The Molecular Evolution And Functional Analysis Of Ubiquitin Of Spodptera Exigua Multi Nucleocapsid Nucleopolyhedrovirus

Advisor: Associate Professor Zhang ZhongxinThis thesis paper includes four chapters.Chapter One is a brief review on recent advances on baculovirology and ubiquitin system. It focuses on the fundamental researches and applied applications about baculovirology and Spotoptera exigua Muti Nucleocapsid Nucleopolyhedrovirus (SeMNPV), and does a systemic review on the ubiquitin system.Chapter Two is about the sequence analysis and prokaryotic expression of ubiquitin of SeMNPV. The gene was 243 bp in length encoding 80 amino acids with a theoretical molecular weight of 9.4 kD. To construct expression vector, the fragment containing ubiquitin gene was inserted into pET-28a expression vector. Expression of the fusion protein was induced by IPTG in E.Coli BL21(DE3).By changing the time induced and the concentration of IPTG, the fusion protein had an optional expression level. The fusion protein was identified by Western blot with a mouse Ab against bovine Ubiquitin. To do the further study, the specific Ab against viral Ubiquitin was produced. In addition, the sequence of the amino acids was analyzed with the software. The result displayed that ubiquitin of baculoviruses had some different amino acid substitutes in some residues and the amino acid sizes varied a lot compared with eukaryotic Ubiquitins. From these results, we suggest that ubiquitins of baculovirus may have a unique way in the gene evolution.Chapter Three is about 3'RACE -PCR amplification and prokaryotic expression of ubiquitin extension gene cDNA of Spodptera exigua. To identify the nucleotide sequence of ubiquitin cDNA of S.exigua, a 513bp cDNA fragment encoding ubiquitin 53aa fusion protein from S.exigua lipid was amplified by 3'RACE-PCR with primers designed on the N-terminal amino acid sequence of Spodptera frugiperda, and the amplified fragment was cloned and sequenced. The cDNA is 513bp in length, including 213bp of 3'untranslated region, encoding 129 amino acids and the molecular weight of the mature peptide is14.8kD. Comparison of the deduced amino acid sequence with Ubiquitins from Bombyx mori,Spodpera. Frugiperda,Drosophila melanogaster,Homo sapienes,SeMNPV and BmMNPV showed that the amino acid homology rates were 96.9%,98.5%,95.3%,93.0%,78.8 %,and 77.2% respectably, and it suggests ubi-53 in eukaryotes may have an evolution way different from virus. The fragment containing ubi-53 gene was inserted into pET-28a expression vector, expression of the fusion protein was induced by IPTG in E.Coli BL21(DE3).The fusion protein was identified by Western blot with a mouse Ab against bovine Ubiquitin. Although eukaryotic ubiquitin has been clearly certified as a protein maker, which can make the ubiquitination protein degraded by proteasome, it is unknown whether ubiquitins of baculovirus can be functional as eukaryotic ubiquitins. Chapter Four did an elementary study on the function of baculovirus ubiquitin. By using yeast two hybrid system, interaction assay in vitro between ubiquitin and IAP2 or IAP3 of SeMNPV has been done. The putative positive clones only existed in the plate of the SD/-Leu/-Trp/X-α-GAL, which suggested that ubiquitin might interact with IAP2 or IAP3 weakly, since ubiquitin may be transferred to IAP2 or IAP3 through E1 and E2 in yeast. To certify the interaction in vitro further, it should be done that ubiquitin and ubiquitin activating enzyme (E1), ubiquitin conjugation enzyme (E2 )and IAP can interact in vitro.