Construction Of Transient Expression System Of Plutella Xylostella Granulovirus And Functional Characterization Of Its Late Expression Factors In Non-permissive Cell-line Sf-9

Baculoviruses are a group of viruses pathogenic for arthropods, which are named of the rod-shaped virons. A common feature of baculoviruses is the division of gene expression into early and late phases. The early phase does not depend on products encoded by baculovirus genes while the late phase is dependent on DNA replication and viral proteins synthesized in the early phase. In Autographa californica multiple nucleopolyhedrovirus (AcMNPV), which is the type species of baculovirus,19 late expression factor (lef) genes have been characterized.Plutella xylostella Granulovirus (PlxyGV), a member of baculoviride, is pathogenic to diamondback moth, which is a major pest of cruciferous crops. It is potentially a useful biological pesticide for the control of the pest. Limited by the lack of permissive cell lines, data on gene expression of PlxyGV is rare. This work is committed to constructing a transient expression system of PlxyGV and carrying out identification and functional analysis of PlxyGV late expression factor genes on the basis of it. Main contents are as follows:(1) A transient expression system of PlxyGV, including a PlxyGV cosmid library, plsmid library of PlxyGV lef genes and reporter plasmid for late expression of PlxyGV, was constructed. Afterwards, five overlapped cosmids covering the whole genome of PlxyGV were identified. According to the genome sequence of PlxyGV reported,15 PlxyGV genes homologous to lef genes of AcMNPV were cloned, and transient expression plasmids of which were constructed. Transient expression assays of late genes of PlxyGV were performed using five cell lines.(2) In transient expression assays, individual lef genes of PlxyGV were used to substitute corresponding lef genes of AcMNPV. It was shown that only lef-2 of PlxyGV can make it in some extent.(3) BLAST on the amino acid sequences encoded by lef genes of 51 baculoviruses sequenced was performed to study evolutional relationship among the members of baculoviruses. And phylogenetic trees of LEF proteins from selected baculoviruses were generated. The data may provide information for insight into association of functions of late expression factors with baculoviruses’evolution.