黄瓜花叶病毒2b蛋白C末端无抑制局部RNA沉默的活性

The C-terminal Residues of the Cucumber Mosaic Virus 2b Protein are Not Responsible for RNA Silencing Suppressor Activity

目的:黄瓜花叶病毒(Cucumber mosaic virus,CMV)编码的2b蛋白具有RNA沉默抑制子的功能,其C末端氨基酸序列非常保守。为了明确2b蛋白C末端保守序列在RNA沉默抑制中的作用,构建了CMV Q株系野生型2b及其C末端缺失突变体2bdelC的植物瞬时表达载体。通过农杆菌共渗滤法对野生型2b及其C末端突变体的沉默抑制子活性进行了分析。结果与结论:烟草接种叶片中野生型2b及其C末端突变体的Western blot检测表明,野生型2b蛋白与其C末端突变体在植物中积累水平变化不大,说明2b蛋白C末端氨基酸残基在维持2b蛋白在植物细胞中的稳定性方面无作用。在整株、细胞和分子水平上分别比较了野生型2b及其突变体2bdelC对共表达GFP的表达量影响,结果表明在所有的测定结果中二者均无明显地差异,说明2b蛋白C末端94-111位氨基酸在抑制局部RNA沉默上无生物学活性,讨论推测C末端应不存在与小RNA结合的结构域。

To gain insights into the function of the conserved residues in the C-terminal of the Cucumber mosaic virus（CMV）-encoded silencing suppressor protein 2b,the 2b protein of Q-CMV,a subgroup II isolate,and 2bdelC,a C terminal truncated mutant of Q 2b,were individually constructed into plant expression vectors.Those two vectors were co-delivered into Tobacco plants（Nicotiana benthamiana）with a Potato virus X-derived expression vector（PVXdt-GFP） by means of an agrobacterium-mediated transient transformation assay and then analyzed for their silencing suppressor activities.Western blotting was performed to monitor the protein level of 2b and its mutants.The results showed that the truncation did not alter the accumulation of Q 2b protein in plant cell and this indicated that the C-terminal residues are not crucial for the stability of 2b protein in vivo.We further show that the co-expressed GFP have indistinguishable expression levels in the presence of Q 2b protein or the Q 2b C-terminal mutant.The results implicate the C-terminal residues neither have effect on the suppressor activity nor contain any small RNA binding domains.