Molecular dissection of species-specific transgene silencing

本研究对龙胆草35 S启动子特异性DNA甲基化的分子机制进行了探讨。在对转基因龙胆中的一个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有序列，龙胆核提取物可以结合到该序列上，并且酵母单杂交筛选确定了结合到该序列上的候选者。此外，采用悬浮培养的转基因龙胆草细胞研究了DNA甲基化与组蛋白修饰的关系，结果表明35 S区域的从头甲基化与组蛋白修饰有关。为了研究35 S启动子特异性DNA甲基化是否存在于其他植物物种中，产生了具有35 S启动子的转基因莴苣植物。结果表明，在转基因莴苣植株中发现了转基因表达的抑制和35 S启动子区域的高甲基化。

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.