黄瓜花叶病毒RNA3基因间隔区的功能研究

Studies on the Functions of Intergenic Regions of Cucumber Mosaic Virus RNA3

黄瓜花叶病毒(Cucumber mosaic virus,CMV)是RNA病毒基因组功能研究的重要模式种,其各个开放阅读框及顺式作用元件的功能已较为清楚,但还缺乏RNA3基因间隔区的功能在植物体水平上的相关研究。本研究采用基因合成和双酶切的方法构建了基因间隔区(Intergenic region,IGR)3’UTR、5’UTR和全序列的缺失突变体侵染性克隆(简称pΔ3’UTR,pΔ5’UTR和pΔAll),并结合农杆菌介导的瞬时表达分析了IGR的功能。接种结果显示,注射pΔ3’UTR侵染性克隆的本氏烟表现出与注射野生型(Wild type,Wt)的本氏烟相似的症状,但要比Wt晚3.5d,而注射pΔ5’UTR、pΔAll的本氏烟没有表现任何症状,直至枯萎。Western Blot结果显示,注射突变体的本氏烟中,只有注射pΔ3’UTR的本氏烟在出现症状后可被检测到外壳蛋白(Coat protein,CP),但远低于Wt中的CP表达量,说明IGR 3’UTR缺失使最终翻译出来的外壳蛋白水平明显降低;Northern Blot结果显示,也只有注射pΔ3’UTR的本氏烟在出现症状后表达了RNA3和RNA4,但表达量始终低于野生型。针对接种叶内RNA3的半定量RT-PCR结果显示,注射Wt、pΔ3’UTR的本氏烟的预期条带亮度都很高,说明RNA3在缺失IGR 3’端序列时仍能正常转录且能正常复制;而注射pΔ5’UTR、pΔAll的本氏烟的预期条带亮度都很低,说明RNA3在缺失IGR 5’端序列或缺失IGR全部序列时,cDNA仍能转录出少量的RNA3,但这些RNA3完全丧失了复制能力。本研究揭示IGR5’UTR含有RNA3复制的必要元件,其缺失将使病毒完全失去复制能力;IGR 3’UTR控制着RNA3复制的效率,其缺失导致RNA3的积累量显著降低;IGR 3’UTR缺失时病毒只翻译出少量的CP,但也使寄主产生严重的症状。

The cucumber mosaic virus(CMV)is an important model for the functional studies of RNA virus genomes. The functions of each open reading frame and cis-acting element are quite clear,but there is little research on the function of the RNA3 intergenic region(IGR)on the plant level. Infectious clones of deletion mutants of the 3’ untranslated region(UTR),5’UTR and full-length within the CMV RNA3 IGR(named as pΔ3’UTR,pΔ5’UTR,and pΔAll,respectively) were constructed by gene synthesis,double digestion,and Agrobacterium-mediated transient expression. Inoculation experiments showed that Nicotiana benthamiana inoculated with pΔ3’UTR showed symptoms similar to those inoculated with the wild type(Wt),but at a delay of 3.5 days in comparison with the Wt,N. benthamiana inoculated with pΔ5’UTR or pΔAll did not show symptoms until their death. Western Blot showed that the coat protein(CP)was detected in N. benthamiana inoculated with pΔ3’UTR and Wt,but not in those inoculated with pΔ5’UTR or pΔAll. However,N.benthamiana inoculated with pΔ3’UTR showed very low CP expression in comparison with those inoculated with Wt,indicating that the IGR 3’UTR deletion reduced the translation of CP significantly. Northern Blot showed that RNA3 and RNA4 were detected in N. benthamiana inoculated with pΔ3’UTR and Wt,but not in those inoculated with pΔ5’UTR or pΔAll. However,N. benthamiana inoculated with pΔ3’UTR gave very low expression of RNA3 or RNA4 in comparison with those inoculated with Wt. Semi-quantitative reverse transcription-polymerase chain reaction of inoculated leaves showed that the expected bands of N. benthamiana inoculated with Wt or pΔ3’UTR exhibited high brightness,and their brightness was very similar,indicating that transcription or replication occurred if RNA3 lacked the 3’UTR of IGR. The expected bands of N. benthamiana inoculated with pΔ5’UTR or pΔAll exhibited very low brightness. This finding indicated that a small amount of RNA3 was transcribed from the cDNA infectious clones when RNA3 lacked the IGR 5’UTR or whole IGR,and RNA3 replication was completely inactive. The present study suggested that IGR 5’UTR contained the necessary elements for RNA3 replication,and its deletion completely disabled the virus-infecting ability;IGR3’UTR controlled the efficiency of RNA3 replication,and its deletion led to a significant decrease in RNA3 expression;if the IGR 3’UTR was absent,the CMV expressed only a small amount of CP,which could continue to cause severe symptoms in the host.