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The expression profile and promoter analysis of β-N-acetylglucosaminidases in the silkworm Bombyx mori

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Abstract

β-N-acetylglucosaminidase (GlcNAcase) is a key enzyme in the chitin decomposition process. In this study, we investigated the gene expression profile of GlcNAcases and the regulation mechanism for one of these genes, BmGlcNAcase1, in the silkworm. We performed sequence analysis of GlcNAcase. Using dual-spike-in qPCR method, we examined the expression of Bombyx β-N-acetylglucosaminidases (BmGlcNAcases) in various tissues of silkworm as well as expression changes after stimulation with ecdysone. Using Bac-to-Bac system and luciferase reporter vectors, we further analyzed the promoter sequence of BmGlcNAcase1. The results showed that these proteins have a highly conserved catalytic domain. The expression levels of the BmGlcNAcase genes varied in different tissues, and were increased 48 h after exposure to ecdysone. BmGlcNAcase1 gene promoter with 5′-end serial deletions showed different levels of activity in various tissues, higher in the blood, skin and fat body. Deletion of the region from −347 to −223 upstream of BmGlcNAcase-1 gene abolished its promoter activity. This region contains the binding sites for key transcription factors including Hb, BR–C Z, the HSF and the typical TATA-box element. These results indicate that BmGlcNAcases are expressed at different levels in different tissues of the silkworm, but all are subjected to the regulation by ecdysone. BmGlcNAcase1 promoter analysis has paved a foundation for further study of the gene expression patterns.

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References

  1. Cohen E (2010) Chitin biochemistry: synthesis, hydrolysis and inhibition, chap. 2. In: Jérôme C, Stephen JS (eds) Advances in Insect Physiology. Academic Press, London, pp 5–74

    Google Scholar 

  2. Wagner G, Lo J, Laine R, Almeder M (1993) Chitin in the epidermal cuticle of a vertebrate (Paralipophrys trigloides, blenniidae, teleostei). Cell Mol Life Sci 49(4):317–319

    Article  CAS  Google Scholar 

  3. Fernandez CW, Koide RT (2012) The role of chitin in the decomposition of ectomycorrhizal fungal litter. Ecology 93(1):24–28

    Article  PubMed  Google Scholar 

  4. Van Leeuwen T, Demaeght P, Osborne EJ et al (2012) Population bulk segregant mapping uncovers resistance mutations and the mode of action of a chitin synthesis inhibitor in arthropods. Proc Natl Acad Sci 109(12):4407–4412

    Article  PubMed Central  PubMed  Google Scholar 

  5. Kramer KJ, Hopkins TL, Schaefer J (1995) Applications of solids NMR to the analysis of insect sclerotized structures. Insect Biochem Mol Biol 25(10):1067–1080

    Article  CAS  Google Scholar 

  6. Kramer KJ, Hopkins TL, Schaefer J (1988) Insect cuticle structure and metabolism. Biotechnology for Crop Protection. American Chemical Society, Washington DC, pp 160–185

    Book  Google Scholar 

  7. Fukamizo T, Kramer KJ (1985) Mechanism of chitin hydrolysis by the binary chitinase system in insect moulting fluid. Insect Biochem 15(2):141–145

    Article  CAS  Google Scholar 

  8. Filho BPD, Lemos FJA, Secundino NFC, Páscoa V, Pereira ST, Pimenta PFP (2002) Presence of chitinase and β-N-acetylglucosaminidase in the Aedes aegypti: a chitinolytic system involving peritrophic matrix formation and degradation. Insect Biochem Mol Biol 32(12):1723–1729

    Article  CAS  PubMed  Google Scholar 

  9. Fukamizo T, Kramer KJ (1985) Mechanism of chitin oligosaccharide hydrolysis by the binary enzyme chitinase system in insect moulting fluid. Insect Biochem 15(1):1–7

    Article  CAS  Google Scholar 

  10. Kramer KJ, Koga D (1986) Insect chitin: physical state, synthesis, degradation and metabolic regulation. Insect Biochem 16(6):851–877

