Sequence And Functional Characterization Of TaMBD9 Encoding Methy-CpG-Binding Domain Protein In Wheat

It is important to explore the molecular mechanism of wheat development regulation for high yield molecular breeding,DNA methylation plays an Important role in the regulation of plant development,CpG binding domain protein(MBD)is a kind of important functional protein which can recognize DNA methylation signal and is directly involved in the development regulation process dependent on DNA methylation.In this study,the transcription and sequence characteristics of methylbinding functions of this gene in the development regulation process preliminarily discussedde.The main findings are as follows:1.Based on the sequence information of TaMBD9 in previous studies,using the wheat genome and transcriptome sequences,a total of 54 transcripts of TaMBD9 gene were obtained,which were divided into chromosome groups 4,5 and 6 located in wheat.the suquence information of different transcripts were analyzed,and the longest transcript of 7 coding proteins was selected as the representative sequence of TaMBD9 gene,named as TaMBD9-6AS、TaMBD9-6BS、TaMBD9-6DS、TaMBD9-4AL、TaMBD9-5BL、TaMBD9-5DL1and TaMBD9-5DL2 according to the location.Genomics analysis indicated that TaMBD9 were composed of 9-12 exons and 8-11 introns.Analysis of proteins encoded in seven transcripts showed that,TaMBD9-6AS/6BS/6DS encoded 1635、1417、1960 and 1536 amino acids,repectively,with the molecular weight of 23 1.86kDa、23 1.63 kDa、232.32 kDa,TaMBD9-4AL/SBL/5DL1/5DL2 encoded 1635、1417、1960 and 1536 amino acids,repectively.with the molecular weight of 183.44 kDa、156.93 kDa、218.16 kDa and 170.57 kDa,with isoelectric point 5.44 to 6.35.Structural analysis of TaMBD9 protein indicated each of seven homeologues had one identical PHD1_Lid2p_like conserved domain,which play important biological functions in the process of chromosome methylation.Three-dimensional structure predictions revealed that sequence consistency of TaMBD9-6AS、-6BS、-6DS、-5DL1and-5DL2 was relatively high,was composed of eleven α-helices、β-sheets and irregular curl.The TaMBD9-4AL has 201 amino acids deletions as compared to TaMBD9-5BL、-5DL1 and-5DL2,the deletions of two β-sheets at the N-terminal were found.Evolutionary tree cluster analysis showed that the homology of the TaMBD9-6AS/6BS/6DS/4AL/5BL/5DL1/5DL2 and AtMBD9 in arabidopsis thaliana was the highest,which belong to the V class of MBD family.The tissue expression of 7 transcripts of TaMBD9 gene were analyed,the result showed that highly expression level of TaMBD9-6AS/6BS/6DS were detected in all tested tissues(seeds,leaves,stems,roots,and spikies);for TaMBD9-4AL,the highest expression levels in seeds and lower expression levels in others tissues.TaMBD9-5BL/5DL1/5DL had the highest expression levels in seed which in filling stage,and lower expression level in roots.The resluts indicated that TaMBD9 family members plays different roles in different wheat tissues.2.In order to confirm the biological function of wheat TaMBD9 gene,the overexpression vectors 1301-TaMBD9-tORF and 1301-TaMBD9-ORF were respectively introduced into the mutant MBD9 gene of arabidopsis thaliana by agrobacterium infection,After screening and identification,six 1301-TaMBD9-tORF and three 1301-TaMBD9-ORF,a total of 9 T2 homozygous transgenic lines,were obtained,Functional recovery of mutant plants after overexpression of TaMBD9 in homozygous transgenic lines was analyzed,the results showed that compared with the mutant control,the flowering time of transgenic lines was delayed by 2-3 days on average after the over-expression of TaMBD9-tORF in arabidopsis mutant mbd9,after overexpression of TaMBD9-ORF,the average delay of transgenic lines was about 1-2 days compared with the mutant control group,It shows that the conservative domain PHD domain,which is absent from TaMBD9-ORF,plays a certain role in functional recovery of mutant.This indicates that the TaMBD9 gene plays a certain role in the methylation dependent flowering regulation process,and further exploration on the regulation mechanism of the flowering process is still in progress.The mutant mbd9 also has the characteristics of multiple lateral branches.In addition to the recovery of different degrees of flowering time,the branch situation of the transgenic strains was observed and it was found that tORF-4-18 strains restored to a single main stem without lateral branches,which was consistent with the wildtype Col.In addition,the leaves of torf-5-22 strain were long,large and flat,while the petioles of torf-2-9 strain were long,the leaves were large,and the whole plant grew strong.This indicates that the insertion and overexpression of the TaMBD9 gene in wheat plays an important role in the growth and development of arabidopsis,and further mechanism of action is still in progress.3.The effects of TaMBD9 gene inhibition on wheat growth and development were analyzed by barley stripe Mosaic virus(BSMV)mediated gene silencing technique.BSMV was inoculated on winter wheat variety "jing 841" and spring wheat variety "yu mai 18",respectively,the expression of TaMBD9 gene was significantly decreased in the plants inoculated with the target gene by qRT-PCR,observation and statistics of wheat growth status and heading time found:Plants inoculated with BSMV-γ-TaMBD9 virus were significantly earlier than those in the control group(those inoculated with BSMV-γ0 virus),The heading time of winter wheat variety"jing 841" was about 13 days earlier than that of spring wheat variety "yu mai 18",and the heading time of spring wheat variety "jing 841" was about 7 days earlier than that of spring wheat variety "yu mai 18",the results indicated that the TaMBD9 gene was involved in the regulation mechanism of wheat flowering.