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Insights into the regulatory characteristics of silkworm fibroin gene promoters using a modified Gal4/UAS system

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Abstract

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.

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References

  • Barber EW (1991) Prehistoric textiles: the development of cloth in the Neolithic and Bronze Ages with special reference to the Aegean. Princeton University Press, Princeton

    Google 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):e1002486

    Article  CAS  Google Scholar 

  • Fischer JA, Giniger E, Maniatis T, Ptashne M (1988) GAL4 activates transcription in Drosophila. Nature 332(6167):853–856

    Article  CAS  Google Scholar 

  • Giniger E, Varnum SM, Ptashne M (1985) Specific DNA binding of GAL4, a positive regulatory protein of yeast. Cell 40(4):767–774

    Article  CAS  Google Scholar 

  • Hara C, Morishita K, Takayanagi-Kiya S, Mikami A, Uchino K, Sakurai T, Kanzaki R, Sezutsu H, Iwami M, Kiya T (2017) Refinement of ectopic protein expression through the GAL4/UAS system in Bombyx mori: application to behavioral and developmental studies. Sci Rep 7(1):11795

    Article  Google Scholar 

  • Hartley KO, Nutt SL, Amaya E (2002) Targeted gene expression in transgenic Xenopus using the binary Gal4-UAS system. Proc Natl Acad Sci USA 99(3):1377–1382

    Article  CAS  Google Scholar 

  • Horn C, Wimmer EA (2000) A versatile vector set for animal transgenesis. Dev Genes Evol 210:630–637

    Article  CAS  Google Scholar 

  • Hui CC, Matsuno K, Suzuki Y (1990) Fibroin gene promoter contains a cluster of homeodomain binding sites that interact with three silk gland factors. J Mol Biol 213(4):651–670

    Article  CAS  Google Scholar 

  • Imamura M, Nakai J, Inoue S, Quan GX, Kanda T, Tamura T (2003) Targeted gene expression using the GAL4/UAS system in the silkworm Bombyx mori. Genetics 165(3):1329–1340

    CAS  PubMed  PubMed Central  Google Scholar 

  • Inoue S, Tanaka K, Arisaka F, Kimura S, Ohtomo K, Mizuno S (2000) Silk fibroin of Bombyx mori is secreted, assembling a high molecular mass elementary unit consisting of H-chain, L-chain, and P25, with a 6:6:1 molar ratio. J Biol Chem 275(51):40517–40528

    Article  CAS  Google Scholar 

  • Inoue S, Kanda T, Imamura M, Quan GX, Kojima K, Tanaka H, Tomita M, Hino R, Yoshizato K, Mizuno S, Tamura T (2005) A fibroin secretion-deficient silkworm mutant, Nd-sD, provides an efficient system for producing recombinant proteins. Insect Biochem Mol Biol 35(1):51–59

    Article  CAS  Google Scholar 

  • Kimoto M, Tsubota T, Uchino K, Sezutsu H, Takiya S (2015) LIM-homeodomain transcription factor Awh is a key component activating all three fibroin genes, fibH, fibL and fhx, in the silk gland of the silkworm, Bombyx mori. Insect Biochem Mol Biol 56:29–35

    Article  CAS  Google Scholar 

  • Kobayashi I, Kojima K, Uchino K, Sezutsu H, Iizuka T, Tatematsu K, Yonemura N, Tanaka H, Yamakawa M, Ogura E, Kamachi Y, Tamura T (2011) An efficient binary system for gene expression in the silkworm, Bombyx mori, using GAL4 variants. Arch Insect Biochem Physiol 76(4):195–210

    Article  CAS  Google Scholar 

  • Laughon A, Gesteland RF (1984) Primary structure of the Saccharomyces cerevisiae GAL4 gene. Mol Cell Biol 4(2):260–267

    Article  CAS  Google Scholar 

  • Ma L, Xu H, Zhu J, Ma S, Liu Y, Jiang RJ, Xia Q, Li S (2011) Ras1 (CA) overexpression in the posterior silk gland improves silk yield. Cell Res 21(6):934–943

    Article  CAS  Google Scholar 

  • Maekawa H, Suzuki Y (1980) Repeated turn-off and turn-on of fibroin gene transcription during silk gland development of Bombyx mori. Dev Biol 78:394–406

    Article  CAS  Google 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):31581–31591

    Article  CAS  Google Scholar 

  • Ornitz DM, Moreadith RW, Leder P (1991) Binary system for regulating transgene expression in mice: targeting int-2 gene expression with yeast GAL4/UAS control elements. Proc Natl Acad Sci USA 88(3):698–702

