The Analysis Of DNA Methylation In MicroRNA Promoters And The Biological Fuction Of AtWRKY30Transcription Factor In Arabidopsis Thaliana

With the rapidly development of genetics, epigenetics become a research hotspot inrecent years as an important genetic subfield studying epigenetic variation. At present,epigenetics research focuses on DNA methylation, histone modification, chromatinremodeling and epigenetically regulated non-coding RNAs. DNA methylation has been foundin both prokaryotes and eukaryotes, and plays a crucial role in the regulation of geneexpression essential for the nomal development of most multicellular organisms.MicroRNAs(miRNAs) are small, non-coding regulatory RNAs of eukaryotic genomes thatmediate target gene silencing by complemetary with the target mRNAs at both transcriptionand posttranscription levels.The WRKY transcription factor superfamily so far is found unique to plants. WRKYtranscription factors are the new type transcriptional regulatory factors in which N-terminalends contain a conserved WRKYGQK amino acids sequences. WRKY transcription factorsregulate the genes expression by binding to (T)(T)TGAC(C/T) sequence in the promoterregions containing the W-box elements of target genes. WRKY transcription factorsparticipate in the plant various kinds defense responses and regulate the plant growth anddevelopment.We analysis the DNA methylation in miRNA promoters and the biological fuction ofAtWRKY30in Arabidopsis thaliana.(1) The Anno-J analysis showed that there were15miRNAs with DNA methylation inthe promoter regions. By BSP-sequecing analysis, there were all three sequence contexts(CpG,CpHpG and CpHpH) of DNA methylation in miRNA156e, miRNA396a, miRNA399c andmiRNA399f promoters.The modification level of DNA methylation in miRNA396apromoters was high while the level was low in the other miRNAs. (2) The expression of miRNA396a increased in methyl-transferase mutants and thetranscript levels of target genes (bHLH74and GRF5) reduced at the same time comparedwith the WT plants which indicated that the methylation of miRNA promoters could regulatethe expression of miRNA itself.(3) The expression of miRNA396a increased under cold, drought and salt stress whileDNA methylation in miRNA396a promoters were slightly reduced. These results suggestedthat the DNA methylation in miRNA promoters could regulate the expression of miRNA inresponse to abiotic stress probably by the RNA-directed DNA methylation pathway.(4)By promoter: β-glucouronidase (GUS) fusions, the expression pattern ofAtWRKY30showed that it mainly expressed in the leaves while less expressed in flower andsilique revealing that WRKY30may play a predominant role during plant development.(5) Both the seed germination and seedling establishment of AtWRKY30RNAi lines wereobviously inhibited by salt stress while the over expression lines were tolerant to salt stress.Besides, the AtWRKY30RNAi lines were sensitive in response to ABA. These results indicatethat WRKY30mediated early Arabidopsis responses to salt stress, most probably throughABA signal pathways.(6)WRKY30may participate in plant hormones-signal pathway. The seed germinationrate of AtWRKYRNAi transgenic plants reduced in response to ABA and MeJA stress. Bytransferring the one week old AtWRKYRNAi transgenic plants from normal media to ABAand MeJA plates, the plants do not sensitive to the stress any more. These results reveal thatWRKY30may participate in plant hormones-signal pathway during seed germination.