The Function And Regulation Pathway Analysis Of Wheat(Triticum Aestivum L.)microRNA021b

MicroRNAs(miRNAs)play essential roles in plant growth and development through regulating target genes negatively.MiR021b was found to be specifically expressed highly in wheat seed embryo.In this thesis,we study miR021b biological functions and regulation pathway deeply.The main results were summarized as follows:1.Three miR021b precursor sequences located on chromosomes 7A,7B,and 7D.And levels of pri-miR021b-7D transcript was much higher than the levels of pri-miR021b-7A and pri-miR021b-7B transcripts.MiR021b starts to accumulate to detectable at 15 days after pollination,with a peak around 25 DAP,which is at its highest level in early germination stages.After 6 hours of imbibition,miR021b levels gradually decreased.2.Analysis of the pri-miR021b-7D promoter sequence identified multiple sequence motifs potentially bound by components of the ABA signaling pathway;these motifs included 6 ABA Response Elements(ABREs),3 Sph(RY)Elements,and 1 Coupling Element 1(CE1)-like.TaVP1 binds two of three RY elements and the unique CE1-like elements.Three out of the six ABREs located in the pri-miR021b-7D promoter are specifically bound by TaABF and TaABI5.The transient transformation assays of wheat immature embryos showed that overexpressing TaVP1,TaABF,or TaABI5 induces a statistically significant increase of endogenous miR021b levels.3.LNCR was miR021b target.Modified RNA ligase-mediated 5'-rapid amplification of cDNA ends(RLM-5'-RACE)further indicated that cleavage of LNCR occurs between the 10th and the 11th nucleotide from the 5' end of miR021b.Analysis of LNCR full-length cDNA showed that this wheat transcript represents a long non-coding RNA.Using the LNCR sequence as a query against the wheat genome sequence database,we identified a single copy sequence,located on chromosome 5B.Expression of LNCR was analyzed in various organs and the results detected that LNCR is only in the embryos of germinating seeds.LNCR accumulates from 15-25 DAP and rapidly disappears at 1-6 HAI,when miR021b is most abundant.4.We observed that miR021b has a U at the 5' position and that all three precursors exhibited asymmetry in their foldback structures in the form of a non-paired nucleotide within their miRNA sequence.These characteristics were consistent with those of classical 22-nt miRNA triggers for biogenesis of phased siRNAs in other species.Accordingly,we searched for the presence of ta-siRNAs from LNCR in the small-RNA sequencing datasets from EGS,DG,SH,15SDAP tissues.The result showed that a series of 21-nt regular phased ta-siRNAs located after the initial miR021b-mediated cleavage.RT-qPCR analysis of the most abundant LNCR-derived phased ta-siRNAs,designated 3'P2-on the basis of its location and antisense polarity,indicates that its expression pattern is identical to that of miR021b.5.The transient experiment in cultured immature embryos indicated that over-expression of miR021b,LNCR,3'P2-inhibited the immature embryo germination.And miR021b,LNCR,3'P2-expression level is negatively correlated with the germination rate of 42 wheat cultivars.MiR021b overexpression in wheat exhibit a statistically significant reduction of the germination percentage and strongly improves resistance to PHS.6.We found that some GA responsive genes,especially the gene encoding a-amylase,are down-regulation.The RNA level of amylase,transporter,peroxidase,glycosyl hydrolase,lipase which were predicted ta-siRNAs putative targets are decreased during germination in seeds from the miR021b overexpressing line,compared with wild-type.MiR021b affects seed germination through regulating the GA homeostasis.Levels of bioactive GA1 and GA3 decrease in miR021b overexpressing line compared to wild-type.And TaCPS,TaKO,TaGA3ox which encoding enzyme catalyzed the GA3 and GA1 biosynthesis,are down-regulated in miR021b overexpressing vs wild-type seeds at the germination timepoints analyzed.