Study On The Relationship Of Exogenous Gene Expression And Methylation In Brich Hybrid Progeny

In this paper,transgenic birch and their F1 were selected as the experiment materials.GUS histochemical staining combined with multiple PCR were used to test the exogenous gene genetic stability,while the real-time PCR technology were used to study the expression quantity of bgt gene.In order to explore the mechanism of gene silence,F1 seedlings were selected to analyze their genetic variation situation of methylation sites by bisulfite DNA sequencing method.It was the first time to isolate methyltransferase gene CMT,MET and DRM from birch genomes,and bioinformatics analysis of methyltransferase gene were studied.The different methyltransferase gene expression was studied between the parents and the F1 to lay the foundation for revealing the genetically modified.The main results were as follows:1 The result of 129 seedlings of different hybrids had shown that the exogenous gene separation appeared in the genetic transfer process,and exogenous gene in some seedlings was silenced.The result bgt gene expression indicated that different hybrids showed significantly different expression quantities,and most of the F1 had a higher expression quantity than their parents,especially the reciprocal cross.2 The analysis of DNA methylation level showed that the seedlings of exogenous gene normal expression had a low ratein bgt and gus gene promoter regions and coding regions,ranging from 0 to 14.29%.Except the seedlings of exogenous gene silencing,other F1 seedlings'methylation rates and methylation sites were highly similar to their parents.In seedlings of gene silencing,the bgt gene and gus gene promoter and coding regions were highly methylated.3 BpDRM and BpCMT were isolated from brich by the degenerate primers amplification method combined with RACE cloning method.The length of BpDRM was 1760bp,which encoded the predicted protein of 585 amino acids and shared the highest homology of 89%with populus trichocarpa.The sequence contained two AdoMet_Mtases super families and some multiple substrate binding sites,DNA binding sites as well as the complementary factor binding sites.There was a DNA 5' cytosine methyltransferase functional area from the 1370 to 1720 bp.The cDNA length of BpCMT was 2928bp,encoding 976 amino acids,and shared the highest homology of 77%with maximum,followed by populus trichocarpa which amino acid homology was 73%compared to brich.Multiple histone binding sites,nine DNA binding sites,six substrate interaction binding sites and one cofactor binding site were predicted from BpCMT gene,while three conserved motifs and four superfamilies sequences were found.4 BpMET gene was isolated from brich using chromosome walking method.BpMET coding sequence information was obtained through blastx.According to the coding regions information,the cDNA conservative primers were designed to clone the cDNA sequence of BpMET gene.For BpMET gene,five steps including one step downstream and four successive steps upstream amplifications generated fragments of 1584,1366,1192,1691 and 1132 bp,with overlapping sequences of 71,42,93,82 and 94 bp to establish a contig sequence of a maximum size of 7424 bp.The coding cDNA sequences of 7424 bp were determined,which encoded the predicted protein of 1571 amino acids and showed the highest amino acid homology of 86%with Vitis vinifera.Six conserved motifs,three superfamilies sequences,five DNA binding sites,four cofactor binding sites and fou substrate interaction binding sites were predicted.Compared BpMET whole sequencing with BpMET coding cDNA sequences,eleven intron regions were found,eight of which had typical GT-AG substructure features.In phylogenetic analysis,MET gene,CMT gene and DRM gene had their own clade of group.Methyltransferase gene of brich had the closer genetic relationship with Populus trichocarpa and Vitis vinifera,and METgene and CMT gene had a closer genetic distance than DRM gene.5 The bioinformatics analysis of the three methyltransferase gene encoding protein showed that CMT,DRM and MET were all hydrophilic proteins,and DRM was stable protein,and CMT and MET were labile proteins.The secondary structure analysis showed that the three methyltransferase all consist of alpha helix,extended strand and random coil,but the rates of the three secondary structures were different among the methyltransferases.MET existed a transmembrane domain,DRM and CMT existed two transmembrane domains.6 Expression of methyltransferase was compared among seedlings by real-time PCR.The results indicated that the F1 expression quantities slightly below their parent,but there were differences from direct crossing and reciprocal crossing.The higher methyltransferase gene expression could cause the high methylation level in order to reduce the expression of exogenous gene,and even be silenced.