    Article  CAS  Google Scholar 

  11. Zen KC, Choi HK, Krishnamachary N, Muthukrishnan S, Kramer KJ (1996) Cloning, expression, and hormonal regulation of an insect beta-N-acetylglucosaminidase gene. Insect Biochem Mol Biol 26(5):435–444

    Article  CAS  PubMed  Google Scholar 

  12. Xia QY, Zhou ZY, Lu C et al (2004) A draft sequence for the genome of the domesticated silkworm (Bombyx mori). Science 306(5703):1937–1940

    Article  PubMed  Google Scholar 

  13. Consortium TISG (2008) The genome of a lepidopteran model insect, the silkworm Bombyx mori. Insect Biochem Mol 38(12):1036–1045

    Article  Google Scholar 

  14. Xia Q, Guo Y, Zhang Z et al (2009) Complete resequencing of 40 genomes reveals domestication events and genes in silkworm (Bombyx). Science 326(5951):433–436

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  15. Nagamatsu Y, Yanagisawa I, Kimoto M, Okamoto E, Koga D (1995) Purification of a chitooligosaccharidolytic beta-N-acetylglucosaminidase from Bombyx mori larvae during metamorphosis and the nucleotide sequence of its cDNA. Biosci Biotechnol Biochem 59(2):219–225

    Article  CAS  PubMed  Google Scholar 

  16. Okada T, Ishiyama S, Sezutsu H et al (2007) Molecular cloning and expression of two novel β-N-acetylglucosaminidases from silkworm Bombyx mori. Biosci Biotechnol Biochem 71(7):1626–1635

    Article  CAS  PubMed  Google Scholar 

  17. Kokuho T, Yasukochi Y, Watanabe S, Inumaru S (2010) Molecular cloning and expression profile analysis of a novel β-d-N-acetylhexosaminidase of domestic silkworm (Bombyx mori). Genes Cells 15(5):525–536

    CAS  PubMed  Google Scholar 

  18. Nomura T, Ikeda M, Ishiyama S et al (2010) Cloning and characterization of a β-N-acetylglucosaminidase (BmFDL) from silkworm Bombyx mori. J Biosci Bioeng 110(4):386–391

    Article  CAS  PubMed  Google Scholar 

  19. Zhang Y, Wei Z, Li Y–Y, Chen Y, Shen W, Lu C (2009) Transcription level of messenger RNA per gene copy determined with dual-spike-in strategy. Anal Biochem 394(2):202–208

    Article  CAS  PubMed  Google Scholar 

  20. Peng R, Zhai Y, Ding H et al (2012) Analysis of reference gene expression for real-time PCR based on relative quantitation and dual spike-in strategy in the silkworm Bombyx mori. Acta Biochim Biophys Sin 44(7):614–622

    Article  CAS  PubMed  Google Scholar 

  21. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22(22):4673–4680

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  22. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28(10):2731–2739

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  23. Zhang Y, Wei Z, Li YY, Chen Y, Shen W, Lu C (2009) Transcription level of messenger RNA per gene copy determined with dual-spike-in strategy. Anal Biochem 394(2):202–208

    Article  CAS  PubMed  Google Scholar 

  24. Léonard R, Rendić D, Rabouille C, Wilson IBH, Préat T, Altmann F (2006) The Drosophila fused lobes gene encodes an N-acetylglucosaminidase involved in N-glycan processing. J Biol Chem 281(8):4867–4875

    Article  PubMed  Google Scholar 

  25. Hogenkamp DG, Arakane Y, Kramer KJ, Muthukrishnan S, Beeman RW (2008) Characterization and expression of the β-N-acetylhexosaminidase gene family of Tribolium castaneum. Insect Biochem Mol Biol 38(4):478–489

    Article  CAS  PubMed  Google Scholar 

  26. Koga D, Funakoshi T, Fujimoto H, Kuwano E, Eto M, Ide A (1991) Effects of 20-hydroxyecdysone and KK-42 on chitinase and β-N-acetylglucosaminidase during the larval-pupal transformation of Bombyx mori. Insect Biochem 21(3):277–284

    Article  CAS  Google Scholar 

  27. Perotti ME, Cattaneo F, Pasini ME, Vernì F, Hackstein JHP (2001) Male sterile mutant casanova gives clues to mechanisms of sperm–egg interactions in Drosophila melanogaster. Mol Reprod Dev 60(2):248–259

