The Structural And Functional Analysis Of A Methl-Binding Domain Protein Gene TaMBD6 In Wheat

As the most important epigenetic modification,DNA methylation is a significant expression regulation mechanism in eukaryotes,involving in growth,differentiation,development,regulation and environment response.MBD proteins are a class of trans-acting factors,which have the capacity of specific binding with DNA methylation.It is significant to explore the biological functions of MBD in wheat growth and regulation.In this study,the complete ORF and corresponding genomic DNA of TaMBD6 were cloned and their sequence were detailed analyzed.The promoter sequences of TaMBD6-A,B,D were isolated and the plant expression vectors were successfully constructed,respectively.The transgenetic plant of Arabidopsis thaliana were identifed.The main results are listed in the following:1.The complete ORF sequences of TaMBD6 three homologous genes were obtained.The sequences Analysis indicated that,22 single nucleotide substitutions were found among the coding region of three homologous genes,while 9 amino acid differences among proteins.TaMBD6 gene encoded a hydrophilic protein with 446 amino acid And the MBD conserved domain was identified from 9 to 76 in amino acid sequence.Theoretical isoelectric point was 4.41.The content of alanine(Ala),glutamic acid(Glu),proline(Pro)and lysine(Lys)occupied more than half of the number of amino acids.2.Expression pattern of TaMBD6-A,B,D were characterized by real-time RT-PCR.The results showed that the lowest expression of TaMBD6-A was observed at 4 day,then the expression was fluctuated and slow increased during the vernalization in the wheat cultivar Jing841.The expression of TaMBD6-B was fluctuated,and the expression of TaMBD6-D totally increased,but decreased from 16 day to 26 day.The expression in two cultivars(luohan2 and yumai 18)were significantly up-regulated during seed development and the highest expression was detected at 5 days after pollination(DAP).There existed some differential expression in two cultivars,but the expression level were both up-regulated at 25 DAP.We proposed that some genes might re-expression by decreased expression of TaMBD6-A,B,D during seed development,and these genes might be involved in seed development or endosperm formation.3.The corresponding genomic DNAs of TaMBD6-A,B,D were isolated respectively.The compared analysis with cDNA indicated thattwo introns were contained in the three TaMBD6 homoeologous genes,which were consistent with the universal rule of GT-AG.Further study showed that the second introns in TaMBD6-A,TaMBD6-B,TaMBD6-D genes were all 108 bp in longth and no significant differences among them were found.While the 3263,3472,3466 bp in length for TaMBD6 three homoeologous gene were identified in the first introns,respectively.Among the first introns,there are almost the same for TaMBD6-B and TaMBD6-D,but significant difference existed in TaMBD6-A compared to the others.The big first introns of three homoeologous genes were all located in the position of 78 bp downstream from the start codon ATG,which might be involved in the gene expression regulation.4.According to the differences in 5' end of TaMBD6-A,TaMBD6-B,TaMBD6-D genes,homeologous genes-specific primers were designed for genome walking PCR.The promoters of three TaMBD6 homoeologous genes were obtained(1573 bp in TaMBD6-A,942 bp in TaMBD6-B,1302 bp in TaMBD6-D).Transcription start sites were confirmed by the analysis of three TaMBD6 homoeologous genes using PLANTCARE database and the TaMBD6 sequence obtained by 5' RACE.The result shows that the distance between transcription start sites(TSS)and translation initiation site(ATG)was less than 100 bp.The TATA boxes were located at 21,21,23 bp upstream from the transcription start site,respectively.The analysis of promoter sequences indicated that upstream region of the transcription start sites spanning from-300 to-1 bp among the three homologous genes were highly similar.There were highly sequence similarities from-750 to-300 bp between TaMBD6-A and TaMBD6-B,while the sequence among TaMBD6-A,B,D were significant differnce.5.The plant expression vectors of pCAMBIA 1301-TaMBD6-A,B,D promoter were constructed,respectively.The wild-type Col was transformed by Agrobacterium infection.The 22,9,and 23 positive plants of pCAMBIA 1301-TaMBD6-A,B,D were identified,and the next generation observation is in progress,which provided a good foundation for the further analysis that expression of TaMBD6-A,B,D promoter-driven GUS reporter gene in transgenic plants under different light conditions and hormonal stimulation.