从表现红心病的潍坊萝卜块根上得到一芜菁花叶病毒(Turnip mosaic virus, TuMV)分离物(TuMV-Ra),通过RT-PCR获得了该分离物的外壳蛋白(CP)基因,对其进行了序列测定,并将其核苷酸序列及推导出的氨基酸序列与GenBank中登录的其它20个TuMV萝卜分离物的相应序列进行比较和分析。结果表明,TuMV-Ra与这20个分离物CP基因核苷酸序列的同源性为89.9%~99.O%,CP氨基酸序列同源性为94.8%~99.7%;其中与中国浙江杭州萝卜的两个分离物HZLB1、HZLB2同源性最高,核苷酸序列的同源性为98.7%和99.0%,氨基酸序列同源性为99.0%和99.7%。TuMV-Ra与1999年从表现相同症状的潍坊萝卜得到的分离物CHINA-WF CP基因核苷酸同源性为93.66%,氨基酸同源性为96.53%,说明病毒发生了比较大的变异,而且变异的位点主要在CP的N末端。将TuMV-Ra CP基因克隆到原核表达载体pET-22b(+),在大肠杆菌BL21(DE3)中表达出分子量为38kD的融合蛋白。Western blotting分析证明TuMV-RaCP在大肠杆菌中得到了正确表达。
A Turnip mosaic virus (TuMV) isolate (TuMV-Ra) was obtained from the radish root with symptom of red flesh in Weifang. The coat protein (CP) gene of TuMV-Ra was amplified by RT-PCR and its complete nucleotide sequence was determined. Homologies of the nucleotide and deduced amino acid sequences were compared between the CP region of TuMV-Ra and those of 20 TuMV radish isolates in GenBank. TuMV-Ra shared identities of 89. 9% - 99.0% and 94. 8% - 99.7% with these 20 radish isolates in nucleotide and corresponding amino acid sequences. It had highest homologies with two isolates from Hangzhou, HZLB1 and HZLB2, which amounted to 98.7% and 99.0% at nucleotide level and 99.0% and 99.7% at amino acid level. The nucleotide and amino acid sequences similarities between TuMV-Ra and CHINA-WF (obtained from the red flesh radish of Weifang in 1999) were 93.66% and 96. 53% respectively. Most of the mutations occurred at the N terminus of CP. The CP gene of TuMV-Ra was cloned into expression vector pET22b ( + ), transferred into E. coli BL21 ( DE3 ) and expressed as a 38 kD fusion protein when induced with IPTG. The result of western blotting assay showed that TuMV-Ra CP gene had been correctly expressed.