Construction of synthetic constitutive promoters and evaluation of their expression strengths in Sf9, HEK293T, and BmN cells.

ObjectivesConstitutive promoters have been widely studied and utilized in silkworm resistance breeding and expression of beneficial proteins, but there is limited research comparing the activities of different promoters across various source cell.MethodsThis study screened three promoters, namely, the ie1 gene promoter (ie1p) of Bombyx mori nuclear polyhedrosis virus (BmNPV), the cytoplasmic actin 3 promoter (A3p) and the cytoplasmic actin 4 promoter (A4p). Enhancer fragments hr3 and hr5 were combined with the three promoters to driving the expression of luciferase (luc⁺) and vacuolar-type ATPase c (Vac). The constructed plasmids were transfected into Sf9, 293 T and BmN cells, and the transient expression of target genes were assessed.ResultsThe luc⁺ activity assays revealed that the enhancer effects of hr3 and hr5 fragments followed a similar trend in both Sf9 and 293 T cell lines. Specifically, hr3 significantly enhanced the ie1p promoter, increasing its activity by 2.44-fold and 2.90-fold, respectively. Similarly, hr5 markedly boosted the A4p promoter, with enhancements of 2.45-fold and 3.00-folds, respectively. There were differences in the relative highest expression levels of Vac gene among the three cell lines, hr5 had a significant effect on all three promoters in Sf9 cells.ConclusionThis research aims to identify highly active promoters in silkworm cells, refine transgenic methodologies and strategies, and lay the experimental groundwork for developing a transgenic silkworm technology platform.