To gain insights into the function of potential post-translational modifications on the activity of the Cucumber mosaic virus (CMV)-encoded silencing suppressor protein 2b, one predicted phosphorylation site (S40) and two predicted ubiquitination/sumoylation sites (K22 and K39) in CMV-Q2b protein were individually or simultaneously mutated by site-directed mutagenesis methods. These Q2b mutants were inserted into plant expression vectors, expressed in plant leaves, and then analyzed for their silencing suppressor activities. The results showed that S40A mutation greatly impaired both the local and systemic silencing suppressor activity, and the K22R mutation has no significant effect on the suppressor activity, while the K22R/K39R double mutation reduced the systemic silencing suppressor activity. To test if the decrease of suppressor activity were due to protein accumulation changes, western blot were performed to moniter the protein level of Q2b mutants. The results indicated that mutations of both K22 and K39 to R or S40 to A all significantly reduced the accumulation of the Q2b protein in plants, while the single mutation of K22 to R did not alter the accumulation of Q2b protein, suggesting that two potential post-translational modification sites, K39 and S40, contribute to the suppressor activity and stability of 2b protein in plant cells.%黄瓜花叶病毒(Cucumber mosaic virus,CMV)编码的2b蛋白具有RNA沉默抑制子功能,为了研究翻译后修饰对2b功能的影响,利用反向PCR定点突变方法对CMV-Q株系2b蛋白的1个预测的磷酸化位点(S40)和2个预测的泛素化/SUMO化位点(K22,K39)进行了点突变,同时将点突变体插入植物表达载体.通过农杆菌共注射法对3个2b突变体的抑制子活性进行了分析,结果证明,当S40突变为A(2bs4OA)后,2b抑制局部和系统沉默的活性均大幅降低;当K22突变为R(2bmK22R)后,2b的抑制子活性无明显变化;而将K22和K39均突变为R(2bK22R/K39R)后,2b抑制系统沉默的活性则大幅减弱.对叶片中2b突变体的蛋白含量的Western blotting检测表明,与野生型2b蛋白相比,2bK22R在植物中积累水平变化不大,而2bS40A和2bK22R/K39R的蛋白积累量则明显降低,说明K39和S40位点在维持2b蛋白的抑制子活性及其在植物细胞中的稳定性方面起重要作用.
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