Functional Analysis Of Open Reading Frame Bm126 Of Bombyx Mori Nucleopolyhedrovirus

The family Baculoviridae is composed of viruses with circular,double-stranded DNAgenomes and rod-shaped,enveloped virions.BmNPV has been completely sequenced.Extensive studies have been done on the function of individual gene.The BmNPV openreading frame 126 (Bm126) encodes a protein which contains a hydrophobic N terminal and aC-terminal peritrophin-A domain.In this thesis,Bm126s were amplified from BmNPVsisolates collected from five different regions of China.Sequence analyses showed that therewere two subtypes of Bm126 in the Chinese isolates,Bm126-SX and Bm126-GD,both weredifferent from that of BmNPV-T3 the genome of which was fully sequenced.The variation ofdifferent Bm126 subtypes occurred in the charged amino acid region in between of theN-terminal hydrophobic domain and the C-terminus C6 motif.A bacmid BmBacJS 13 whichwas originally derived from a BmNPV harboring Bm126-SXwas used as a backbone for studythe function of Bm126.The thesis contains six chapters.Chapter 1 is a general introduction of baculovirus.It contained a general introduction ofbiology,molecular biology and application of baculovirus.The molecular biology of BmNPVand advances of BmNPV baculovirus expression vector system (BEVS) were summarized.The BM126 phenotype and Bm126 gene were introduced and the aim of the thesis waspresented.In chapter 2,the Bm126 was amplified from BmNPVs isolated from five differentregions of China.Sequence analysis showed that the Chinese isolates had two differentsubtypes of Bm126,Bm126-SX and Bm126-GD,both were different from that of BmNPV T3strain.All the BM126s contain a hydrophobic N terminus and a C6 motif at C terminus,butthe sequence in between varies.The amino acid alignment of baculoviral BM126 was studied.We also used an in vitro chitin-binding assay to test whether GST-126 is able to bind to chitin,and these results were negative.In chapter 3,the transcription,location and expression of Bm126 in BmNPVinfected-insect cells were analyzed,3'RACE revealed that the transcript of the Bm126 genewas first detected at 6 hours post infection,and lasted to the end of the infection.These indicated that BmNPV Bm126 gene is an early gene.The Bm126-SX gene was cloned andexpressed in bacteria cells with either a His-tag or GST-tag.The His-tagged BM126 orGST-tag BM126 protein was purified and immunized rabbit to generate antiserum againstBmNPV BM126.The obtained antiserum was found to react specifically to BmNPV BM126.The subcellular localization of the BM126-SX and BM126-GD were examined usingC-terminal fusion to egfp and egfp alone as a negative control.BmN cells were transfectedwith different plasmid DNA,the superinfection were performed at 12 hour post transfection.The fluorescence was examined by confocal laser scanning microscopy at 60 h posttransfetion.The EGFP protein was uniformly present throughout the cytoplasm and nucleuseither expression alone or superinfection.However,EGFP was linked to either theBM126-SX or BM126-GD,fluorescence was uniformly present in the cytoplasm andfluorescenc were observed shallow in the presence of vBmBacJS13-ph infection.Butfluorescenc were not observed in the nuclei of the infected-cells.In chapter 4,to delete the Bm126 gene of BmBacJS 13,a transfer vector containing theBm126 flanking regions for homologues recombination was constructed in which the Bm126was replaced with chloramphenicol resistance gene (Cm^R) under itself promoter and an egfpgene under the hsp70 (Drosophila heat shock 70) promoter.A linear 3.4 kb fragmentcontaining Cm^R and egfp in between of Bm126 flanking regions was transformed into E.coliBW25113 which contain BmBacJS13 DNA and helper plasmid pKD46 to construct aBm126-null bacmid.The resulted bacmids were identified by PCR and restrictionendonucleases mapping and the correct bacmid was named BmBacΔ126.To compare theproperties of different Bm126,the promoter and coding sequences were amplified fromBmNPV-SX and BmNPV-GD isolates.The PCR products were cloned into donar plasmid.Bytransposition,Bm126 and polh were reintroduced into the BmBacΔ126 to generate repairedbacmids.The recombinant bacmids were confirmed by PCR and restriction enzymedigestions and designed as BmBacΔ126-SX-ph and BmBacΔ126-GD-ph.For control,the polhgene was inserted into BmBacJS13 or BmBacΔ126 to generate BmBacJS13-ph orBmBacΔ126-ph.In chapter 5,the recombinant viruses were obtained by transfection and infection theBmN cells.The deletion of Bm126 did not affect the BV infectivity in BmN cells indicatingthat Bm126 is not an essential gene for viral replication.Deletion of Bm126-SX resulted insuccessful oral infection and had no obvious effect on the mean lethal dose (LC₅₀) of the virus,but with a delayed ST₅₀ of the recombinant virus and this delay was rescued by repairing thegene.Different phenotype was observed when different subtype of Bm126 was repaired intoBm126-deleted virus.The delay of ST₅₀ caused by deletion of Bm126-SX could be rescued by the reparation of Bm126-SX but not by Bm126-GD,however,the Bm126-GD126 repairedvirus showed a significant increase of OBs production both in vitro and in vivo.In ourexperiments,the cell infected with Bm126-GD-repaired virus showed different phenotype,such as cell survived a much longer time,in comparison to other virus infected cell.All theresults show Bm126 can facilitates virus infection or replication and variants of Bm126 inwild-type BmNPV isolates exhibit functional differences.Chapter 6 gives general conclusions of the thesis.