3 Biotech

, 8:214 | Cite as

Molecular cloning and characterization of Pif gene from pearl mussel, Hyriopsis cumingii, and the gene expression analysis during pearl formation

  • Rui Zhang
  • Mengting Qin
  • Jie Shi
  • Lu Tan
  • Jiamin Xu
  • Zhenyan Tian
  • Yuhui Wu
  • Yuxuan Li
  • Yitian Li
  • Ning Wang
Original Article


In the present study, the Pif gene of the freshwater pearl aquaculture mussel, Hyriopsis cumingii (HcPif) was successfully cloned and functionally characterized. The full sequence of HcPif gene consists of 3415 base pairs, which putatively encode two proteins, HcPif90 and HcPif80. A sequence analysis revealed that HcPif contained a von Willebrand factor type A domain and a chitin-binding domain, and shared many functional residues with other Pif homologues. A highly conserved sequence, FKGLDEIELML, at the C-terminus of Pif80s was identified as the key functional site. The corresponding peptide fragment markedly modified the morphology of calcite crystallites in CaCO3 crystallization assay and might play an essential role in the interactive binding between HcPif80 and CaCO3. Moreover, real-time PCR results showed that HcPif gene was dominantly expressed in the pearl secreting tissues and its expression changed in response to the different development status of the pearl sac during pearl aquaculture. The gene expression of HcPif was maximum 7 days after mantle grafting and declined to about the control level on day 30. Our in vitro and in vivo experimental data indicated that HcPif gene possessed the inherent characteristics of a nacre formation gene and its expression might faithfully reflect the pearl secretion status of the pearl mussels examined. Our findings may extend the understanding of the biomineralization mechanism of nacre formation and provide a potential biomarker for pearl farming.


Pif Hyriopsis cumingii Biomineralization Pearl aquaculture 



This study was supported by the Natural Science Foundation of Jiangsu province (Grant no BK20130496), Natural Science Foundation of the Jiangsu Higher Education Institutions of China (Grant no 13KJB240001), Scientific Research Foundation for Returned Scholars, Ministry of Education of China and Startup Foundation for Advanced Talents of Jiangsu University (Grant no 13JDG006). Dr. Rui Zhang was supported by the National Natural Science Foundation of China (Grant no 81300673), Startup Foundation for Advanced Talents of Jiangsu University (Grant no 13JDG004), and Cultivation Project for Young Core Teacher of Jiangsu University.

Compliance with ethical standards

Conflict of interest

The authors declare that there is no conflict of interest regarding the publication of this article.

Supplementary material

13205_2018_1233_MOESM1_ESM.pdf (86 kb)
Supplementary material 1 (PDF 85 kb)


