| Summary: | 碩士 === 國立成功大學 === 生物學系 === 88 === Hz-1 virus is an unclassified insect virus. It is the only insect virus that can infect insect cell lines persistently with temporal gene expressions between productive and persistent infections, and thus is a good model virus for studies of viral persistent infection. Previously, it was difficult to clone the HA, HD and HI genomic segments which came from Hz-1 viral genome digested by HindIII, and eventually theses fragments were cloned by using a low-copy number vector, pWSK29. With the Northern hybridization data, it was shown that there were several genes expressed in these three genomic segments. It was presumed that these genes encoded some transcription factors or toxin genes that influenced the viral infection and replication. In this study, the genomic segments of HA, HD and HI were sequenced and gene predicted and annotated. Finally, the predicted genes were assigned the corresponding bands on the previous Northern hybridization data to confirm the viral gene expressions.
Three sequencing strategies were used to finished genomic segments sequencing in this study, including random shotgun cloning and sequencing, end sequencing of subclones and primer walking sequencing. These genomic segments contain 41,733 bp with a G+C content of 41.55 %. Total 108 open reading frames (ORFs) encoding at least 50 amino acids were predicted and patterns of transcriptional signals were searched. Then the full-length nucleotide sequences of these segments and peptide sequences of predicted ORFs were uploaded to Genbanks for searching alignments by BLASTP, BLASTX and FASTA programs to find potential genes and the similarity between Hz-1 virus and other viruses. The results of alignments showed that there were two significant genes contained in Hz-1 viral genome, they are the transposase (Tn) and the thymidilate synthease (Ts) respectively. The transposase was presumed that coming from contamination of Escherichia coli genomic DNA during experimental artifacts. The Hz-1 virus thymidilate synthease was aligned to herpesviruses ones with high similarities, and the gene was presumed to be obtained from its host, lepidopteran insects.
In this study, the random shotgun sequencing strategy was used in genomic segment sequencing and can be considered to be used in genome project of Hz-1 virus in the future. The genomic sequences and alignment data can be afforded new data in Genbank and assist the studies in Hz-1 viral infection and classification.
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