| To get more information for bioinsecticide developing and understand the baculovirus replication and transcription mechanism, genome of ClanGV and CaLGV and proteomics of ClanGV were determined and analyzed.1. Genomic sequencing and analysis of the Clostera anachoreta D. granulovirusThe complete genome of the Clostera anachoreta D. granulovirus (ClanGV) from a pivotal pest of poplar, Clostera anachoreta D., is 101,487 bp in size with C+G content of 44.4%. It is predicted to contain 123 putative open reading frames (ORFs), which covers 93% of whole genome sequence.111 ClanGV ORFs are homologues of the previously reported baculovirus genes; 2 ORFs only exist in NPV; 10 ORFs are unique to ClanGV accounting for 3.9% of the genome in total. The average amino acid sequence identities between ClanGV and other GV homologues ranged from 55.5%(PiraGV) to 43.1%(HearGV and PsunGV). ClanGV includes 16 DNA replication and transcription related genes, which are ie-1, dnapol, helicase, lef-1, lef-2, lef-3, lef-4, lef-5, lef-6, lef-8, lef-9, lef-10, lef-11,39K, p47 and vlf-1, respectively. Seventeen conserved structural genes and two types of apoptosis suppression proteins (P35 and LAP) have also been identified. To date, ClanGV is the second identified GV containing the p35 gene which is present in 8 NPVs and ChocGV. Chitinase and cathepsin involved in the liquefaction of the host were found in the ClanGV genome, which is consistent with the typical insecticidal manner of ClanGV. In ClanGV genome there are four homologous repeat regions, each one of which contains only 2-3 direct repeats. Phylogenetic analysis, based on the 29 core baculovirus genes, indicates that ClanGV has a close relationship with PhopGV, AdorGV, CpGV, CrleGV, PiraGV and ChocGV, which is identical with the results of homology analysis and Gene parity plot analysis.2. Comparative analysis of the genomes of Clostera anastomosis L. and Clostera anachoreta D. granulovirusesThe Clostera anastomosis L. granulovirus (CaLGV) and Clostera anachoreta D. granulovirus (ClanGV) are both capable of infecting each other’s native host insects. Despite this we have little information on their genetic relationship. The complete nucleotide sequence of CaLGV was determined and compared with the genome of ClanGV. The circular, double stranded DNA of CaLGV genome had a G+C content of 46.7% and was 101,818 bp in size (GenBank accession no. KC179784) (331bp larger than the ClanGV genome). Overall the CaLGV was found to be 90% identical for nucleotides to that of ClanGV. It contained a total of 123 CaLGV ORFs, 119 of which had ClanGV homologues with identical transcription direction and ORF organization.75 of the 119 ORFs showed 90% or greater identity to ClanGV homologues. CaLGV contained only a single identifiable homologous region (hr) (similar to ClanGV hr4). The mean frequency of nucleotide substitutions in the CaLGV/ClanGV coding regions was 8.33%. CaLGV contained four unique ORFs (CaL23, CaL39, CaL48 and CaL92). Eight ORFs found in both CaLGV and ClanGV have no homologues in other baculoviruses. The length of total intergenic regions of CaLGV and ClanGV occupied 6.6% and 7% of their respective genomes. CaLGV appears closer phylogenetically to ClanGV than to any other baculoviruses.3. Proteomics of the ODV of Clostera anachoreta D. granulovirusThe ClanGV Occlusion Bodies (OBs) were purified and then detected by the Transmission Electron Microscope (TEM). We prepared two protein samples for this experiment, one from OB and one from ODV. By LC-MS/MS method,74 ODV construtral proteins were identified. This is the first time that so many proteins were found in one baculovirus. These ODV proteins made up 60.2% of the all ClanGV ORFs, which was highest in all analyzed baculoviruses. Mose of the 74 proteins gathered in two regions and formed two protein cluster, ORF7-ORF24 (including 15 proteins, no ORF10, ORF16 and ORF21) and ORF63～ORF91 (including 24 proteins, no ORF67, ORF75, ORF77, ORF80 and ORF85).We compared the ClanGV ODV proteins to those of other 5 analyzed baculoviruses. It indicated that 11 proteins (P49, PIF-2, P74, P6.9, P33, VP39, ODV-E27, VP91, vlf-1, GP41, P1054) were identified in other baculovirus ODV; 13 (P49, PIF-2, ODV-EC43, P74, P6.9,38K, P33, VP39, ODV-E27, VP91, vlf-1, GP41, VP1054) were ODV, BV or ODV/BV proteins; 12 were just identified in ODV of GV (pepl, Clan22, Clan27, pep/p10, pep-2, Clan69, Clan83, Clan84, Clan90, Clan94, Clan116, fgf-3); others were just existed in ODV, BV or ODV/BV of some baculoviruses.21 ClanGV ODV proteins (Clan17, Clan18, Clan20, P13, Clan32, Clan37, Clan39, Clan42, P35, P47, P38.7, Clan63, Clan64, DBP, LEF-5, Clan79, TLP-20, Clan93, Clan108, LEF-8, Clan117) were identified for the first time in baculovirus. Most of these 21 protiens were unknown, and 4 of them were just existed in ClanGV and CalGV.To further verify the LC-MS/MS results,7 ClanGV ODV protein genes have been cloned and 3 of them were expressed by prokaryotic system and purified. Next we will express all of the remainder proteins and comfirm their location in ClanGV. |
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