The Role Of MiRNA1139in Mediating Plant Tolerance To Low-Pi Stress And The Genetic Transformation Of Promoter Of Wheat Phosphate Transporter Gene TaPT2-1

Through interacting with the target genes via sequence complementary mechanism, miRNAs, thesmall molecules of RNA, regulate the roles of the target genes at the molecular levels ofpost-transcription and translation. MiRNAs in plants play an important role in diverse biologicalprocesses, such as to be involved in regulation of plant development and resistance of manyenvironmental stresses. In this study, the molecular characterization, expression patterns under low-Pistress, the role in mediating plant tolerance to low-Pi of TaMIR1139, a miRNA to be response to thedeficient-Pi in wheat, were investigated. In addition, recombinant DNA technology was used toconstruct a series of binary recombinant plasmids that fused the truncated promoter fragments ofTaPT2-1, a phosphorus transport gene in wheat. Transgenic tobacco lines integrated the binary plasmidswere established. The main research results are as follows:1. Based on the international miRNA database,32released miRNAs were obtained in wheat. UsingChinese Spring and Shixin828as the experimental material, a wheat miRNA referred to TaMIR1139was confirmed to be up-regulated expression under low-Pi stress.2. It was revealed that TaMIR1139is up-regulated in both roots and leaves. More transcripts of thismiRNA were detected in Shixin828, a high-P use efficiency wheat variety, than variety Chinese Spring.These results suggest that TaMIR1139is possibly involved in mediating plant tolearanve to low-Pistress through regulating the target genes at the post-transcription level and the translation level.3. Using an online tool associated with prediction of the target genes interacted by miRNAs,7putative target genes of TaMIR1139were identified. Functional prediction analysis of these target genessuggested they are classified into follow groups, including transcription regulation, protein functionmodification, cellular defense, nucleotide degradation, and protein degradation as well as unknownfunction. TaMyb1, a target gene of TaMIR1139, was further subjected to analysis of the molecularcharacteristics and system evolution characteristics.4. Using the recombinant DNA technology, the binary expression plasmids fusing the sequences ofTaMIR1139with sense orientation and anti-sense orientation were constructed. The plasmids weresubjected to transformation of tobacco using Agrobacterium-mediated approach. The typical transgeniclines with up-regulated expression of TaMIR1139were subjected to low-Pi stresses as well as stressesof low-N, drought, and salt. Compared with the control, the transgenic lines with up-regulated TaMIR1139expression displayed improved plant morphological characteristics under stresses of low-Pi,low-N, drought, and salt. However, no obvious phenotypic variations were observed between thetransgenic lines with down-regulated TaMIR1139expression and the control under stresses of low-Pi,low-N, drought and salt.5. The plant fresh and dry weights of the transgenic lines with up-regulated TaMIR1139expressionshowed significantly higher than those of the control under low-Pi stress, so did under stresses of low-N,drought, and salt. Therefore, TaMIR1139can be acted as an important resource in evaluation andcreation of elite germplasms with strong tolerances to stresses of low-Pi, low-N, drought and salt.6. Transcription analysis indicates that the expression of the reporter gene under the control ofTaPT2-1, a promoter of wheat phosphate transporter gene TaPT2-1, showed an expression pattern to beup-regulated by low-Pi stress, predominant expression in leaves, and induced by photoperiod. UsingDNA recombinant technique, a series of binary expression plasmids integrating the truncated TaPT2-1promoter fragments were constructed.7. Based on the genetic transformation approach mediated by Agrobacterium-tumefaciens, thetransgenic tobacco plants that integrate the various binary plasmids of TaPT2-1truncated promoterregions were generated. The molecular identifications of the transgenic lines were performed. Thesetransgenic plants can be used to determine the cis-acting regulatory elements that are involved in low-Pistress responding and in regulating gene expression with a pattern of aging leaf predominant.