Study On Biology Of Recombinant AcMNPV With P7-2 From RBSDV

In this study,P7-2 gene of Rice black streaked dwarf virus?RBSDV?that could induce cell apoptosis was inserted into the genome of AcMNPV to enhance the viral virulence and provide a new idea to construct recombinant baculovirus for better biocontrol.The polyhedrin gene?ph?,ie-1 promoter and RBSDV P7-2 gene fused to a Flag-tag at the C-terminus were inserted into the multiple cloning site?MCS?of the donor plasmid pFastBac-HTB.The ph gene was initiated by the the promoter of the donor plasmid while P7-2 gene was droven by ie-1 promoter.Ph,P7-2 and promoters were inserted into the polyhydrin location of bacmid bMON14272 by transposition to construct recombinant bacmid Ac-P7-2.By measuring the viral growth curve and DNA replication curve,differences of proliferation and DNA replication capacity between recombinant virus?Ac-P7-2?and wild type virus?Ac-wt?were compared.The third instar Spodoptera exigua larvae were infected with Ac-P7-2 and Ac-wt to test the insecticidal efficacy of recombinant virus.Following were major conclusions:1.Recombinant baculovirus Ac-P7-2 was successfully constructed and P7-2gene can express correctly.2.Sf9 cells were transfected with 1?g bacmid Ac-P7-2 and Ac-wt.The formation of polyhedra was observed in different time.The results showed that Ac-P7-2 can be packed into normal virions.The formation and release of polyhedra of Ac-P7-2 was faster than Ac-wt from 48 to 144 h.p.t..The amount of dead cells caused by recombinant virus was significantly more than those caused by wild type virus at 48,72,96 and 120h.p.t.,respectively.The recombinant virus had a short lethal time against host cells.3.The viral growth curve indicated that multiplication rate of BVs of Ac-P7-2was faster than Ac-wt after 48 h.p.t..The viral DNA replication curve indicated that replication capacity of Ac-P7-2 was stronger than Ac-wt at 72,96 and 120 h.p.t..The third instar Spodoptera exigua larvae were used to test LD₅₀,LD₉₀,LT₅₀ and LT₉₀.The results showed that the LD₅₀ value of Ac-P7-2 was about 1/6 of Ac-wt,the LD₉₀0 value of Ac-P7-2 was about 1/5 of Ac-wt,the LT₅₀ value of Ac-P7-2 was 2.4 days less than Ac-wt and LT₉₀ value of Ac-P7-2 was 3.4 days less than Ac-wt,which indicated that Ac-P7-2 has higher virulence and faster killing speed.