Palmgren G, Mattson O, Okkels F T
Institute of Plant Physiology, University of Copenhagen, Denmark.
Plant Mol Biol. 1993 Feb;21(3):429-35. doi: 10.1007/BF00028801.
We have studied the effect of the demethylating agent azacytidine (azaC) on expression of a beta-glucuronidase (GUS) gene transferred to tobacco leaf disks by Agrobacterium-mediated transformation. In a system where no selection was performed, where shoot formation was partially repressed, and where Agrobacterium does not express the GUS gene, we were able to follow the early events of transient and stable expression. Two days after inoculation, 8% of the cells expressed GUS but this proportion rapidly decreased to near zero in the following week. Treatment of leaf disks with azaC just after transformation retarded this inactivation to some extent, while treatment of Agrobacterium prior to transformation increased the frequency of transient expression. Three weeks after inoculation the number of GUS-expressing cells increased 4- to 6-fold in the leaf disks treated with azaC and in the leaf disks transformed with azaC-treated bacteria, while the control remained low. These data suggest that DNA methylation is involved in transgene inactivation and that a large number of silent but potentially active transgenes become integrated.
我们研究了去甲基化剂氮杂胞苷（azaC）对通过农杆菌介导转化转移到烟草叶盘上的β-葡萄糖醛酸酶（GUS）基因表达的影响。在一个未进行选择、芽形成部分受到抑制且农杆菌不表达GUS基因的系统中，我们能够追踪瞬时表达和稳定表达的早期事件。接种两天后，8%的细胞表达GUS，但在接下来的一周内，这一比例迅速降至接近零。转化后立即用azaC处理叶盘在一定程度上延缓了这种失活，而在转化前处理农杆菌则增加了瞬时表达的频率。接种三周后，用azaC处理的叶盘以及用经azaC处理的细菌转化的叶盘中，表达GUS的细胞数量增加了4至6倍，而对照组仍然很低。这些数据表明DNA甲基化参与了转基因失活，并且大量沉默但潜在活跃的转基因被整合。
