Characterization Of Six Novel MiRNAs And Their Target Genes Associated With Grain Development In Wheat

The process of grain development is the key period of yield and quality formation in wheat(Triticum aestivum L.).It`s significant to study the molecular mechanisms of wheat grain development for wheat molecular breeding focused on high yield and great quality.Some reports indicated that miRNAs play important roles in developmental regulation of cereal grain.Based on our previous studies on miRNA expression profiling,six novel miRNAs associated with wheat grain development were identified and their characteristics were also systematically analysed.Additionally,the biological function of partial miRNAs which involed in the developmental regulation of wheat grains were disscussed.The main results were as follows:1.The characterization of six novel miRNAs showed that both of Ta-mi R154-5p and Ta-mi R021-1-5p is 22 bp in length and there is an identical region covering 19 bp,as well as,the Ta-mi R007-5p and Ta-mi R007-3p are complementary and generated from the same stem of Ta-mi R021-1-5p precursor.The sequence of six pre-miRNA could fold into typical hairpin structures.In total,880 target genes of these six miRNAs were predicted and gene ontonology annotation indicated that several pathways,including redox regulation,stress regulation,cell metabolism,protein degradation and signal reansduction were involved.Interestingly,some targets are potentially regulated by 2 or 3 miRNAs tested.According to their functional annotations,several important candidates,encoding Nucleoredoxin,MYB transcription factor,F-box like protein,RPP13 protein and E3 ubiquitin ligase,were selected for further analysis.The sequence analysis of candidate target genesrevealed that the binding sites for most of miRNAs were located in the region of 3`UTR and were potentially regulated by miRNA mediated cleavage of m RNA.Furtherly,the sequence characteristics and expression patterns of partial target genes were analysed by q RT-PCR and the results showed that expression of TaNRX1 was gradually down-regulated during grain development,which was contrary to the expression pattern of Ta-mi R154-5p.Ourresults indicated that TaNRX1 is potentially regulated by Ta-mi R154-5p indeveloping wheat grains.2.The spatio-temporal expression characterization of six novel miRNAs showed that the expression trend of Ta-mi R154-5p and Ta-mi R021-1-5p were up-regulated along with the grain development,while Ta-mi R034-5p decreased firstly and then rised,as well as,the expression level of Ta-mi R007 was down-regulated during grain filling stage.The tissue specific expressing detection showed that these tested novel miRNAs were differentially expressedin different cultivars,as well as cultivars.Ta-mi R154-5p,Ta-mi R021-1-5p and Ta-mi R034-5p were highly expressed in peduncle,internode and roots,while lowly in the flag leaves.In addition,the expression patterns of candidate miRNAs underdrought and salt stress indicated that Ta-mi R154-5p and Ta-mi R021-1-5p might involved in the molecular regulation of osmotic stress responding in wheat.3.The sequence characterization of TaNRX1,a key target gene of Ta-mi R154-5p,showed that the c DNA sequence of three partial homeologues were highly identical(>98%);Characterization of amino acid sequence indicated that three Try X_like_Try X_NRX conserved domains were detected in TaNRX1 and it was classed into the subgroup I of NRX family.The tridimensional structure analysis demonstrated that TaNRX1 can fold into a C-shape patial structure including 4 subunits,which were folded by anti-parallel beta sheets sandwiched between alpha helices.The q RT-PCR based spatio-temporal expression analysis showed that the TaNRX1 is generally down-regulated during the process of grain development.Additionally,TaNRX1 is differentially expressed significantly between different tissues,which showed the highest expression level in internode and peduncle,while the lowest is mature seeds.Under the drought stress,TaNRX1 was downregulated,while the response is weak in leaf tissues of Chinese Spring.Under the osmotic stress,the expression level of TaNRX1 was induced by stress and played important roles during the processes of grain development and osmotic stress responding.Interestingly,repression of TaNRX1 results to an increasing of thousand grain weight with ratio of about 22%,which indicated that TaNRX1 is associated with grain development in wheat.4.To functionally validate the regulating roles of Ta-mi R154-5p in wheat grains,the expression of endogenous Ta-mi R154-5p was down-regulated by STTM technology and the repression of Ta-mi R154-5p was obvious at the time points of 15 days and 20 days after spike inoculation.Furtherly,it was detected that the length and width of Ta-mi R154-5p repressed grains were significantly decreased with the ratios of 11.39% and 16.07%,respectively,as well as the thousand grain weight was reduced by 23.67%.The results indicated that Ta-mi R154-5p plays an important role during the regulation of wheat grain development.