Methylation pattern analysis of double-transformed tobacco plants with antisense-oriented MET1 gene.

DNA methylation in plants is related to a number of epigenetic phenomena. Heavy methylation of cytosine residues plays an important role in gene expression, and significant differences in cytosine methylation levels have been observed among various tissue types, which can be explained as one of the regulatory mechanisms during development and differentiation. Tobacco (Nicotiana tabaccum cv. Havana SR1) was transformed with Agrobacterium tumefaciens LBA4404 containing a rescue cloning vector, pRCV2. The T-DNA tags were isolated by rescue cloning. These intact T-DNA tags, obtained by rescue cloning from transgenic lines, were fully sequenced to analyze the actual insertion site of T-DNA in tobacco. In the case of mutant line single transformant (ST) #5-1 and 2, blast search of plant flanking DNA were shown homology with N. tabaccum RENT family: tCUP promoter. Flanking plant DNA of ST #2-3 and #8-0 clones showed significant aligriments to H2 trans-silencing gene and enoyl-acyl carrier protein reductase, respectively. Transgenic plants transformed with pRCV2 were retransformed with A. tumefaciens EHA105 carrying pFTM vector. Restriction enzyme digestion patterns of RENT repetitive sequence were analyzed using DNA extracted from the STs and the double transformants (DTs) in order to determine whether transcriptional gene silencing (TGS) of MET1 affected DNA methylation. As a result, STs with dsMET1 decreased methylation in all DTS. This result means that DT-MET1 transformants causes hypomethylation of the STs, presumably due to TGS of MET1.