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以大豆花叶病毒北京分离株(SMV-BJ)RNA为模板,随机六聚核苷酸为引物,合成cDNA。根据SMV-BJ外壳蛋白基因序列和国外已报道的SMV,WMV-Ⅱ的有关序列,设计寡聚核苷酸引物。通过聚合酶连锁反应,得到了SMV NIb基因的全长cDNA克隆,并测定了其全序列。所测SMV NIb基因序列与WMV-Ⅱ(USA)株系比较,其核苷酸同源性达80.3％,由核苷酸序列所推导出的氨基酸序列同源性达到91.3％。综合SMV-BJ基因组3′端非编码区、外壳蛋白基因和NIb基因的资料,我们认为:WMV-Ⅱ可能是SMV的一个西瓜株系。实验中发现一些含NIb基因的重组克隆在3′端区域有大的序列变异,并讨论了这种变异的原因。在完成基因拚接、改造的基础上,构建了SMV-BJ NIb基因的高等植物表达载体。
The SMV-BJ RNA of soybean mosaic virus was used as template and random hexamers were used as primers to synthesize cDNA. According to the sequence of SMV-BJ coat protein gene and the reported sequence of SMV and WMV-Ⅱ in foreign countries, oligonucleotide primers were designed. The full-length cDNA clone of SMV NIb gene was obtained by polymerase chain reaction and its full sequence was determined. Compared with the strain of WMV-Ⅱ (USA), the nucleotide sequence of SMV NIb was 80.3% homologous to that of WMV-Ⅱ (USA). The deduced amino acid sequence homology was 91.3%. Based on the data of 3 ’non-coding region, coat protein gene and NIb gene of SMV-BJ genome, we think that WMV-Ⅱ may be a watermelon line of SMV. In the experiment, some recombinant clones containing NIb gene were found to have large sequence variation in the 3 ’end region, and the reasons for this variation were discussed. Based on the completion of gene splicing and transformation, the higher plant expression vector of SMV-BJ NIb gene was constructed.