    Article  CAS  Google Scholar 

  • Patel CV, Gopinathan KP (1991) Development stage-specific expression of fibroin in the silkworm Bombyx mori is regulated translationally. Indian J Biochem Biophys 28(5–6):521–530

    CAS  PubMed  Google 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):8941–8946

    Article  CAS  Google Scholar 

  • Scheer N, Campos-Ortega JA (1999) Use of the Gal4-UAS technique for targeted gene expression in the zebrafish. Mech Dev 80(2):153–158

    Article  CAS  Google Scholar 

  • Takiya S, Kokubo H, Suzuki Y (1997) Transcriptional regulatory elements in the upstream and intron of the fibroin gene bind three specific factors POU-M1, BmFkh and FMBP-1. Biochem J 321(Pt 3):645–653

    Article  CAS  Google Scholar 

  • Tan A, Tanaka H, Tamura T, Shiotsuki T (2005) Precocious metamorphosis in transgenic silkworms overexpressing juvenile hormone esterase. Proc Natl Acad Sci USA 102(33):11751–11756

    Article  CAS  Google Scholar 

  • Tatematsu K, Kobayashi I, Uchino K, Sezutsu H, Iizuka T, Yonemura N, Tamura T (2010) Construction of a binary transgenic gene expression system for recombinant protein production in the middle silk gland of the silkworm Bombyx mori. Transgenic Res 19(3):473–487

    Article  CAS  Google Scholar 

  • Tsutsumi M, Muto H, Myoba S, Kimoto M, Kitamura A, Kamiya M, Kikukawa T, Takiya S, Demura M, Kawano K, Kinjo M, Aizawa T (2016) In vivo fluorescence correlation spectroscopy analyses of FMBP-1, a silkworm transcription factor. FEBS Open Bio 6(2):106–125

    Article  CAS  Google Scholar 

  • Wang G, Xia Q, Cheng D, Duan J, Zhao P, Chen J, Zhu L (2008) Reference genes identified in the silkworm Bombyx mori during metamorphism based on oligonucleotide microarray and confirmed by qRT-PCR. Insect Sci 15(5):405–413

    Article  Google Scholar 

  • Wang F, Xu H, Yuan L, Ma S, Wang Y, Duan X, Duan J, Xiang Z, Xia Q (2013) An optimized sericin-1 expression system for mass-producing recombinant proteins in the middle silk glands of transgenic silkworms. Transgenic Res 22:925–938

    Article  CAS  Google Scholar 

  • Xu H, O’Brochta DA (2015) Advanced technologies for genetically manipulating the silkworm Bombyx mori, a model Lepidopteran insect. Proc Biol Sci 282(1810):20150487

    Article  Google Scholar 

  • Xu PX, Fukuta M, Takiya S, Matsuno K, Xu X, Suzuki Y (1994) Promoter of the POU-M1/SGF-3 gene involved in the expression of Bombyx silk genes. J Biol Chem 269(4):2733–2742

    CAS  PubMed  Google Scholar 

  • Zhao XM, Liu C, Jiang LJ, Li QY, Zhou MT, Cheng TC, Mita K, Xia QY (2015) A juvenile hormone transcription factor Bmdimm-fibroin H chain pathway is involved in the synthesis of silk protein in silkworm, Bombyx mori. J Biol Chem 290(2):972–986

    Article  CAS  Google Scholar 

Download references

Acknowledgements

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.

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Correspondence to Hanfu Xu.

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Fig. S1

Fluorescence detection of EGFP expression in cocoons of Gal4/UAS transgenic silkworms. The cocoon shells of HG4/UEGFP and LG4/UEGFP were peeled from outside to inside, and four thin cocoon pieces were obtained and are indicated by Arabic numerals. Images were acquired under white light or under a fluorescence microscope. (TIFF 2301 kb)

Fig. S1

Fluorescence detection of EGFP expression in Gal4/UAS transgenic pupae. Day 3 pupae of HG4/UEGFP and LG4/UEGFP were collected, and images were acquired under white light or under a fluorescence microscope. WT was used as a control. (TIFF 1083 kb)

Table S1

Identification of piggyBac integration sites using inverse PCR.(DOCX 15 kb)

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Liu, R., Zeng, W., Tan, T. et al. Insights into the regulatory characteristics of silkworm fibroin gene promoters using a modified Gal4/UAS system. Transgenic Res 28, 627–636 (2019). https://doi.org/10.1007/s11248-019-00175-w

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  • DOI: https://doi.org/10.1007/s11248-019-00175-w

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