The Elementary Research On Structures And Functions Of Ie0,ie1 And 25k Gene In Spodoptera Litura Multicapsid Nucleopolyhedrovirus (SpltMNPV) Japanese Strain

Spodoptera litura (Lepidoptera: Noctuidae) multicapsid nucleopolyhedrovirus(SpltMNPV) isolated firstly from cadavers of Spodoptera litura in Guangzhou, belongs tothe subgroup A of Nucleopolyhedrovirus. Spodoptera litura, the host of the SpltMNPV is akind of polyphagous insect which is harmful to economic crop and vegetables includingalimentary crop, cotton, oil plants and so on. in addition it is also one of the primaryinsects pests around the world. Generally, SpltMNPV is considered to be the most effectiveinsecticide against the insect. Therefore, the studies on it are becoming more and morepopular. Recently, some studies concerned the virus including the sequence determination,the analysis of its gene structure, its function and regulation of expression, have beengreatly improved on the analysis of the structure of some important genes which hascontributed to the selecting of the strain with stronger virulence, the improving anddeveloping of this viral insecticide, and the constructing of the baculovirus expressionvector. In the present study, we explored the structure and fuction of the ie0/ie1 and 25kgenes using Splli-SpltMNPV model. The main complishments are as follows:1. The ie0 gene was isolated and cloned from SpltMNPV JapanoC3 strain genome (theaccession, numbers in NCBI GenBank is AY 727987), which is 864bp long and canencode potentially a polypeptide with predicted molecular mass of 33124Da. Aminoacid sequence analysis showed that there were abundant acidic and hydrophobic amino acidresiduals of from the 23th to 47th sites and from the 111th to 126th sites in N-terminus side ofSpltMNPV IE0, and SpltMNPV IE0 was characterized by a typical double zinc-finger structure at225th to 271th sites in C-terminus side, which was conservative in IE0 of the nine comparative.insect baculoviruses. We costructed E. coli expression vector to express highly the ie0gene was highly expressed with induction of IPTG., identified the expression of it byWestern Blotting.2. To study the cell apoptosis induced by ie0 and ie1, two novel baculovirus transfervectors pSplt-ie0 and pSplt-ie1,which earn the marker of GFP and the promoter that identified directly and early by the host cell, were constructed. Combining the effects of threeinhibitors on the cell apoptosis, we explored the mechanism underlying IE0 and IE1 mediated cellapoptosis. Our results showed that the cell apoptosis can be induced by IE0 and IE1protein and those abilities influenced by the amount of IE0 and IE1 products.The IE0and IE1 products might lead to the host cell DNA replication.and induce cell apoptosis.3. A few polyhedrin mutant has been found in SpltMNPVs. The 25k gene including in threestrains (A, B and C) of SpltMNPV from Japan have been amplified. Compared with Chinese type,the B type has several losts and inserts in nucleic acids sequence, leading to 21 amino acids changeof encoded protein and getting a few polyhedrin phenotype. And the B phenotype also cannot leadto the host degradation after 144h p.i.The results suggested that the 25k gene product could notinhabit normal expression of cystein protease gene and chitin protease.