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Molecular characterization and expression analysis of chitinase (Fcchi-3) from Chinese shrimp, Fenneropenaeus chinensis

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Abstract

Complementary DNA (cDNA) and genomic DNA, including flanking regions of the chitinase gene (Fcchi-3) of Fenneropenaeus chinensis, were cloned and sequenced. Fcchi-3 was found to have 92.0 and 91.4% identity at the cDNA level to that of Litopenaeus vannamei and Marsupenaeus japonicus, respectively. The predicted amino acid sequence had an overall similarity with a comparable region of L. vannamei (96.8%) and M. japonicus (93.4%). Based on the cDNA sequence, the genomic structure of the gene was characterized. Sequence analysis revealed that the Fcchi-3 gene was composed of seven exons with 411, 252, 186, 132, 171, 117 and 135 bp and six introns with 232, 196, 121, 90, 159 and 157 bp. Analysis by RT-PCR revealed that Fcchi-3 was a hepatopancreas specific gene. Semi-quantitative RT-PCR analysis revealed that Fcchi-3 transcript was down-regulated significantly in response to the challenge of WSSV at 5 h post-injection and then came back to normal level at 37 h. A fusion protein containing Fcchi-3 was produced and the purified recombinant protein exhibited similar biological function. The result of identification through LC-ESI-MS showed that three peptide fragments (-MAADPVLR-, -ATIDPAYNVPELSK- and -AILAVGGWNEGSPK-) of the recombinant protein were identical to the corresponding sequence of L. vannamei chitinase-3. The recombinant Fcchi-3 could degrade the colloid chitin confirming that the recombinant protein is actually the chitinase.

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References

  1. Arakane Y, Zhu QS, Matsumiya M, Muthukrishnan S, Kramer K (2003) Properties of catalytic, linker and chitin-binding domains of insect chitinase. Insect Biochem Mol Biol 23:631–648. doi:10.1016/S0965-1748(03)00049-3

    Article  Google Scholar 

  2. Bachère E, Chagot D, Grizel H (1988) Separation of Crassostrea gigas hemocytes by density gradient centrifugation and counterflow centrifugal elutriation. Dev Comp Immunol 12:549–551. doi:10.1016/0145-305X(88)90071-7

    Article  PubMed  Google Scholar 

  3. Bairoch A, Bucher P, Hofmann K (1996) The PROSITE database, its status in 1995. Nucleic Acids Res 24(1):189–196. doi:10.1093/nar/24.1.189

    Article  CAS  PubMed  Google Scholar 

  4. Boot RG, Blommaart EF, Swart E, Ghauharali-van der Vlugt K, Bijl N, Moe C, Place A, Aerts JM (2001) Identification of a novel acidic mammalian chitinase distinct from chitotriosidase. J Biol Chem 276(9):6770–6778. doi:10.1074/jbc.M009886200

    Article  CAS  PubMed  Google Scholar 

  5. He NH, Qing QW, Xu X (2005) Differential profile of genes expressed in hemocytes of white spot syndrome virus-resistant shrimp (Penaeus japonicus) by combining suppression subtractive hybridization and differential hybrization. Antiviral Res 66:39–45. doi:10.1016/j.antiviral.2004.12.010

    Article  CAS  PubMed  Google Scholar 

  6. Joo GJ (2005) Purification and characterization of an extracellular chitinase from the antifungal biocontrol agent Streptomyces halstedii. Biotechnol Lett 27(19):1483–1486. doi:10.1007/s10529-005-1315-y

    Article  CAS  PubMed  Google Scholar 

  7. Kincaid HL, Krueger CC, May B (1993) Preservation of genetic variation in the Green Lake strain lake trout derived from remnant domestic and feral populations. N Am J Fish Manage 13:318–325. doi:10.1577/1548-8675(1993)013<0318:POGVIT>2.3.CO;2

    Article  Google Scholar 

  8. Kono M, Matsui T, Shimizu C, Koga D (1990) Purifications and some properties of chitinase from the liver of a prawn, Penaeus japonicus. Agric Biol Chem 54(8):2145–2147

    CAS  Google Scholar 

  9. Kramer KJ, Muthukrishnan S (1997) Insect chitinases: molecular biology and potential use as biopesticides. Insect Biochem Mol Biol 27:887–900. doi:10.1016/S0965-1748(97)00078-7

    Article  CAS  PubMed  Google Scholar 

  10. Kumar S, Tamura K, Nei M (2004) MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 5:150–163. doi:10.1093/bib/5.2.150

    Article  CAS  PubMed  Google Scholar 

  11. Kurokawa T, Uji S, Suzuki T (2004) Molecular cloning of multiple chitinase genes in Japanese flounder, Paralichthys olivaceus. Comp Biochem Physiol B 138:255–264. doi:10.1016/j.cbpc.2004.03.015

    Article  PubMed  Google Scholar 

  12. Liu CH, Cheng W, Kuo CM, Chen JC (2004) Molecular cloning and characterisation of a cell adhesion molecule, peroxinectin from the white shrimp Litopenaeus vannamei. Fish Shellfish Immunol 17:13–26. doi:10.1016/j.fsi.2003.11.002

    Article  CAS  PubMed  Google Scholar 

  13. Mali B, Mohrlen F, Frohme M, Frank U (2004) A putative double role of a chitinase in a cnidarian: pattern formation and immunity. Dev Comp Immunol 28(10):973–981. doi:10.1016/j.dci.2004.04.002

    Article  CAS  PubMed  Google Scholar 

  14. Oshima H, Miyazaki R, Ohe Y, Hayashi H, Kawamura K, Kikuyama S (2002) Isolation and sequence of a novel amphibian pancreatic chitinase. Comp Biochem Physiol B 132:381–388. doi:10.1016/S1096-4959(02)00046-5

