An Exploration On The Construction Of Replication Controllable AcMNPV Vector Used For Gene Expression

Most of the lepidopteran insects are agricultural pests. The larvae of them are destructive to agricultural crops and forest trees every year. Until recently, chemical pesticides have been the primary control agent for these pests. Due to the lacking of high efficient research tools and methods, the molecular mechanism of these pests has not been deeply characterized, therefore we still lack better approaches to control them.Autographa californica multiple nucleopolyhedrovirus(Ac MNPV), the hosts of which are primarily lepidopteran insect larvae, is the most widely studied one among the numerous baculovirus. To better study the molecular mechanism of lepidopteran insects, we try to develop a recombinant Ac MNPV vector, the replication of Ac MNPV will be controllable after we modify a replication essential gene of it, the modified Ac MNPV can be used for carrying foreign genes for overexpression, RNAi mediated gene silence and protein epitope tagging.First, the function of DNA polymerase(dnapol) gene of Ac MNPV was confirmed as an essential gene required for baculovirus DNA replication. Then we knocked out dnapol and inserted the rt TA2s-M2, a trans element of the Tet-on Advanced Inducible Gene Expression Systems, by using Red recombination system at the same locus. Then the dnapol expression cassette under the control of Ptight(an inducible promoter of the Tet-on system) and enhanced green fluorescent protein(EGFP) gene were transferred to the polyhedrin(polh) locus in the modified Ac MNPV genome through Bac-to-Bac Baculovirus Expression System. After that, Sf9 cells were transfected with the recombinant Ac MNPV genome and the replicative controllability of this vector was studied.In this study, multiple different modified Ac MNPV genomes were constructed. The results show that after the dnapol sequence is transferred to the polh locus, the sequence at dnapol locus will return to its original sequence based on the replication of the modified genome of Ac MNPV, but the phenomenon will disappear after the dnapol sequence at polh locus is mutated. The results also show that the basal expression of dnapol is closely related to the expression quantity of rt TA2s-M2. Finally, a recombinant vector was constructed, in which the dnapol sequence at polh locus was mutated and the promoter of rt TA2s-M2 was replaced to reduce its expression level. Although not too much, brighter EGFP fluorescence and increased Ac MNPV DNA amount can be detected in Sf9 cells, showing that the DNA replication of the vector is induced in the presence of Doxycycline(Dox). However, the titer of Ac MNPV can not be induced upon addition of Dox. This may suggest that the Ac MNPV virus package is not linearly related to the DNA replication, although the DNA replication and virus package is tightly coordinated in many DNA virus including Ac MNPV, and there is a buffer between these two processes. Again, there is a low basal expression in this system, we can still detect replication of the vector in the absence of Dox. We need to optimize the vector to reduce the basal expression.The replication controllable Ac MNPV vector can be used as a tool to study the molecular mechanism of lepidopteran insects and accelerate the research process of it, and then better approaches used to control these pests will be developed. The construction of this vector has important meaning in agricultural and forestry production.