黄瓜花叶病毒丹参株系外壳蛋白基因的克隆和序列分析

The Cloning and Sequence Analysis of Coat Protein Gene of Cucumber Mosaic Virus Isolate from Salvia Miltorrhiza Bge

从河北安国具有典型花叶症状的大田丹参叶片中分离到病毒病原 ,经过生物学接种、电镜观察和酶联免疫吸附测定表明该离物与CMV有较近的亲缘关系 (记为CMV DS)。从叶片中提取了该病毒的总RNA ,按照CMV外壳蛋白 (CP)基因前后保守区序列 ,设计合成了 1对特异引物 ,采用RT PCR法扩增了 780bp的全长CP基因的cDNA序列 ,将其克隆到pGEM T载体上 ,并进行序列分析。结果重组克隆序列长为 778bp ,含 1个开放阅读框架 (ORF) ,长度为 65 7nt,编码 2 1 8个氨基酸组成的蛋白。与国内外报道的CMV株系序列比较 ,CMV DS和亚组Ⅰ株系的核苷酸序列和氨基酸序列的同源性为 93 3%～ 99 4%和95 9%～ 1 0 0 % ;而与亚组Ⅱ株系的核苷酸和氨基酸序列同源性仅为为 78 4%～ 79 0 %和81 2 %～ 83 0 % ,表明CMV DS与CMV亚组Ⅰ的株系较亚组Ⅱ株系同源关系更密切 ,将其划归为CMV亚组IB中 ,这是首次在丹参上发现的CMV株系。

A virus named CMV-DS isolated from Danshen (Salvia miltorrhiza Bge.) leaves with typical mosaic symptoms was collected in Anguo county of Hebei Province,which is very similar to Cucumber mosaic virus (CMV)in biological inoculation?electron micrography and Double-antibody Sandwich Enzyme-linked immunosorbert assay (DAS-ELISA).Total virus RNA was extracted from infected Danshen leaves,a pair of DNA primers were synthesized based on the forward and behind region of the CMV coat protein (CP) gene nucleotide sequence. The 780 bp cDNA product of CP gene was amplified by reverse-transcriptase polymerase chain reaction (RT-PCR) and it was cloned into the pGEM-T vector and sequenced. The results showed that the cloned segment was 778 bp and contained one open reading frames (ORF) which was composed of 657 nucleotides encoding a polypetide of 218 amino acids. Comparison of the nucleotide and amino acid sequences of the full length of CP gene of CMV-DS with the corresponding sequences of several CMVⅠisolates showed that the homology are 93.3%～99.4% at nucleotide level,and 95.9%～100% at amino acid level,on the other hand,it were 78.4%～79.0% at nucleotide level,and 81.2%～83.0% at amino acid level with the strains of CMVⅡ,which suggested that the CMV-DS belongs to CMVⅠother than CMVⅡ,so it was sort to CMV IB. This is the first reported CMV isolate from Danshen in China.