黄瓜花叶病毒反式遗传复制体系的构建

Construction of reverse genetic replication system for Cucumber mosaic virus in Nicotiana benthamiana

为了确定复制酶1a在寄主植物中的含量对黄瓜花叶病毒(Cucumbermosaic virus,CMV)基因组复制的影响,在本氏烟中瞬时表达CMV复制酶1a和2a,构建CMV的反式复制遗传体系,分析1a蛋白不同表达水平对基因组R3-gfp的影响。结果显示:瞬时表达载体pCB301在寄主植物中表达1a和2a蛋白能复制CMV基因组R3-gfp;在1a2aR3-gfp侵染本氏烟中RNA3和RNA4含量低于R1R2R3-gfp,1a2aR3-gfp复制的效率低于R1R2R3-gfp;在1a2aR3-gfp浸润接种的植物中,1a浓度接种提高,CMV基因组RNA3和RNA4复制受到抑制。该构建反式遗传复制体系为进一步研究CMV复制的分子机理提供实验材料以及理论依据。

In order to determinethe effect of the content of replicase1 a in the host plant on genome replication of Cucumber mosaic virus(CMV),replicase 1 a or 2 a were transiently expressed in Nicotiana benthamiana,and reverse replication genetic system of CMV was constructed to analyze the effect of different expression level of 1 a protein on genome R3-gfp.The results indicated that 1 a and 2 a protein expressed from transient expression vector pCB301 could drive the replication of CMV genomic R3-gfp;RNA3 and RNA4 content in N.benthamiana infected by 1 a2 aR3-gfp was lower than that infected by R1 R2 R3-gfp.The replication efficiency of 1 a2 aR3-gfp was lower than that of R1 R2 R3-gfp.In the plants infiltrated and inoculated by 1 a2 aR3-gfp,1 a concentration increased.RNA3 and RNA4 replication were inhibited.The reverse genetic replication system provides the experimental material and theoretical basis for further studying the molecular mechanism of CMV replication.