Institute of Biotechnology, Shiraz University, Shiraz, Iran.
Department of Plant Protection, School of Agriculture, Shiraz University, Shiraz, Iran.
Mol Biol Rep. 2021 Oct;48(10):6719-6728. doi: 10.1007/s11033-021-06662-w. Epub 2021 Aug 21.
DNA methylation is one of the most important epigenetic event that regulates gene expression. In addition to DNA methylation, transgene copy number may induce gene silencing. Therefore, the study of these cases is useful for understanding of gene silencing regulation.
In this study, the methylation pattern of 35S promoter was investigated in the second generation of MAP30 transgenic tobacco lines. Therefore, the genomic DNA melting curve changes were investigated before and after bisulfite treatment by real time PCR. To determine the exact position of methylation, the samples were sequenced after bisulfite treatment. Observation of decrease in DNA melting curve of expressing line in comparison with silenced line confirmed the presence of DNA methylation in silenced line. In order to induce the MAP30 expression, the silenced line was treated using different concentrations of Azacytidine and green tea extracts. The results showed that all concentrations of green tea extracts for 6 days and the concentrations of 3 and 10 μM Azacytidine for 10 and 3 days could induce the expression of MAP30 in silenced line respectively. Finally, the transgene copy number was estimated using real time PCR, as silenced line contained more than two copies while the lines expressing MAP30 contained only one or two copies.
Finally, we found that the presence of DNA methylation and also multiple gene copy numbers in silenced line have been led to gene silencing. Moreover, the effect of green tea extract on DNA methylation showed incredible results for the first time.
DNA 甲基化是调控基因表达的最重要的表观遗传事件之一。除了 DNA 甲基化之外，转基因拷贝数也可能诱导基因沉默。因此，研究这些情况有助于理解基因沉默的调控机制。
本研究调查了 MAP30 转基因烟草系第二代中 35S 启动子的甲基化模式。因此，通过实时 PCR 研究了亚硫酸氢盐处理前后基因组 DNA 熔解曲线的变化。为了确定甲基化的确切位置，在用亚硫酸氢盐处理后对样品进行测序。与沉默系相比，表达系 DNA 熔解曲线的下降表明沉默系存在 DNA 甲基化。为了诱导 MAP30 的表达，沉默系用不同浓度的氮杂胞苷和绿茶提取物进行处理。结果表明，绿茶提取物的所有浓度处理 6 天，以及浓度为 3 和 10 μM 的氮杂胞苷处理 10 和 3 天，分别可以诱导沉默系中 MAP30 的表达。最后，使用实时 PCR 估计转基因拷贝数，沉默系中含有超过两个拷贝，而表达 MAP30 的系中仅含有一个或两个拷贝。
最后，我们发现沉默系中 DNA 甲基化的存在以及多个基因拷贝数导致了基因沉默。此外，绿茶提取物对 DNA 甲基化的影响是首次发现的令人难以置信的结果。
