马铃薯卷叶病毒56kD蛋白基因及3'端非编码区克隆和序列分析

MOLECULAR CLONING AND NUCLEOTIDE SEQUENCE ANALYSIS OF POTATO LEAFROLL VIRUS 56kD PROTEIN GENE AND 3NON-CODING 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，反转录合成了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－Ｃｈ５６ｋＤ蛋白基因由１５２７个核苷酸组成，编码５０８个氨基酸，３＇端非编码区由１４１个核苷酸组成，与国外报道的苏格兰ＰＬＲＶ－Ｓ、荷兰ＰＬＲＶ－Ｎ、加拿大ＰＬＲＶ－Ｃ、澳大利亚ＰＬＲＶ－Ａ各株系核苷酸序列和氨基酸序列有很高的同源性。５６ｋＤ蛋白基因核苷酸同源率分别为９７．１％、９７．８％、９７．１％和９３．９％，推测的氨基酸同源率分别为９６．２％、９６．４％、９５．８％和９４．６％，非编码区核苷酸同源率分别为９７．９％、９７．２％、９８．６％和９８．６％。

Based on the reported genomic RNA sequence of PLRV,two specific primers were synthesized.The cDNA of the 56kD protein gene and its 3non-coding region of PLRV-Ch was synthesized by RT-PCR reaction and further cloned in plasmid pUC19 which was reconfirmed to be required by PCR test and restriction mapping.The sequencing data showed that the 56kD gene consists of 1527nt encoding 508 amino acids,with a 3 non-coding region of 141nt.The 56kD sequence homologies,if compared with the corresponding region of PLRV-S,-N,-C and -A,were 97.1%,97.8%,97.1%,and 93.9% in nucleotide,and 96.2%,96.4%,95.8% and 94.6% in amino acid while the 3 non-coding region were 97.9% ,97.2%,98.6% and 98.6% respectively.