Molecular dissection of species-specific transgene silencing

本研究对龙胆中35S启动子特异性DNA甲基化的分子机制进行了研究。在对转基因龙胆的整个T-DNA片段进行甲基化分析后，发现了两个不同的从头甲基化区域。这两个区域都包含两个共同的序列，龙胆核提取物可以与之结合，酵母单杂交筛选确定了与这些序列结合的候选序列。此外，利用转基因龙胆细胞悬浮培养研究了DNA甲基化与组蛋白修饰的关系，结果表明35S区域发生了与组蛋白修饰有关的从头甲基化。为了研究35S启动子特异的DNA甲基化是否存在于其他植物中，我们获得了含有35S启动子的转基因生菜植株。结果表明，转基因生菜中转基因表达受到抑制，35S启动子区域高甲基化。

Molecular mechanism of the 35S promoter-specific DNA methylation in gentian was investigated in this study. Two distinct de novo methylated regions were identified after the methylation analysis of an entire T-DNA segment in transgenic gentian. Both of the regions contain two consensus sequences, to which gentian nuclear extracts could bind, and yeast one-hybrid screening identified candidates for binding to the sequences. Additionally, relationship between DNA methylation and histone modification was investigated using transgenic gentian cell suspension culture, and the results showed that de novo methylation of the 35S region occurred in connection with histone modification. To investigate whether the 35S promoter-specific DNA methylation exists in other plant species, transgenic lettuce plants having 35S promoter were produced. As the result, suppression of the transgene expression and hypermethylation of the 35S promoter region were found in the transgenic lettuce plants.