    Article  CAS  PubMed  Google Scholar 

  28. Cattaneo F, Ogiso M, Hoshi M, Perotti ME, Pasini ME (2002) Purification and characterization of the plasma membrane glycosidases of Drosophila melanogaster spermatozoa. Insect Biochem Mol Biol 32(8):929–941

    Article  CAS  PubMed  Google Scholar 

  29. Cattaneo F, Pasini M, Intra J et al (2006) Identification and expression analysis of Drosophila melanogaster genes encoding β-hexosaminidases of the sperm plasma membrane. Glycobiology 16(9):786–800

    Article  CAS  PubMed  Google Scholar 

  30. Lehmann R, Nüsslein-Volhard C (1987) Hunchback, a gene required for segmentation of an anterior and posterior region of the Drosophila embryo. Dev Biol 119(2):402

    Article  CAS  PubMed  Google Scholar 

  31. Fernandes M, Xiao H, Lis JT (1994) Fine structure analyses of the Drosophila and Saccharomyces heat shock factor-heat shock element interactions. Nucleic Acids Res 22(2):167–173

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  32. Morton EA, Lamitina T (2012) Caenorhabditis elegans HSF‐1 is an essential nuclear protein that forms stress granule-like structures following heat shock. Aging Cell 12(1):112–120

  33. Dottorini T, Persampieri T, Palladino P, Spaccapelo R, Crisanti A (2012) Silencing of the Hsf gene, the transcriptional regulator of A. gambiae male accessory glands, inhibits the formation of the mating plug in mated females and disrupts their monogamous behaviour. Pathogens Glob Health 106(7):405–412

    Article  CAS  Google Scholar 

  34. Emery IF, Bedian V, Guild GM (1994) Differential expression of Broad-Complex transcription factors may forecast tissue-specific developmental fates during Drosophila metamorphosis. Development 120(11):3275–3287

    CAS  PubMed  Google Scholar 

  35. Talbot WS, Swyryd EA, Hogness DS (1993) Drosophila tissues with different metamorphic responses to ecdysone express different ecdysone receptor isoforms. Cell 73(7):1323–1337

    Article  CAS  PubMed  Google Scholar 

  36. Tzolovsky G, Deng WM, Schlitt T, Bownes M (1999) The function of the broad-complex during Drosophila melanogaster oogenesis. Genetics 153(3):1371–1383

    PubMed Central  CAS  PubMed  Google Scholar 

  37. Roeder R (1998) Role of general and gene-specific cofactors in the regulation of eukaryotic transcription. Cold Spring Harbor Symposia on Quantitative Biology, Cold Spring Harbor Laboratory Press, New York, pp 201–218

    Google Scholar 

Download references

Acknowledgments

This work was supported by the foundation of post scientist in National Sericultural System (CARS-22-ZJ0305).

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Authors and Affiliations

  1. School of Basic Medicine and Biological Sciences, Soochow University, Suzhou, 215123, Jiangsu, People’s Republic of China

    Yuan-fen Zhai, Ming-xia Huang, Yu Wu, Guo-dong Zhao, Jie Du, Bing Li, Wei-de Shen & Zheng-guo Wei

  2. National Engineering Laboratory for Modern Silk, Soochow University, Suzhou, 215123, Jiangsu, People’s Republic of China

    Bing Li, Wei-de Shen & Zheng-guo Wei

Authors
  1. Yuan-fen Zhai
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  2. Ming-xia Huang
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  3. Yu Wu
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  4. Guo-dong Zhao
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  5. Jie Du
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  6. Bing Li
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  7. Wei-de Shen
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Correspondence to Wei-de Shen or Zheng-guo Wei.

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Yuan-fen Zhai and Ming-xia Huang have contributed equally to this work.

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Zhai, Yf., Huang, Mx., Wu, Y. et al. The expression profile and promoter analysis of β-N-acetylglucosaminidases in the silkworm Bombyx mori . Mol Biol Rep 41, 6667–6678 (2014). https://doi.org/10.1007/s11033-014-3550-6

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