Screening Of Proteins Binding With Polh Promoter Of BmNPV By Yeast One-hybrid System

Polyhedrin gene promoter plays an essential role in regulating foreign gene expression in BEVS, but the high-level transcription mechanism is still unknown. One-hybrid screening in yeast is a powerful method to rapidly identify heterologous transcription factors that can interact with polyhedron promoter DNA sequence. Total RNA was extracted from silkworm fat body of fifth instar larvae infected by BmNPV for five days, cDNA for silkworm gene expression library construction was generated by RT-PCR technique. Bait sequence of core element of polyhedron promoter was synthesized to generate the bait yeast strain which was used to screen the one-hybird cDNA libray.12 positive yeast colonies were obtained from SD/-Leu/AbA plates, sequencing analysis showed that they belong to 3 different protein cDNA colonies. Furthermore, positive colonies were underwent bioinformatics analysis. They were known as ribosomal proteins:RPSA and RPL5, nuclear polyhedrosis virus DNA-binding protein (DBP). In order to further verify the regulation function of RPSA and DBP, RPSA and DBP gene were inserted into transient expression vector pSK-IE, respectively. Then recombinant vector was transfected to BmN cells,24 hours later, which have been infected by a recombinant virus of Bacmid-Luc. Our results showed that transient expression of RPSA and DBP could up-regulated the transcription level of luciferase gene. RNAi assay results also indicated that interference referred to the inhibition of RPSA and DBP expression could down-regulated the transcription level of luciferase gene.This work studied the high-level transcription mechanism of polyhedron promoter and it will provide a new way for further study baculovirus transcriptional mechanism.