Strict de novo methylation of the 35S enhancer sequence in gentian.
Strict de novo methylation of the 35S enhancer sequence in gentian.
复制标题
龙胆中 35S 增强子序列的严格从头甲基化。
DOI：
10.1371/journal.pone.0009670
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发表时间：
2010-03-23
期刊：
影响因子：
3.7
通讯作者：
Nishihara M
中科院分区：
文献类型：
--
作者：
Mishiba K;Yamasaki S;Nakatsuka T;Abe Y;Daimon H;Oda M;Nishihara M
在观赏植物龙胆（Gentiana triflora × G. scabra）中发现了一种新的转基因沉默现象，其中引入的花椰菜花叶病毒（CaMV）35S启动子区域被严格甲基化，与转基因拷贝数和整合位点无关。具有相同载体的转基因烟草没有表现出沉默行为。在单拷贝转基因龙胆品系中，不仅未修饰的，而且包含35S增强子序列的修饰的35S启动子也被发现高度甲基化。引入35S核心启动子(-90)的转基因品系显示出小程度的甲基化，这意味着35S增强子序列参与了甲基化机制。严格的沉默现象使我们能够并行分析许多转基因系中的甲基化，从而发现了从头甲基化的共有目标区域，其中包含不对称胞嘧啶（CpHpH；H是A、C或T）序列。因此，在每个（修饰的）35S启动子序列中检测到了不同的从头甲基化足迹，并且增强子区域（-148至-85）被确定为从头甲基化的关键靶标。电泳迁移率变动分析（EMSA）显示，龙胆核提取物中-149至-124和-107至-83区域探针形成的复合物与烟草核提取物中形成的复合物不同，表明这些复合物可能有助于从头甲基化。我们的结果提供了对高等植物中序列和物种特异性基因沉默现象的见解。
A novel transgene silencing phenomenon was found in the ornamental plant, gentian (Gentiana triflora × G. scabra), in which the introduced Cauliflower mosaic virus (CaMV) 35S promoter region was strictly methylated, irrespective of the transgene copy number and integrated loci. Transgenic tobacco having the same vector did not show the silencing behavior. Not only unmodified, but also modified 35S promoters containing a 35S enhancer sequence were found to be highly methylated in the single copy transgenic gentian lines. The 35S core promoter (−90)-introduced transgenic lines showed a small degree of methylation, implying that the 35S enhancer sequence was involved in the methylation machinery. The rigorous silencing phenomenon enabled us to analyze methylation in a number of the transgenic lines in parallel, which led to the discovery of a consensus target region for de novo methylation, which comprised an asymmetric cytosine (CpHpH; H is A, C or T) sequence. Consequently, distinct footprints of de novo methylation were detected in each (modified) 35S promoter sequence, and the enhancer region (−148 to −85) was identified as a crucial target for de novo methylation. Electrophoretic mobility shift assay (EMSA) showed that complexes formed in gentian nuclear extract with the −149 to −124 and −107 to −83 region probes were distinct from those of tobacco nuclear extracts, suggesting that the complexes might contribute to de novo methylation. Our results provide insights into the phenomenon of sequence- and species- specific gene silencing in higher plants.
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影响因子：
11.4
作者：
Mette, MF;Kanno, T;Matzke, AJM
通讯作者：
Matzke, AJM
影响因子：
4
作者：
Mathieu, O;Bender, J
通讯作者：
Bender, J
DOI：
10.1007/bf00285449
发表时间：
1994-08-02
期刊：
MOLECULAR & GENERAL GENETICS
影响因子：
--
作者：
MATZKE, AJM;NEUHUBER, F;MATZKE, MA
通讯作者：
MATZKE, MA
影响因子：
56.9
作者：
BENFEY, PN;CHUA, NH
通讯作者：
CHUA, NH
影响因子：
7.2
作者：
HIEI, Y;OHTA, S;KUMASHIRO, T
通讯作者：
KUMASHIRO, T