Insights into the regulatory characteristics of silkworm fibroin gene promoters using a modified Gal4/UAS system
- 169 Downloads
The silkworm Bombyx mori is a valuable insect that synthesizes bulk amounts of fibroin protein in its posterior silk gland (PSG) and weaves these proteins into silk cocoons. The mechanism by which the fibroin protein is efficiently synthesized and precisely regulated is an important aspect that has yet to be fully elucidated. Here, we describe the regulatory characteristics of the promoters of fibroin protein-encoding genes, namely, fibroin heavy chain (fibH) and fibroin light chain (fibL), using an optimized Gal4/UAS binary system. We found that (1) UAS-linked enhanced green fluorescent protein (EGFP) was effectively activated in the PSGs of Gal4/UAS transgenic silkworms, and fluorescence was continuously detected in the PSGs after complete formation of silk glands. (2) In the PSGs of fourth- and fifth-instar larvae of transgenic silkworms driven by fibL-Gal4 (LG4) or fibH-Gal4 (HG4), EGFP mRNA was detected in only day-3 to day-6 fifth-instar larvae, while the EGFP protein could be detected at each day of both larval stages. (3) High-level expression of Gal4 and UAS-linked EGFP caused a delay in PSG degradation in Gal4/UAS transgenic silkworms. (4) At the early pupal stage, EGFP fluorescence was also detected in fat bodies of Gal4/UAS transgenic silkworms, indicating that the PSG-specific EGFP was transported into fat bodies during PSG degeneration; however, the underlying mechanism needs to be further elucidated. This study provides a modified Gal4/UAS system used for efficient tissue-specific expression of target genes in the PSGs of silkworms and provides new insights into the regulatory characteristics of the promoters of key fibroin protein-encoding genes.
KeywordsSilkworm Gal4/UAS binary system Fibroin protein-encoding gene Promoter Regulatory characteristics
This work was supported by the National Natural Science Foundation of China (Grant No. 31872291) and grants from the Chongqing Science and Technology bureau (Nos. cstc2017jcyjBX0041, cstc2017jcyj-yszx0009, cstc2016jcyjA0237). The English in the manuscript was polished by American Journal Experts.
- Barber EW (1991) Prehistoric textiles: the development of cloth in the Neolithic and Bronze Ages with special reference to the Aegean. Princeton University Press, PrincetonGoogle Scholar
- Daimon T, Kozaki T, Niwa R, Kobayashi I, Furuta K, Namiki T, Uchino K, Banno Y, Katsuma S, Tamura T, Mita K, Sezutsu H, Nakayama M, Itoyama K, Shimada T, Shinoda T (2012) Precocious metamorphosis in the juvenile hormone-deficient mutant of the silkworm, Bombyx mori. PLoS Genet 8(3):e1002486CrossRefGoogle Scholar
- Ohno K, Sawada J, Takiya S, Kimoto M, Matsumoto A, Tsubota T, Uchino K, Hui CC, Sezutsu H, Handa H, Suzuki Y (2013) Silk gland factor-2, involved in fibroin gene transcription, consists of LIM homeodomain, LIM-interacting, and single-stranded DNA-binding proteins. J Biol Chem 288(44):31581–31591CrossRefGoogle Scholar
- Sakudoh T, Sezutsu H, Nakashima T, Kobayashi I, Fujimoto H, Uchino K, Banno Y, Iwano H, Maekawa H, Tamura T, Kataoka H, Tsuchida K (2007) Carotenoid silk coloration is controlled by a carotenoid-binding protein, a product of the Yellow blood gene. Proc Natl Acad Sci U S A 104(21):8941–8946CrossRefGoogle Scholar