Comparison Of Helicoverpa Armigera NPV VHA273 Wild Isolate With Its Clone Isolate And Sequences Analysis On The Chitinase Gene Of Wild Isolate

This thesis includes three chapters.Chapter one is a general introduction of the research on baculovirus which includes the structures, functions of baculovirus, together with recent advances in this area as well as the overview and the aim of this research.Chapter two includes a comparison between Wild HaNPV-VHA273 Multi-nucleocapsid NPV and its clone Single-nucleocapsid NPV H9 on their shape, structure, biological activity, restriction pattern and structural polypeptide. The research indicated that bioactivities of the two isolates were similar but their structural polypeptides were obviously different and restriction endonuclases fragments demonstrated certain differences between VHA273 and H9 isolate. With VHA273 and H9 isolates separately infecting early third instar H.armigera larvae, the LC50 values were 2.987+104 PIBs/ml and 1.647+104PIBs/ml respectively; the LT50 values for 2+107PIBs/ml concentration were 4.866 and 4.797 days respectively. This fact suggested that there was little difference in the infecting ability between the two isolates, and at the same time showed that plaque-purification did not affect the bioactivities of the baculovirus. Assay by SDS-PAGE indicated that there were losses of some structural polypeptides, and gains of some new structural polypeptides between clone and wild isolates. It also showed that there are many differences in their protein's quantity. The experience of digestion of genomes by restriction endonucleases showed that VHA273 and H9 genomes had some differences in their fragmentation profiles in that there were losses of some fragmentations and gains of others. We predicated that in this different restriction endonuclases fragments and structural polypeptide, some genes and proteins might be directly related to the nucleiocapsid's type of nuclear polyhedrosis virus, which perhaps is the reason for the change of multi-nucleocapsid NPV into single-nucleocapsid NPV. This discovery will be helpful for revealing the factor that caused the formation of Multi-nucleocapsidNPV and Single-nucleocapsid NPV at the molecular level.Chapter three reported the PCR amplification and sequences analysis of thechitinase of Helicoverpa armigera Multi-nucleocapsid NPV VHA273 Wild Isolateand homology comparison of 21 baculoviral and prokaryotic chitinases. Resultindicated that the ORF of the chitinase gene of Helicoverpa armigeraMulti-nucleocapsid nucleopolyhedrovirus VHA273 isolate was 1713 nucleotidesin length which encoded a protein of 570 amino acids with a molecular mass of 60kDa. Sequences analysis of amino acids demonstrated baculoviral chitinases genewas a high conservative gene. The amino acids sequences of baculoviral andprokaryotic chitinases contained similarly three domains. Domain I, namely Nterminal domain, was the signal peptide region of prokaryotic and baculoviralchitinases. Domain II, the central section of chitinases sequence, was chitinases'active site. This domain contained catalyze site of the chitinase family 18. It hadhigh homology of chitinases ammo acid sequences and acted as the catalyzingregion. Domain III, namely the C terminal domain, was the ER-attaching regionof baculoviral chitinases. This domain included the ER-located motifs(KDELsequences). High homology between prokaryotic and baculoviral chitinases(especially bacterium chitinases) suggested that baculovirus and bacterium hadcloser evolutional relations, and perhaps baculoviral chitinases had the sameancestors with bacterium's chitinases.