Mechanism And Functional Analysis Of A Tobacco MAR On Stability Of Transgene Expression

Gene transfer technology is becoming a fundamental tool to enhance agronomic performance or improve quality traits in a wide variety of crop species. But gene transfer technology are sometimes severely handicapped by transgene silencing and instability of gene expression. Transgene expression is frequently lost after one or a few sexual generations. So it is important to adopt efficient strategies to overcome gene silencing and increase transgene expression level for optimizing transgenic technology.Matrix Attachment Regions (MARs) are the DNA sequences with rich A/T nucleotides that may be involved in anchoring DNA/chromatin to the nuclear matrix. By the interaction with the binding proteins, MARs play an important role in the maintance and modification of the DNA/chromatin structure and the regulation of the gene expression. They have been seen as fundamental tools to reduce or eliminate some forms of transgene silencing because of their capacity to increase transgene expression levels or reduce the variance between transgenic events when flanking a transgene cassette in both plants and animals.TM6 is a matrix attachment region isolated from the genomic DNA of tobacco, which can strongly bind to the tobacco nuclear matrix in vitro. In this study, we investigated the effect of TM6 on stability of transgene expression in the progeny of the primary stably transformed tobacco and the function of TM6 in Arabidopsis. We also investigated the effect of TM6 on the DNA methylation status of promoter regions. The main results were as follows: 1. TM6 can decrease transgene silence of backcross progeny in tobacco, and increase stability of transgene expression in the progeny of the primary tansformed tobacco. This results make it possible for the application of TM6 in transgene plants to increase stability of transgene expression.2. According to the DNaseI accessibility analysis, the CaMV35S promoter adjacent to the TM6 is degraded more rapidly than the control without MAR. Considering the increasing accessibility to DNaseI would result in the decrease of PCR products for the regions of interest, the difference reveals that TM6 plays a role in decreasing DNA methylation of the promoter regions. This maybe one mechanism of TM6 decrease transgene silence and increase gene expression.3. we also detect DNA methylation status of the CaMV35S region and NOS region in transformed tobacco with and without TM6. In our results, there is almost no DNA methylation of CaMV35S regions in transformed tobacco with TM6 but there is 13%～37% DNA methylation of CaMV35S regions in transformed tobacco without TM6. In NOS region, frequence of DNA methylation is 24%～51% in transformed tobacco with TM6 compared 9%～14% those without TM6. This results indicate that TM6 can decrease the DNA methylation in both CaMV35S regions and NOS regions.4. Although TM6 is isolated from tobacco, it can also enhance transgene expression in arabidopsis. We use different type promoters (CaMV35S, PNZIP, COR) to test the influence of TM6 in transgenic arabidopsis, and find that TM6 can enhance gene expression in the transgenic Arabidopsis with three promoters. This indicated TM6 can widely enhance gene expression in different kinds of plants, not change the gene expression pattern.