Infect Garlic Linear Plant Virus Coat Protein Gene Of Prokaryotic Expression, Antiserum Preparation And Detection Applications

The virus diseases of garlic are widespread throughout the world, and cause severe loss of yield and poor quality.The plant viruses that infect garlic crop include two Potyviruses(Leek yellow stripe virus, LYSV;Onion yellow dwarf virus, OYDV),two Carlaviruses (Shallot latent virus, SLV;Garlic common latent virus, GarCLV),one Potxvirus (Scallion visus x, ScaVX) and several members of Allexiviruses (Garlic virus A , GarV-A;Garlic virus B , GarV-B;Garlic virus C , GarV-C;Garlic virus D , GarV-D;Garlic virus E, GarV-E;Garlic virus X, GarV-X etc).Specific primers were designed to amplify the complete coat protein(CP) genes of GarV-A , GarV-B , GarV-C , GarV-D , GarV-E ,GarV-X, OYDV and LYSV according to the published sequence of these viruses. The CP genes were then inserted into pGEM-T vector and then were sequenced to confirm that they were correct. The plasmids containing the CP genes of these viruses for prokaryotic expression were constructed and then expressed in Escherichia coli BL21 plys S strain respectively. The expressed proteins were purified by 12%SDS-PAGE firstly and subsequently 5%-20% gradient SDS-PAGE. The antisera against the CPs were raised in mouse(ICR) .Western blot analysis showed each antiserum reacted strongly to its homologous CP but not to the controls.Western blot analysis of the expressed coat proteins of allexiviruses probed with the polyclonal antiserum raised to GarV-A CP shows serological relationship between GarV-A CP and other CPs. The same method was used for testing the serological relationships among GarV-B , GarV-C , GarV-D , GarV-E ,GarV-X. The results suggested that GarV-D and GarV-E were closely related serologically and that there were weaker, but detectable, relationships between some other pairs, particularly GarV-E/GarV-A, GarV-X/GarV-A, GarV-X/GarV-B and GarV-X/GarV-C.OYDV was distantly serological related to LYSV. Indirect ELISA was used to detect the occurrence of LYSV and OYDV in some Chinese garlic samples. It was suggested that these two viruses were widely spread and commonly existed as theform of co-infection. Their distantly serological relationship influenced little to results of previous investigations, and ELISA method established in this study is practicable for detection of garlic viruses in the fields.Specific primers were designed to amplify four segments from previous constructed GarV-X cDNA clones. The full-length cDNA clone of GarV-X was constructed using ligation strategy. T7 promoter sequence was added to the upstream of 5'terminus of genome. The infectivity of the full-length cDNA clone of GarV-X is to be further proved.