Cloning And Sequences Analysis Of Coat Protein Genes Of Turnip Mosaic Virus Isolates From Shandong

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 submittedto 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.