马铃薯卷叶病毒基因间隔区的克隆及序列分析

The cDNA Cloning and Nucleotide Sequence Analysis of Potato Leafroll Virus Intergenic Region

根据已报道的马铃薯卷叶病毒基因组序列．设计合成一对特异性引物，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，经ＰＣＲ扩增后克隆于ｐＵＣ１９质粒中，进一步用ＰＣＲ鉴定、限制酶切分析和序列分析，结果表明：ＰＬＲＶ－Ｃｈ基因间隔区由１９７个核苷酸组成，与国外报道的荷兰ＰＬＲＶ－Ｎ加拿大ＰＬＲＶ－Ｃ，澳大利亚ＰＬＲＶ－Ａ，苏格兰ＰＬＲＶ－Ｓ各株系核苷酸序列具有很高的同源性，同源率依次为９９％、９８％、９３％、９８％。

wo specific primers were designed and synthesized according to the genomic sequence ofPLRV reported in literature,The first strand of cDNA was synthesized by reverse-transcriptionusing RNA of PLRV Chinese isolate(PLRV-Ch)as a template,followed by PCR amplification.The synthesized cDNA was cloned into plasmid pUC19 in DH5α.The cDNA clone was further i-dentified by PCR,restriction enzymes analysis and nucleotide sequence analysis.Tests show thatthe intergenic region of PLRV-Ch isolate consists of 197 nucleotides,and there is a high homologyin nucleotide sequence in comparision with PLRV Netherland isolate(PLRV-N),PLRV Canadianisolate(PLRV-C),PLRV Austrilian isolate (PLRV-A)and PLRV Scottish (PLRV-S)The rate ofnucleotide sequence homobgy is 99%,98%, 93%and 98%,respectively.