Evaluation Of A Novel Polyadenylation Signal From The First Bidensovirus BmBDV

Bombyx mori bidensovirus（BmBDV）was the only virus belonging to the Bidnaviridae family,which was established by the International Committee on Taxonomy of Viruses in 2012.This new bidensovirus is a kind of pathogeny that can infect the columnar cells of the midgut epithelium of silkworms,leading to chronic densonucleosis disease.The genome of Bm BDV consists of two single-stranded linear DNA molecules（VD1 and VD2）,which are respectively encapsidated into separate virions.The gene of VD1 encoding MCP and pPolB overlaps partially at the 3’untranslated regions（UTRs）,which forms a dual function polyA signal（BDVpA）to complete post-transcriptional modification and terminate the protein expression.In this study,we evaluated the effect of this short BDVpA sequence on the regulation of foreign gene expression.Fluorescent microscopy was used to detect the expression of visual protein GFP,and the activity of BDVpA（63）was analyzed qualitatively in contrast with SV40polyA.We constructed instantaneous expression vectors with BDVpA（63）containing overlap region,which was fused to the 3’-terminal sequence of reporter gene gfp（pT-p5-gfp-BDVp A（63）and pT-ie1-gfp-BDVp A（63））.Besides,gfp transient plasmids without polyA were constructed as negative control groups.Then these fusion plasmids were respectively transfected into three insect cells（Hi5、BmN and Sf9）.All cells were observed by fluorescence inverted microscope.The exogenetic gfp from recombinant plasmids with BDVpA（63）signal could be expressed in transfected cells.Compared with the SV40 polyA,more transfected cells producing green fluorescent were detected and brighter fluorescence appeared in each cell.The results showed that BDVpA（63）was more conducive to the expression of gfp reporter gene in insect cells than SV40 polyA.Through detection of relative expression of firefly luciferase by dual luciferase reporter system,the activity of BDVpA（63）was analyzed quantitatively in comparison with SV40 polyA.Firefly luciferase plasmids with BDVpA（63）were constructed（pGL₃-p5-BDVpA（63）and pGL₃-ie1BDVp A（63））,then transfected into two insect cells（Hi5 and BmN）and detected dual luciferase activity.Compared with SV40 polyA,the relative expression of foreign proteins was higher in all transfection groups when BDVpA（63）was used.In Hi5 cells,the fold activity of BDVpA（63）regulated by promoter p5 and ie1 was 6.5 times and 21.7 times as much as that of SV40 polyA,respectively.Meanwhile,the fold activity of BDVpA（63）in BmN cells was up to 2.7-fold and 12.6-fold compared with that of SV40 polyA,separately.Thus,the data indicated that BDVpA（63）was more beneficial to the expression of firefly luciferase gene than SV40 polyA.Real-time qPCR was used to test the relative content of gfp mRNA driven by different promoters in different cell lines,then the influence of BDVp A（63）on the transcription level of foreign gene was assessed.When BDVpA（63）was inserted into downstream of gfp,the relative content of mRNA was higher in contrast with SV40polyA.In Hi5 cells,under the control of two promoters（p5 and ie1）,the relative content of gfp transcripts after insertion of BDVpA（63）was 2.8 times and 4.9 times higher than that of SV40 polyA,separately.Whereas the relative content in BmN cells was respectively 2.8-fold and 4.9-fold as much as that of SV40 polyA.Therefore,BDVpA（63）integrated into the downstream of foreign gene could promote the level of gene transcription.The relative activity of firefly luciferase containing different lengths of BDVp A was measured by dual luciferase reporter assay system to identify BDVpA core functional areas.The characteristic sequences of BDVpA were intercepted and a series of firefly luciferase plasmids with BDVpA of different lengths were constructed,Then these plasmids were separately transfected into Hi5 and BmN cells.The discrepancies between the fold activity of BDVpA were measured.The results suggested that the relative activity of firefly luciferase with intact BDVpA（92,63,50,38）containing AAUAAA motif and GU-rich regions was higher than that with incomplete BDVpA,which had only one of these two characteristic sequences.However,the relative activity of firefly luciferase with insertion of BDVpA（50）was the highest among the whole transfections.Hence,BDVpA（50）composed of AAUAAA motif,GU-rich regions and downstream 12 nts was the core functional region identified in this study.In conclusion,BDVpA is a promising characteristic polyA signal from insect virus,which could not only promote the expression of multiple foreign genes in insect cell lines,but also potentially increase the loading amount of plasmid or viral vectors to alleviate the restrictions of inserted fragments size.