| The watermelon mosaic virus is one member of potyviruses, transmitted by aphids with nonpersistent type,extensively spreaded over the world, and mainly cause mosaic diseases of watermelon and melon. In the field, the virus is transmitted by the aphid vectors,mostly by Myzus persicae (Sultzer) and Aphis gossypii Glover. WMV belongs to Potyvirus, when which are transmiting, virus-coded nonstructural protein helper component-proteinase (HC-Pro) first bind with the aphids stylets'receptors, then combine with the coat protein(CP) of virion particles, sequentially whose aphid transmission is finished. Therefore,virus-coded structural protein CP and constructural protein helper component-proteinase HC-Pro of the member of Potyvirus are indispensible protein in process aphid transmission.In this text, CP of WMV Shaanxi isolate was cloned, a recombinant gene segment, in which full-length sequence of WMV CP was fused to 5-terminus of GFP applying the gene splicing overlay extension by PCR method, was inserted into the pGEX-4T-1 vector and transformed into E.coli BL21(DE3) pLysS, and could inducibly-express a great mount of GST-CP-GFP under the optimized conditions.We attained a GST-CP-GFP fusion protein with the lysis procedure. The bacterially by inducibly-expressed emit strong green-fluorescence under blue or UV light, which indicate the fusion protein does not lose biological activity.The cells solution of E.coli BL21(DE3)pLysS with pET-30a-HC expressed-vector was lysed with the lysis procedure. We fed Myzus persicae and non-vector aphid-Radish aphid and Rhopalosiphum padi with HC-Pro protein sucrose,CP-GFP protein sucrose, and mixture of CP-GFP protein and HC-Pro protein sucrose in different orders. M. Persicae was fed on HC-Pro, CP-GFP fusion protein successively or the mixture of HC-Pro and CP-GFP fusion protein and their distal part of stylets emit the green-fluorescence under the blue and UV compound light, rather than M. Persicae fed in other orders and non-vector aphid in every orders. The experiment results show : The aphid stylet binds with watermelon mosaic virus HC-Pro first,then binds with CP through HC-Pro. The experimentation further confirmed the "bridge" hypothesis. |