  1. Baeuerlein E (2007) Handbook of biomineralization. Wiley, Weinheim, pp 273–286CrossRefGoogle Scholar
  2. Bahn S, Jo B, Hwang B, Choi Y, Cha H (2015) Role of Pif97 in nacre biomineralization: in vitro characterization of recombinant Pif 97 as a framework protein for the association of organic-inorganic layers in nacre. Cryst Growth Des 15:3666–3673CrossRefGoogle Scholar
  3. Belcher AM, Wu XH, Christensen RJ, Hansma PK, Stucky GD (1996) Control of crystal phase switching and orientation by soluble mollusc-shell proteins. Nature 381:56–58CrossRefGoogle Scholar
  4. Blank S, Arnoldi M, Khoshnavaz S, Treccani L, Kuntz M, Mann K, Grathwohl G, Fritz M (2003) The nacre protein perlucin nucleates growth of calcium carbonate crystals. J Microsc 212:280–291CrossRefGoogle Scholar
  5. Blay C, Parrad S, Cabral P, Aiho V, Ky C (2016) Correlations between cultured pearl size parameters and PIF-177 biomarker expression in Pinctada margaritifera families reared in two contrasting environments. Estuar Coast Shelf Sci 182:254–260CrossRefGoogle Scholar
  6. Chang EP, Evans JS (2015) Pif97, a von Willebrand and peritrophin biomineralization protein, organizes mineral nanoparticles and creates intracrystalline nanochambers. Biochemistry 54:5348–5355CrossRefGoogle Scholar
  7. Chen X, Liu X, Bai Z, Zhao L, Li J (2017) HcTyr and HcTyp-1 of Hyriopsis cumingii, novel tyrosinase and tyrosinase-related protein genes involved in nacre color formation. Comp Biochem Physiol B Biochem Mol Biol 204:1–8CrossRefGoogle Scholar
  8. Du Y, Chang H, Yang S, Huang S, Tsai Y, Huang JJ, Chan JCC (2016) Study of binding interaction between Pif80 protein fragment and aragonite. Sci Rep 6:30883CrossRefGoogle Scholar
  9. Falini G, Albeck S, Weiner S, Addadi L (1996) Control of aragonite or calcite polymorphism by mollusk shell macromolecules. Science 271:67–69CrossRefGoogle Scholar
  10. Ferre F, Clote P (2005) Disulfide connectivity prediction using secondary structure information and diresidue frequencies. Bioinformatics 21:2336–2346CrossRefGoogle Scholar
  11. Fu G, Valiyaveettil S, Wopenka B, Morese DE (2005) CaCO3 biomineralization: acidic 8-kDa proteins isolated from aragonitic abalone shell nacre can specifically modify calcite crystal morphology. Biomacromolecules 6:1289–1298CrossRefGoogle Scholar
  12. Giles R, Manne S, Mann S, Morse DE, Stucky GD, Hansma PK (1995) Overgrowth of aragonite on molluscan nacre examined by atomic force microscopy. Bio Bull 188:8–15CrossRefGoogle Scholar
  13. Huang X, Madan A (1999) CAP3: a DNA sequence assembly program. Genome Res 9:868–877CrossRefGoogle Scholar
  14. Joubert C, Piquemal D, Marie B, Manchon L, Pierrat F, Zanella-Cleon I, Cochennec-Laureau N, Gueguen Y, Montagnani C (2010) Transcriptome and proteome analysis of Pinctada margaritifera calcifying mantle and shell: focus on biomineralization. BMC Genom 11:613CrossRefGoogle Scholar
  15. Kawabata S, Nagayama R, Hirata M, Shigenaga T, Agarwala KL, Saito T, Cho J, Nakajima H, Takagi T, Iwanaga S (1996) Tachycitin, a small granular component in horseshoe crab hemocytes, is an antimicrobial protein with chitin-binding activity. J Biochem 120:1253–1260CrossRefGoogle Scholar
  16. Kroger N (2009) The molecular basis of nacre formation. Science 325:1351–1352CrossRefGoogle Scholar
  17. Kumar S, Tamura K, Nei M (2004) MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 5:150–163CrossRefGoogle Scholar
  18. Liu C, Li S, Kong J, Liu Y, Wang T, Xie L, Zhang R (2015) In-depth proteomic analysis of shell matrix proteins of Pinctada fucata. Sci Rep 5:17269CrossRefGoogle Scholar
  19. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25:402–408CrossRefGoogle Scholar
  20. Mao Y, Jiang B, Peng Q, Liu W, Lin Y, Xie D, He X, Li S (2017) Cloning and characterization of WRKY gene homologs in Chiehqua (Benincasa hispida Cogn. var. Chieh-qua How) and their expression in response to fusaric acid treatment. 3 Biotech 7:86CrossRefGoogle Scholar
  21. Marie B, Joubert C, Tayale A, Zanella-Cleon I, Belliard C, Piquemal D, Cochennec-Laureau N, Marin F, Gueguen Y, Montagnani C (2012) Different secretory repertoires control the biomineralization processes of prism and nacre deposition of the pearl oyster shell. Proc Natl Acad Sci USA 109:20986–20991CrossRefGoogle Scholar
