马铃薯卷叶病毒中国分离株外壳蛋白基因及其上游先导序列的cDNA克隆和序列分析

The cDNA Cloning and Nucleotide Sequence Analysis of PLRV-Ch Coat Protein Gene and Its Leader Sequence

按照马铃薯卷叶病毒（ＰＬＲＶ）核苷酸序列，针对ＣＰ基因及其上游基因间隔区全长约０．８ｋｂ的区段设计合成两个特异性引物，以马铃薯卷叶病毒中国分离株（ＰＬＲＶ－Ｃｈ）的ＲＮＡ为模板，反转录合成ＣＤＮＡ第一条链，再经ＰＣＲ扩增合成ｃＤＮＡ，将ＣＤＮＡ克隆于ｐＵＣ１９质粒．限制性酶切分析和核苷酸序列测定表明克隆的ＰＬＲＶ－Ｃｈ外壳蛋白（ＣＰ）基因及其上游基因间隔区的全长ＣＤＮＡ共８２４个核苷酸，与国外报道的４个ＰＬＲＶ分离株的核苷梳序列相比具有高度同源性．ＰＬＲＶ的外壳蛋白基因序列与其上游基因间隔区相比保守性更强．

Two specific primers were designed and synthesized according to the genomic sequence reported by Mayo (1989). The first strand of cDNA was synthesized by reverse-transcription usingRNA of potato leaf roll virus Chinese isolate (PLRV-Ch) as a template. The cDNA was amplified by polymerase chain reaction and cloned into plasmid pUC19. The cDNA clone was further identified by restriction mapping analysis and nucleotide sequence analysis. The results show that the coat protein gene and its upstream intergenic segtlence of PLRV-Ch consists of 824 nucleotides and are highly homologous to the CP genes and their leader sequences of PLRV-N.PLRV-C.PLRV-A adn PLRV-S. The nucleotide sequence of PLRV coat protein gene is more conserved than that of intergenic sequence.