The Expression Patterns Of Wheat MiRNAs And Genetic Transformation Of Part Members Of TaMIRs Together With Their Interacting Target Genes

As important regulators, the microRNAs (miRNAs) control dramatically thetranscripts and translated proteins of their interacting target genes, via post transcriptionalor translation repression mechanisms by combining the reverse complement zone of thetarget genes. In this study, using38miMRAs in wheat (TaMIRs) as the basis, theexpression patterns of the TaMIRs under conditions of various N supply and droughtwere analyzed. The putative target genes interacted by part of TaMIRs that were low N-and drought-responsive were further identified. Based on DNA recombinant and genegenetic transformation approaches, the transgenic tobacco lines fused the sense-andantisense-orientation sequences of TaMIR1129, a low N-responsive TaMIR, weregenerated. The capacity of this TaMIR in mediating plant tolerance to nitrogendeprivation was determined. In addition, the binary expression vectors fused TaCAM2-2and TaCAM2-3, two calmodulin genes in wheat acted as the target genes of TaMIR1118,another low N-responsive TaMIR, were constructed. The tobacco lines integrated thesetarget genes were generated. The main results are as follows:1. Among the38TaMIRs, seven members including TaMIR156, TaMIR399,TaMIR444, TaMIR1118, TaMIR1129, TaMIR1133, and TaMIR1136were responsive tolow N stress. Of these, TaMIR399and TaMIR1133were down-regulated and otherswere up-regulated. Seven members including TaMIR156, TaMIR408, TaMIR119,TaMIR1129, TaMIR1133, TaMIR1136, and TaMIR1139were drought-responsive. Ofwhich, TaMIR1136was up-regulated and others were down-regulated.2. The TaMIRs responding to low N stress and drought possessed various numbersof putative target genes. These target genes could be classified into groups such as signaltransduction, transcriptional regulation, RNA metabolism, primary metabolism,secondary metabolism, trafficking, development, and defense response based on thebiological function.3. Using RT-PCR technique, the primary sequence of TaMIR1129, a lowN-responsive TaMIR, was cloned. The binary expression vectors fused the sense-andantisense-orientation sequences were constructed. Furthermore, the transgenic tobacco lines with overexpression and down-regulated expression of TaMIR1129were generatedvia genetic transformation mediated by Agrobacterium-tumafeciens.4. Using the typical transgenic tobacco lines and the wild type (CK) as the materials,the plant responses to external N supplies mediated by TaMIR1129were evaluated.Under sufficient-N condition, the plant phenotypes, fresh and dry weight per plant weresimilar each other between the transgenic plants and CK. Under low N stress condition,compared with CK, the plant morphological characterization in TaMIR1129overexpressing plants were improved together with significantly increase of plant dryweight per plant. However, there were no obvious variations on plant morphology andplant dry weight between the tobacco plants with down-regulated expression ofTaMIR1129and CK. These results suggest that TaMIR1129has application potentials inevaluation of low N stress tolerance in genotypes (cultivars) in wheat and other crops aswell as in generation of novel crop germplasms with high nitrogen usage efficiency.5. The responding to low N stress in TaCAM2-2and TaCAM2-3exhibited a reversepattern in comparison with TaMIR1118, the interacting TaMIR with above target genes,suggesting that these calmudolin genes are trascriptionally regulated by this wheatmiRNA member. Using RT-PCR and DNA recombinant technology, the binaryexpression vectors fused these target genes were constructed and the transgenic tobaccoplants integrated these genes were generated. These work provides a solid basis forfurther elucidating the functions of these genes on mediating plant tolerance to low Nstress and for understanding the corresponding molecular mechanisms.