  22. Marie B, Arivalagan J, Matheron L, Bolbach G, Berland S, Marie A, Marin F (2017) Deep conservation of bivalve nacre proteins highlighted by shell matrix proteomics of the Unionoida Elliptio complanata and Villosa lienosa. J R Soc Interface 14:20160846CrossRefGoogle Scholar
  23. McGinty EL, Zenger KR, Jones DB, Jerry DR (2012) Transcriptome analysis of biomineralisation-related genes within the pearl sac: host and donor oyster contribution. Mar Genom 5:27–33CrossRefGoogle Scholar
  24. Norton JH, Lucas JS, Turner I, Mayer RJ, Newnham R (2000) Approaches to improve cultured pearl formation in Pinctada margaritifera through use of relaxation, antiseptic application and incision closure during bead insertion. Aquaculture 184:1–17CrossRefGoogle Scholar
  25. Nudelman F (2015) Nacre biomineralisation: a review on the mechanisms of crystal nucleation. Semin Cell Dev Biol 46:2–10CrossRefGoogle Scholar
  26. Orme CA, Noy A, Wierzbick A, McBride MT, Grantham M, Teng HH, Dove PM, DeYoreo JJ (2001) Formation of chiral morphologies through selective binding of amino acids to calcite surface steps. Nature 411:775–779CrossRefGoogle Scholar
  27. Suzuki M, Saruwatari K, Kogure T, Yamamoto Y, Nishimura T, Kato T, Nagasawa H (2009) An acidic matrix protein, Pif, is a key macromolecule for nacre formation. Science 325:1388–1390CrossRefGoogle Scholar
  28. Suzuki M, Iwashima A, Kimura M, Kogure T, Nagasawa H (2013) The molecular evolution of the Pif family proteins in various species of mollusks. Mar Biotechnol 15:145–158CrossRefGoogle Scholar
  29. Takahashi K, Yamamoto H, Onoda A, Doi M, Inaba T, Chiba M, Kobayashi A, Taguchi T, Okamura T, Ueyama N (2004) Highly oriented aragonite nanocrystal–biopolymer composites in an aragonite brick of the nacreous layer of Pinctada fucata. Chem Commun 8:996–997CrossRefGoogle Scholar
  30. Tuckwell D (1999) Evolution of von Willebrand factor A (VWA) domains. Biochem Soc Trans 27:835–840CrossRefGoogle Scholar
  31. Wang G, Yuan Y, Li J (2007) SSR analysis of genetic diversity and phylogenetic relationships among different populations of Hyriopsis cumingii from the five lakes of China. J Fish China 12:12–18Google Scholar
  32. Wang N, Lee Y, Lee J (2008) Recombinant perlucin nucleates the growth of calcium carbonate crystals: molecular cloning and characterization of perlucin from disk abalone, Haliotis discus discus. Comp Biochem Physiol B Biochem 149:354–361CrossRefGoogle Scholar
  33. Wang N, Kinoshita S, Riho C, Maeyama K, Nagai K, Watabe S (2009) Quantitative expression analysis of nacreous shell matrix protein genes in the process of pearl biogenesis. Comp Biochem Physiol B Biochem Mol Biol 154:346–350CrossRefGoogle Scholar
  34. Watabe S (2013) The importance of total genome databases in research on Akoya pearl oyster. Zool Sci 30:781–782CrossRefGoogle Scholar
  35. Whittaker CA, Hynes RO (2002) Distribution and evolution of von Willebrand/integrin A domains: widely dispersed domains with roles in cell adhesion and elsewhere. Mol Biol Cell 13:3369–3387CrossRefGoogle Scholar
  36. Yao HB, Ge J, Mao LB, Yan YX, Yu SH (2014) 25th anniversary article: artificial carbonate nanocrystals and layered structural nanocomposites inspired by nacre: synthesis, fabrication and applications. Adv Mater 26:163–187CrossRefGoogle Scholar
  37. Zhang J (2007) Disulfide-bond reshuffling in the evolution of an ape placental ribonuclease. Mol Biol Evol 24:505–512CrossRefGoogle Scholar
  38. Zhang R, Wang M, Xia N, Yu S, Chen Y, Wang N (2016) Cloning and analysis of gene expression of interleukin-17 homolog in triangle-shell pearl mussel, Hyriopsis cumingii, during pearl sac formation. Fish Shellfish Immunol 52:151–156CrossRefGoogle Scholar
  39. Zhao M, He M, Huang X, Wang Q (2014) A homeodomain transcription factor gene, PfMSX, activates expression of Pif gene in the pearl oyster Pinctada fucata. PLoS One 9:e103830CrossRefGoogle Scholar
  40. Zhu W, Fan S, Huang D, Liu B, Bi X, Yu D (2015) Highly expressed EGFR in pearl sac may facilitate the pearl formation in the pearl oyster, Pinctada fucata. Gene 566:201–211CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.School of MedicineJiangsu UniversityZhenjiang CityChina
  2. 2.School of Food and Biological EngineeringJiangsu UniversityZhenjiang CityChina

Personalised recommendations