transgenic and WT plants indicated that totally 3540 genes were shown to be differentially expression in the Pi deprivation-treated transgenic plants, in comparison with the WT plants. These differentially expressed genes included that 1745 were up-regulated and 1973 were down-regulated. Therefore, similar to Ta MIR1139, Ta MIR167 may also act as an important regulator in mediating plant reponse to the low-Pi stress through its effects on modifying transcriptions of vast amount of the downstream genes.4. As an important target gene interacted by Ta MIR167, Ta ARF6 was identified in a root c DNA subtractive suppression hybridization(SSH) library enriching differentially expressed genes under the low-Pi stress condition. Ta ARF6 has a full-length c DNA of 3655 bp, containing an open reading frame(ORF) of 2328 bp that encodes a 775-aa polypeptide. The expression patterns of Ta ARF6 in wheat roots under various Pi-supply conditions were analyzed. The results indicated that the expression of Ta ARF6 was down-regulated by the low-Pi stress. Using DNA recombinant technology, the binary expression cassette fused the Ta ARF6 ORF was constructed and the transgenic tobacco plants over-expressing Ta ARF6 were generated via Agrobacterium tumefaciens-based genetic transformation method.5. Using transgenic lines with over-expressing Ta ARF6 and WT as materials, the function of Ta ARF6 in responses to the low-Pi stress were studied. Under low-Pi stress, the transgenic plants exhibited much more improved root architecture system and larger aboveground tissue phenotype, showing longer length of the lateral roots and higher fresh and dry weights per plant than WT. These behaviors were closely in consistent with the higher antioxidant enzymatic activities of SOD, CAT, POD and lower content of MDA in the transgenic plants. Combined the results of its increased expressions of several cellular antioxidant enzyme genes and the improved plant growth under Pi deprivation in the transgenic plants, it is suggested that Ta ARF6 play an important role in mediating plant response to the low-Pi stress.6. Ta MIR1136 was identified based on searching analysis against the mi RBase database(Release 10.1, http://microrna.sanger.ac.uk/ sequences/index.shtml). It has been revealed that Ta MIR1136 shares a precursor sequence of 107 nt, with a mature sequence of 5’- uugucgcagguauggauguaucua- 3’. Target prediction analysis suggested that Ta MIR1136 interatcts 14 genes that are involved in different biological function groups, including signal transduction, transcriptional regulation and transporters, etc. Based on the cloned precursor sequence, Ta MIR1136 was integrated into the binary expression vector p CAMBIA3301 and subjected to generation of transgenic tobacco plants via an Agrobacterium tumefaciens-based genetic transformation method. These generated transgenic plants are valuable for exploring the functions of this wheat mi RNA member in regulating plant growth, development, and in mediating plant responses to abiotic stresses in future.