Analysis of replication of tobacco mosaic virus by means of an in vitro transcription system

烟草花叶病毒（TMV）的基因组是由约6，400个核苷酸组成的单链信使RNA。TMV RNA编码130k、180k、30k蛋白和外壳蛋白。130k和180k蛋白由相同的起始密码子翻译，后者通过通读130k蛋白基因的琥珀终止密码子合成。本研究的目的是为了更好地了解TMV的复制。首先，我们建立了一个体外转录系统，从克隆的全长cDNA中提供感染性TMV RNA。利用该系统构建了多种TMV突变体RNA，并对其进行了分析。结果表明，130k和180k蛋白均参与复制，只有180k蛋白是复制所必需的蛋白，30k和外壳蛋白基因序列中不存在顺式作用元件，这两种蛋白都是复制所必需的，推测的复制酶（130K和180K蛋白）与3 ′非编码序列之间的关系不是那么严格。除此之外，还发现130k和180k蛋白强烈定位于感染特异性形成的细胞质结构中。在研究过程中，我们发展了一种改进的电穿孔方法，利用这种方法可以用少量的TMV RNA高效地感染烟草原生质体。

The genome of tobacco mosaic virus (TMV) is a single-stranded, messenger-sense RNA composed of about 6,400 uncleotides. TMV RNA encodes 4 proteins: 130k, 180k, 30k and coat proteins. The 130k and 180k proteins are translated from the same initiatin codon and the latter is synthesized by read-through of the amber termination codon of the 130k protein gene. The purpose of this research is to lead to a bettwe understanding of TMV replication. First, we estabilished an in vitro transcription system that provides infectious TMV RNAs from cloned full-length cDNAs. By using this system, we constructed verious mutant TMV RNAs and analyzed their characters. Results showe that both the 130k and 180k proteins are involved in replication, only the 180k protein is the indispensable protein for replication, there are no cis-acting elements in the 30k and coat protein gene sequences, these two proteins are dispensable for replication, and that the relationship between the putative replicase (the 130k and 180k proteins) and 3' non-coding sequence is not so stringent. Besides these results, it was reveald that the 130k and 180k proteins are strongly localized in a cytplasmic structure formed specific to infection. In the course of the research, we developed an improved electroporation method by which tobacco protoplasts can be efficiently infected with a small amount of TMV RNA.