摘要： 为从分子水平鉴定山东聊城的一表现明显花叶、黄化、蕨叶及果实畸形的南瓜病毒病的病原.采用RT-PCR的方法,用TMV外壳蛋白(CP)基因的特异引物,对该样品进行了检测,克隆到的基因序列进行分析,并使其在大肠杆菌中表达.结果表明,从该样品中扩增到了TMV的CP基因,说明该样品受到TMV的侵染,首次发现TMV自然侵染南瓜.对CP基因序列测定及分析表明,与GenBank上其他TMV分离物CP基因核苷酸序列的同源性为86.%～99.0%,推导的氨基酸序列同源性为93.7%～99.4%.根据完整CP 基因核苷酸序列构建的系统进化树显示:32个TMV分离物可分为个组,其中TMV-liaocheng与Nakron Pathom、Fujian、017等分离物属于Ⅳ组.TMV-liaocheng可能发生过重组.将TMV-liaocheng CP基因与原核表达载体pET-22b(+)连接,在大肠杆菌BL21(DE3)pLysS诱导表达出分子量约20 kDa的融合蛋白,并用Ni2+ -NTA His · Bind(R)树脂纯化,纯化后的蛋白可直接用于制备特异性的抗血清,为准确、快速地检测TMV奠定了基础.

关键词: 南瓜, 烟草花叶病毒, CP基因, 序列分析, 原核表达

Abstract: Tobacco mosaic virus(TMV) is responsible for considerable economic losses in the growing of plant. There is no report that TMV infects pumpkin plant naturally. TMV was detected from one pumpkin(Cucurbita moschata) plant in Liaocheng,Shandong Province. The nucleotide sequences of coat protein (CP) gene of TMV isolate (TMV-liaocheng) was determined from RT-PCR derived cDNA clones.It was the first report that TMV naturally infected pumpkin. The obtained CP gene sequences were compared with sequences of TMV that are available in the GenBank. Results showed that the CP gene of TMV-liaocheng shared similarity of 86. 5%-9. 0% with those of other TMV isolates at nucleotide acid level,and identity of 93. 7%-99. 4% at amino acid level. The phylogenetic tree constructed with the complete nucleotide sequence of the CP genes showed that 32 TMV isolates were divided into 5 groups,and TMV-liaocheng,Nakron,Pathom,Fujian and 017 isolates belonged to group Ⅳ. There were probably recombination event during the evolution of TMV-liaocheng. It was the first report that TMV infected pumpkin. The CP gene of TMV-liaocheng was inserted to expression vector pET22b(+ ) and transferred into E. coli BL21(DE3) pLysS. SDS-PAGE showed that TMV-liaocheng CP gene was expressed as a 20 kDa fusion protein when induced with IPTG. The expressed fusion protein was purified through Ni2 +-NTA His. Bind column,and it could be used to immunize the mice for preparing the specific the antibody.

Key words: Pumpkin, Tobacco mosaic virus, CP gene, Sequence analysis, Prokaryotic expression

中图分类号: 

• Q78

• S642.4

• S432.4+1