| Plutella xylostella granulovirus (PlxyGV) belongs to betabaculovirus genus. It is an important biological agent to control the population of Plutella xylostella in the ecosystem. Although PlxyGV has been registered as a commercial biopesticide to kill Plutella xylostella, little is known about the molecular information of this insect virus, duo to inavailability of stable permissive cell lines for in vitro replication of PlxyGV. PlxyGV bacmids were constructed by using Bacterial Artificial Chromosome (BAC). The PlxyGV bacmid provides a fast and efficient method for the manipulation of PlxyGV genome in E. coli. This will overcome the rate-limiting step associated with the lack of highly permissive GV susceptible cell lines, allowing the study of PlxyGV gene expression analysis and in vitro replication.(1) The bacmid vPxG contains the complete genome sequence of PlxyGV, a mini-F sequence enabling replication of the bacmid in E. coli, a kanamycin resistant marker, a lacZa and an attachment site for the bacterial transposon Tn7within the lacZa by using cloning method and the phage lambda-derived Red recombination system. To confirm the insertion of the BAC sequence in the PlxyGV genome, the bacmids were subjected to restriction analysis. To facilitate detection of viral gene expression and replication in cells, the egfp was used as a reporter gene being inserted into vPxG through Tn7-mediated transpostion at the gran locus, making four reporter bacmids, in which the egfp was put under control of predicted promoters of PlxyGV iel (vPxGPiel-gfp), vp39(vPxGPvp39-gfp),gran (vPxGPgran-gfp), or AcMNPV iel(vPxGPaciel-gfp). The fluorescence was seen in Hi5cells transfected with vPXGPaciel-gfp or vPXGPiel-gfp and in the Sf9cells transfected with vPxGPaciel-gfp. Fluorescence was never observed in any of the cells transfected with vPxGPvp39-gfp or vPxGPgran-gfp. No infectious BV was produced in the cells transfected by vPxGPiel-gfp, vPxGPvp39-gfp, vPxGPgran-gfp, or vPxGPaciel-gfp.(2) The reporter plasmids were constructed, in which the egfp or luc was put under control of predicted promoters of PlxyGV iel, exon0, dnapol, lef1, lef9, orf105, vp39and gran. The cells were transfected with the individual reporter plasmids together with vPxG or bMON14272. LUC activities were determined at48h.p.t. Flurescence was observed in the Hi5cells transfected by pPiel-gfp or pPdnapol-gfp.Cells expression flurescence were observed in all the cultures transfected with each individual reporter plasmid together with bMON14272. Significant levels of LUC activity, were detected in extracts of Hi5cells transfected with all six individual early gene reporter plasmids. The levels of LUC activity detected in extracts of the Sf9cells transfected with individual early gene reporter plasmids were much lower than those detected in Hi5cells transfected under the same conditions. LUC activity was largely reduced in the Hi5and Sf9cells individually transfected with the six early reporter plasmids, together with vPxG. LUC activities were elevated in all the three kinds of cells transfected with the lated reporter plasmid pPvp39-luc or pPgran-luc, together with vPxG. LUC activities were increased greatly in the cells transfected with the individual reporter plasmids, together with bMON14272, except the cells transfected with pPiei-luc and bMON14272.(3) The reporter plasmids containing PlxyGV hr were constructed, in which the luc was put under control of predicted promoters of PlxyGV iel, dnapol, lef1, and lef9. In the PxE2cells, cis-linked PlxyGV hr1, hr2, hr4, and AcMNPV hr5had an enhancing effect on expression from the PlxyGV early promoters. In the Sf9cells, cis-linked P1xyGV hrl, hr4, and AcMNPV hr5had an enhancing effect on expression from the PlxyGV early promoters. In the Hi5cells, cis-linked P1xyGV hr1, hr3, hr4, and AcMNPV hr5had an enhancing effect on expression from the PlxyGV early promoters and cis-linked P1xyGV hr2had an enhancing effect on expression from some PlxyGV early promoters(Piei, Pdnapol. and Plefl).(4) Addition of pPxiel caused a reduction in LUC activity in all of the transfected cells, either with the reporter plasmids containing PlxyGV hr1or the plasmids without hr. The transactivator gene iel of AcMNPV stimulated expression of PlxyGV dnapol, lef1, and lef9, and the stimulation effects were further enhanced by hrl.(5) The gp64-null AcMNPV bacmid containing egfp and cat were constructed. Gene encoding F protein from PlxyGV were cloned and transposed into the polh locus of the gp64-null AcMNPV bacmid. Transfection-infection assays of the recombinant bacmids indicated that PlxyGV F could not rescue the infectivity of gp64-null AcMNPV. The p35-null AcMNPV bacmid containing cat were constructed. Gene encoding IAP protein from PlxyGV were cloned and transposed into the polh locus of the p35-null AcMNPV bacmid. Transfection assays of the recombinant bacmids indicated that PlxyGV iapl or iap2could not rescue the infectivity of p35-null AcMNPV.(6) Seven PlxyGV bacmids containing one, two, or three of the AcMNPV genes (p35, iel, and gp64) were constructed. The expression of reporter gene driven by the PlxyGV vp39promoter was elevated to a detectable level in the Hi5cells by the addition of AcMNPV p35, and AcMNPV iel enhanced the stimulatory effect of p35. Cooperation of AcMNPV p35, iel, and gp64further increased the reporter gene expression. AcMNPV iel and gp64together stimulated reporter gene expression slightly. Cultures of PxE2, Sf9, and Hi5cell lines transfected with vpxGPvp39-gfp-aciel-p35-gp64, which contained AcMNPV p35, iel, and gp64; and the numbers of the fluorescence-containing cells in each case were more than those observed in the cultures transfected with the bacmid containing the p35alone, or p35together with iel or gp64. It should be mentioned that, although infectious BV was produced in the cells transfected by the bacmid containing AcMNPV p35, iel, and gp64, the viral productivity was very low, so that it was continuously propagated at low levels in the cells. |
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