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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。
Objective: 2b protein encoded by cucumber mosaic virus (CMV) has the function of RNA silencing suppressor, and its C-terminal amino acid sequence is very conservative. In order to clarify the role of the conserved sequence of 2b protein in the silencing of RNA, we constructed a plant transient expression vector of CMV Q strain wild-type 2b and its C-terminal deletion mutant 2bdelC. The silencing inhibitory activity of wild-type 2b and its C-terminal mutant was analyzed by Agrobacterium co-diafiltration. RESULTS AND CONCLUSION: Western blot analysis of wild-type 2b and its C-terminal mutant in tobacco inoculated leaves showed that the level of accumulation of wild-type 2b protein and its C-terminal mutant did not change much in plants, suggesting that the C-terminal amino acid residues of 2b It is ineffective to maintain the stability of the 2b protein in plant cells. The effects of wild-type 2b and its mutant 2bdelC on co-expression of GFP were compared at the whole plant, cell and molecular levels, respectively. The results showed that there was no significant difference in all the assay results, indicating that the 2b protein C-terminal The 94-111 amino acids have no biological activity in inhibiting local RNA silencing. It is hypothesized that there should be no C-terminal binding domain with small RNA.