潍坊萝卜中芜菁花叶病毒分离物的分子特性及CP基因的表达

 从表现红心病的潍坊萝卜块根上得到一芜菁花叶病毒( Turnip m osaic 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% ～99.0% , 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38 kD的融合蛋白。Western blotting分析证明TuMV-Ra CP在大肠杆菌中得到了正确表达。

Molecular Characterization and Coat Protein Gene Expression of a Turnip mosaic virus Isolate from Radish in Weifang

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.