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.