Abstract
Silk glands are specialized in the synthesis of several secretory proteins. Expression of genes encoding the silk proteins in Bombyx mori silk glands with strict territorial and developmental specificities is regulated by many transcription factors. In this study, we have characterized B. mori sage, which is closely related to sage in the fruitfly Drosophila melanogaster. It is termed Bmsage; it encodes transcription factor Bmsage, which belongs to the Mesp subfamily, containing a basic helix–loop–helix motif. Bmsage transcripts were detected specifically in the silk glands of B. mori larvae through RT-PCR analysis. Immunoblotting analysis confirmed the Bmsage protein existed exclusively in B. mori middle and posterior silk gland cells. Bmsage has a low level of expression in the 4th instar molting stages, which increases gradually in the 5th instar feeding stages and then declines from the wandering to the pupation stages. Quantitative PCR analysis suggested the expression level of Bmsage in a high silk strain was higher compared to a lower silk strain on day 3 of the larval 5th instar. Furthermore, far western blotting and co-immunoprecipitation assays showed the Bmsage protein interacted with the fork head transcription factor silk gland factor 1 (SGF1). An electrophoretic mobility shift assay showed the complex of Bmsage and SGF1 proteins bound to the A and B elements in the promoter of fibroin H-chain gene(fib-H), respectively. Luciferase reporter gene assays confirmed the complex of Bmsage and SGF1 proteins increased the expression of fib-H. Together, these results suggest Bmsage is involved in the regulation of the expression of fib-H by being together with SGF1 in B. mori PSG cells.
Highlights
The silkworm, Bombyx mori, which is a key economic insect often used as a model for lepidopteran insects, has huge economic value for its potential applications in industry and medical technology [1,2,3], as well as for studying the fundamental mechanisms underlying gene regulation and organ differentiation [4]
The B. mori silk gland is a differentiated silk-producing organ composed of the anterior silk gland (ASG), middle silk gland (MSG) and posterior silk gland (PSG)
The Bmsage basic helix– loop–helix domain (bHLH) domain showed 74%, 74%, 50%, 44.8%, 48.3% and 48.3% identity to those of Gmsage, Dmsage, Aasage, DrMesp, BfMesp and MmMesp, respectively (Fig. S2, Table S1). Based on this similarity and the phylogenetic tree, we concluded that Bmsage belongs to the Mesp-related subfamily of bHLH transcription factors
Summary
The silkworm, Bombyx mori, which is a key economic insect often used as a model for lepidopteran insects, has huge economic value for its potential applications in industry and medical technology [1,2,3], as well as for studying the fundamental mechanisms underlying gene regulation and organ differentiation [4]. The B. mori silk gland is a differentiated silk-producing organ composed of the anterior silk gland (ASG), middle silk gland (MSG) and posterior silk gland (PSG). The fib-H gene is highly expressed in PSG cells but is repressed in MSG cells. Details of the mechanism underlying the ability to be synthesized effectively in silk glands and the precise regulation of how fib-H is highly expressed in PSG cells but is repressed in MSG cells is not fully understood
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