Quantification and extension of transient GFP expression by the co- introduction of a suppressor of silencing
Quantification and extension of transient GFP expression by the co- introduction of a suppressor of silencing
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DOI：
10.1007/s11248-008-9192-5
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发表时间：
2008-12-01
影响因子：
3
通讯作者：
Finer, John J.
中科院分区：
文献类型：
--
作者：
Chiera, Joseph M.;Lindbo, John A.;Finer, John J.
利用粒子轰击法，将含有绿色荧光蛋白（GFP）报告基因的DNA表达载体导入植物细胞。GFP基因的表达是瞬时的;导致在引入后约24小时达到峰值GFP表达，此后迅速下降。基因表达的这种众所周知的下降以前被归因于涉及引入的DNA丢失或细胞死亡的整合前DNA事件。在这里，我们表明，转录后基因沉默（PTGS）也参与其中。单独引入GFP表达载体导致30 h后瞬时表达迅速下降。然而，如果GFP表达为与来自烟草蚀纹马铃薯Y病毒的RNA沉默抑制蛋白HCPro的翻译融合物，则转基因表达延长至远超过100小时。突变体分析表明，一个功能性的HCPro蛋白质是必需的瞬时表达的这种扩展。各种缺失和翻译融合分析证实，蛋白质的C-末端区域是重要的抑制活性和整个蛋白质的最佳抑制宿主沉默。粒子轰击过程中基因表达的瞬时性似乎是由PTGS的诱导引起的，这可以通过沉默抑制子的存在来减轻。RNA沉默抑制蛋白的使用可能使粒子轰击介导的瞬时表达试验更有助于评估影响基因表达的因素。
Using particle bombardment, a DNA expression vector containing the green fluorescent protein (GFP) reporter gene was introduced into plant cells. Expression of the GFP gene was transient; resulting in peak GFP Expression about 24 h post introduction and a rapid decline thereafter. This well known decline in gene expression has previously been attributed to pre-integrative DNA events that involved the loss of introduced DNA or cell death. Here, we show that post-transcriptional gene silencing (PTGS) is also involved. Introduction of a GFP expression vector alone resulted in a rapid decline in transient expression after 30 h. However, if GFP was expressed as a translational fusion to the RNA silencing suppressor protein HCPro from tobacco etch potyvirus, transgene expression was extended to well over 100 h. Mutant analyses of HCPro showed that a functional HCPro protein was required for this extension of transient expression. Various deletion and translational fusion analyses confirmed that the C-terminal region of the protein was important for suppressor activity and the entire protein was required for optimal suppression of host silencing. The transient nature of gene expression during particle bombardment appears to result from induction of PTGS, which can be mitigated by the presence of a suppressor of silencing. The use of RNA silencing suppressor proteins may make particle bombardment-mediated transient expression assays more useful for evaluating factors that effect gene expression.