摘要
芜菁花叶病毒（TuMV）是植物病毒中最大的属——马铃薯Y病毒属的一个重要成员，在世界范围内广泛分布，能侵染43科、156属的300多种植物，其为害仅次于黄瓜花叶病毒（CMV）。本研究分离纯化了芜菁花叶病毒的山东分离物，对其生物学、血清学、细胞病理学和粒子形态学特征进行了研究。利用RT-PCR方法克隆了TuMV山东分离物的外壳蛋白(CP)基因，测定了它们的核苷酸序列、并将其与已报道的序列进行同源性比较和分析，最终确定了其归属地位，具体研究结果如下：
1. 从山东省3地市感病的白菜和萝卜上分离到芜菁花叶病毒的6个分离物，分别命名为TuMV-SD1、TuMV-SD2、TuMV-SD3、TuMV-SD4、TuMV-SD5和TuMV-SD6。寄主反应特性表明，TuMV-SD1～6能侵染3科10种植物，TuMV-SD1在寄主细胞内形成风轮状内含体，外壳蛋白为3组分，分子量分别为45kd、38kd和14kd；提纯的病毒粒体为长线条状。体外抗性测定表明：TuMV-SD1～6稀释限点为1：3500～4000，体外存活期为96小时，失毒钝化温度为55℃～60℃。
2．应用改进的芜菁花叶病毒的提取方法从病叶中提取病毒粒体，应用蛋白酶K法从病毒粒体中提取病毒RNA基因组，根据已报道的TuMV的CP基因序列，设计并合成了一对特异引物，利用RT-PCR法克隆了6个分离物的外壳蛋白基因，与克隆载体pUC19连接后通过热激法转化大肠杆菌DH5α。序列测定结果表明：TuMV-SD1～6的CP基因均为867个碱基，编码289个氨基酸。将测定序列递交GenBank，登录号分别为AF539407、AF539408、AF539409、AF539410、AF539411、AF539412。所得6个分离物的CP基因的核苷酸序列同源性较高，在97.0%～99.4%之间，系统进化分析表明：它们与Brassica（B）致病型分离物中的world-B组分离物的同源性最高（91.8%～100%），亲缘关系最近，从而从分子水平上将TuMV山东分离物划分为world-B组。
Turnip mosaic virus (TuMV) is a member of the Potyvirus genus. It occurs worldwide and can infect more than 300 species in 156 genera of 43 plant families. It was found to be one of the most important (only second to cucumber mosaic virus). In this research, the isolates of TuMV from Shandong were separated and purified. Their biological, serological, cytopathological and particle morphological properties were studied. RT-PCR was used to clone the coat protein (CP) genes of TuMV-SD1～6. Meanwhile, these six coat protein genes were sequenced and compared with other homologous sequences in GenBank. The strain designation of the six isolates of TuMV was finally determined. The results are as the following:
1.Six isolates of turnip mosaic virus named TuMV-SD1, TuMV-SD2, TuMV-SD3,TuMV-SD4,TuMV-SD5 and TuMV-SD6 were respectively acquired from infected Chinese Cabbages and radishes in 3 cities (Taian, Yantai and Zaozhuang) of Shandong province. Different hosts' response suggested that TuMV-SD1 could infect plants of 10 species in 3 families. TuMV-SD1 formed pine-wheel inclusion bodies in plant cells. The coat protein of the TuMV-SD1 contains 3 components whose estimated molecular weight are 45kd, 38kd and 14kd respectively. The virons are filament-like particle. In vitro resistance tests showed that the dilution end point is 1：3500～4000,the longevity was 96 hours and the inactivation temperature is 55℃～60℃.
2.An up-dated method was employed to purify TuMV in this research. Using the protease K method, we acquired the viral genome-RNA. A pair of specific primers was designed and synthesized based on the nucleotide sequences of TuMV coat protein genes reported before and RT-PCR was used to clone the CP genes of the six TuMV isolates. The PCR product was then cloned into pUC19 vector, which was used to transform E.coli DH5α through heat shock. Sequence analysis showed that the CP genes of TuMV-SD1～6 included 867bp and encoded 289 amino acids. The sequences were submitted
to GenBank and the accession number were AF539407, AF539408, AF539409, AF539410, and AF539411and AF539412 respectively. These six sequences had a high homology to each other, which was between 97% and 99.4%. They shared the highest homology to 'world-B' group isolates in Brassica (B) pathotype (between 91.8% and 100%). Phylogenetic analysis based on the CP gene sequences of TuMV strains or isolates showed that these 6 TuMV isolates from Shandong belonged to 'world-B' group.
引文
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