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.

中文翻译：

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Actin4的内含子启动子主要在蚕的翅膀和表皮中介导高水平的转基因表达。

家蚕（Bombyx mori）的胞质肌动蛋白基因Actin4（A4）是20年前分离的，在第一个外显子的上游有一个远端启动子，在第一个内含子内有一个近端启动子。然而，启动子如何调节基因表达尚未完全阐明。在这里，我们通过分析转基因Gal4 / UAS家蚕A4IP-Gal4 / UAS-EGFP来表征近端启动子（命名为A4IP）的功能和表达。我们证明了A4IP驱动Gal4的表达，从而激活了从成虫的第3天胚胎开始的转基因蚕中UAS连锁的EGFP。进一步的检测表明，EGFP在气管，脂肪体和中肠等组织中低水平表达，但在转基因蚕的翼盘/翼和内表皮中高表达。通过蛋白质印迹分析未在其他组织中检测到EGFP信号。有趣的是，EGFP荧光在转基因幼虫的表皮上具有斑点状分布。这些发现与由肌动蛋白（B. mori）的另一种细胞质肌动蛋白基因Actin3（A3）的启动子驱动的转基因家蚕的观察结果完全不同。这些发现揭示了A4IP启动子的表达谱，并提供了对家蚕细胞质肌动蛋白基因调控机制的新见解。