The intronic promoter of Actin4 mediates high-level transgene expression mainly in the wing and epidermis of silkworms

The cytoplasmic actin gene Actin4 (A4) in silkworm (Bombyx mori) was isolated 20 years ago and has a distal promoter upstream of the first exon and a proximal promoter within the first intron; however, how the promoter regulates gene expression has yet to be fully elucidated. Here, we characterized the function and expression of the proximal promoter (named A4IP) by analyzing transgenic Gal4/UAS silkworms, A4IP-Gal4/UAS-EGFP. We demonstrated that A4IP drives the expression of Gal4 and thereby activates UAS-linked EGFP in transgenic silkworms beginning in day-3 embryos through adults. Further detection revealed that EGFP was expressed at a low level in tissues including the trachea, fat body and midgut but was highly expressed in the wing disks/wings and inner epidermis of transgenic silkworms. No EGFP signals were detected in other tissues by western blot assay. Interestingly, EGFP fluorescence had a spot-like distribution on the epidermis of transgenic larvae. These observations are quite different from those in transgenic silkworms driven by the promoter of Actin3 (A3), another cytoplasmic actin gene in B. mori. These findings reveal the expression profiles of the A4IP promoter and provide new insights into the regulatory mechanism of cytoplasmic actin genes in silkworms.