| Bombyx mori bidensovirus(BmBDV)is the only species in the family Bidnaviridae so far.It mainly replicates in the columnar cells of the midgut of the silkworm,causing the nucleus of the columnar cells to expand and causing serious flacherie disease.It is one of the main causes of death in silkworms.BmBDV belongs to a single-strand linear bipart DNA virus,and the genome includes two single-stranded DNA segments packaged in separated capsid,wherein VD1 is about6.6 kb and VD2 is about 6.0 kb.The two open reading frames(ORFs)of VD2 encode the non-structural protein NS3 and the minor structural protein P133.The promoter regulating the p133 is called P89.In our previous study,the core functional region of the P89 promoter was characterized by the dual luciferase system,a possible silencer element was found in the region of-143 nt to-268 nt of P89 to inhibit the activity of the promoter.Through further bioinformatics analysis and truncation analysis,the range of this element was located in the region of-195 nt and-205 nt.In this study,the luciferase assay system,deletion mutation and gel retardation EMSA were used to further identify the silencer element that may exist in the BmBDV P89 promoter,and analyze its characteristics and mechanism.First,the-205 nt to-195 nt sequence in the P89 promoter was deleted to construct the reporter vector pGL3-ΔS~+-P89.Based on this,the deletion sequence was cloned into the upstream of the P89 promoter to construct the plasmid pGL3-U-ΔSp89.These two plasmid were transfected to insect cells respectively and the expression of luciferase was measured.The results showed that the activity of P89 promoter was significantly up-regulated after deletion of nt205 to nt195,and restored its initial activity after the ectopic replacement of nt205 to nt195,indicating that the BmBDV P89nt205-195 sequence is a silencer.To investigate whether the BmBDV P89nt205-195 silencer has promoter specificity,the sequence was cloned into the upstream of the BmBDV ns1/ns2 gene promoter P5 and the BmNPV early promoter Pie1 to construct reporter vectors pGL3-S~+-P5 and pGL3-S~+-Pie1.The results showed that BmBDV P89nt205-195 can inhibit the activity of P5 and Pie1,and the expression of reporter gene can be down-regulated in insect cells BmN and Hi5.Studies on the mode of action of this silencer indicate that its negative regulation is independent of its insertion direction and insertion site.When this sequence is inserted upstream of the promoter of the target gene or after the downstream of the reporter gene in the direction of nt205-195 or nt195-205,it was observed that the transcription of the target gene was significantly inhibited.Therefore,this silencer has no promoter specificity and does not have directional dependence and positional dependence.Gel retardation experiments indicate that there is a specific protein or protein complex in Hi5 cells that interacts with the silencer,and the identification of this specific protein is still ongoing.The silencer sequences were mutant to construct silencer mutant luciferase reporter plasmids using overlapping PCR.The results of luciferase expression show that mutation of the"AAAATG"sequence in this silencer significantly reduces the function of the silencer.EMSA indicates that mutation of this region causes the protein(s)to fail to bind,further demonstrating that"AAAATG"are the key bases of this silencer.In conclusion,BmBDV P89nt205-195 has the function of a silencer,which interacts with specific protein(s)to inhibit the expression of genes independent of the position and orientation of the silencer and without promoter specificity.The function of the last six bases of the silencer is critical.Our research facilitates an in-depth understanding of the expression regulation mechanism of the BmBDV gene and lays a foundation for the regulation of gene expression using this silencer element. |
PDF Full Text Request