Department of Horticultural Biotechnology, Kyung Hee University, 1732 Deogyoung-daero, Giheung-gu, Yongin-si 17104, Gyeonggi-do, Korea.
Genes (Basel). 2021 Sep 30;12(10):1563. doi: 10.3390/genes12101563.
Plant tissue culture is an in vitro technique used to manipulate cells, tissues, or organs, and plays an important role in genetic transformation. However, plants cultured in vitro often exhibit unintended genetic and epigenetic variations. Since it is important to secure the stability of endogenous and exogenous gene expressions in transgenic plants, it is preferable to avoid the occurrence of such variations. In this study, we focused on epigenetic variations, exclusively on methylation level changes of DNA, in transgenic Chinese cabbage ( ssp. ) plants. To detect these methylation level changes of DNA, bisulfite sequencing was performed and the obtained sequences were compared with the 'CT001' reference genome. Differentially methylated regions (DMRs) of DNA between the non-transgenic and transgenic lines were detected by bisulfite sequencing, and ten DMRs located in exonic regions were identified. The regions with methylation variations that were inherited and consistently maintained in the next generation lines were selected and validated. We also analyzed the relationship between methylation status and expression levels of transformant-conserved DMR () genes by quantitative reverse transcription-PCR. These results suggested that the changes in methylation levels of these DMRs might have been related to the plant transformation process, affecting subsequent gene expression. Our findings can be used in fundamental research on methylation variations in transgenic plants and suggest that these variations affect the expression of the associated genes.
植物组织培养是一种体外技术，用于操纵细胞、组织或器官，在遗传转化中起着重要作用。然而，在体外培养的植物往往表现出意想不到的遗传和表观遗传变异。由于确保转基因植物中外源和内源基因表达的稳定性非常重要，因此最好避免这种变异的发生。在这项研究中，我们专注于转基因白菜（ ssp. ）植物的表观遗传变异，仅研究 DNA 的甲基化水平变化。为了检测这些 DNA 的甲基化水平变化，我们进行了亚硫酸氢盐测序，并将获得的序列与“CT001”参考基因组进行了比较。通过亚硫酸氢盐测序检测到非转基因和转基因系之间的 DNA 差异甲基化区域（DMR），并鉴定出 10 个位于外显子区域的 DMR。选择并验证了在下一代系中遗传并持续保持甲基化变化的区域。我们还通过定量逆转录 PCR 分析了转化体保守 DMR（）基因的甲基化状态与表达水平之间的关系。这些结果表明，这些 DMR 甲基化水平的变化可能与植物转化过程有关，影响后续基因表达。我们的研究结果可用于研究转基因植物中甲基化变异的基础研究，并表明这些变异会影响相关基因的表达。