    Article  PubMed  Google Scholar 

  15. Pan D, He N, Yang Z, Liu H, Xu X (2005) Differential gene expression profile in hepatopancreas of WSSV-resistant shrimp (Penaeus japonicus) by suppression subtractive hybridization. Dev Comp Immunol 29(2):103–112. doi:10.1016/j.dci.2004.07.001

    Article  CAS  PubMed  Google Scholar 

  16. Shevchenko A, Jensen ON, Podtelejnikov AV, Sagliocco F, Wilm M, Vorm O, Mortensen P, Shevchenko A, Boucherie H, Mann M (1996) Linking genome and proteome by mass spectrometry: large-scale identification of yeast proteins from two dimensional gels. Proc Natl Acad Sci USA 93(25):14440–14445. doi:10.1073/pnas.93.25.14440

    Article  CAS  PubMed  Google Scholar 

  17. Skinner DM (1965) Amino acid incorporation into protein during the molt cycle of the land crab, Gecarcinus lateralis. J Exp Zool 160:225–233. doi:10.1002/jez.1401600210

    Article  CAS  PubMed  Google Scholar 

  18. Tan SH, Degnan BM, Lehnert SA (2000) The Penaeus monodon chitinase 1 gene is differentially expressed in the hepatopancreas during the molt cycle. Mar Biotechnol NY 2(2):126–135

    CAS  PubMed  Google Scholar 

  19. Terwisscha van Scheltinga AC, Kalk KH, Beintema JJ, Dijkstra BW (1994) Crystal structures of hevamine, a plant defence protein with chitinase and lysozyme activity, and its complex with an inhibitor. Structure 2(12):1181–1189. doi:10.1016/S0969-2126(94)00120-0

    Article  CAS  PubMed  Google Scholar 

  20. Tjoelker LW, Gosting L, Frey S, Hunter CL, Trong HL, Steiner B, Brammer H, Gray PW (2000) Structural and functional definition of the human chitinase chitin-binding domain. J Biol Chem 275(1):514–520. doi:10.1074/jbc.275.1.514

    Article  CAS  PubMed  Google Scholar 

  21. Wang B, Li FH, Dong B, Zhang XJ, Zhang CS, Xiang JH (2006) Discovery of the genes in response to white spot syndrome virus (WSSV) infection in Fenneropenaeus chinensis through cDNA microarray. Mar Biotechnol NY 8(5):491–500. doi:10.1007/s10126-005-6136-4

    Article  PubMed  Google Scholar 

  22. Watanabe T, Kono M (1997) Isolation of a cDNA encoding a chitinase family protein from cuticular tissues of the Kuruma prawn Penaeus japonicus. Zoolog Sci 14(1):65–68. doi:10.2108/zsj.14.65

    Article  PubMed  Google Scholar 

  23. Watanabe T, Kono M, Aida K, Nagasaw H (1998) Purification and molecular cloning of a chitinase expressed in the hepatopancreas of the penaeid prawn Penaeus japonicus. Biochim Biophys Acta 1382:181–185

    CAS  PubMed  Google Scholar 

  24. Watanabe T, Kono M, Aida K, Nagasaw H (1996) Isolation of a cDNA encoding a putative chitinase precursor in the Kuruma prawn Penaeus japonicus. Mol Mar Biol Biotechnol 5:299–303

    CAS  PubMed  Google Scholar 

  25. Xiang JH (2002) Over 10,000 expressed sequence tags from Fenneropenaeus chinensis. International aquaculture conference & exposition, Abstract. p 837

  26. Zhang JQ, Li FH, Wang ZZ, Zhang XJ, Zhou Q, Xiang JH (2006) Cloning, expression and identification of ferritin from Chinese shrimp, Fenneropenaeus chinensis. J Biotechnol 125(2):173–184. doi:10.1016/j.jbiotec.2006.03.010

    Article  CAS  PubMed  Google Scholar 

  27. Zou E, Bonvillain R (2004) Chitinase activity in the epidermis of the fiddler crab, Uca pugilator, as an in vivo screen for molt-interfering xenobiotics. Comp Biochem Physiol C Toxicol Pharmacol. 139:225–230. doi:10.1016/j.cca.2004.11.003

    Article  PubMed  Google Scholar 

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Acknowledgments

This work was supported by Major State Basic Research Development Program of China (973 Program) (Grant No. 2006CB101804), National High-Tech Research and Development Program of China (863 Program) (Grant No. 2006AA100311) and Open-end Funds of Jiangsu Key Laboratory of Marine Biotechnology, Huaihai Institute of Technology (Grant No. 2008HS006).

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Authors and Affiliations

  1. Institute of Oceanology, Chinese Academy of Sciences, Qingdao, 266071, People’s Republic of China

    Jiquan Zhang, Fuhua Li, Bingxin Huang & Jianhai Xiang

  2. Jiangsu Key Laboratory of Marine Biotechnology, Huaihai Institute of Technology, Lianyungang, 222005, People’s Republic of China

    Yuying Sun

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  1. Jiquan Zhang
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  3. Fuhua Li
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Correspondence to Jianhai Xiang.

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Zhang, J., Sun, Y., Li, F. et al. Molecular characterization and expression analysis of chitinase (Fcchi-3) from Chinese shrimp, Fenneropenaeus chinensis . Mol Biol Rep 37, 1913–1921 (2010). https://doi.org/10.1007/s11033-009-9633-0

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  • DOI: https://doi.org/10.1007/s11033-009-9633-0

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