Molecular cloning, characterization and mRNA expression of copper-binding protein hemocyanin subunit in Chinese mitten crab, Eriocheir sinensis

Fish Shellfish Immunol. 2012 Nov;33(5):1222-8. doi: 10.1016/j.fsi.2012.09.023. Epub 2012 Sep 29.

Abstract

Hemocyanin is a copper-binding protein and plays a crucial role in the physiological processes in crustacean. In this study, the cDNA encoding hemocyanin subunit from Chinese mitten crab Eriocheir sinensis (EsHc) was cloned by using EST analysis and rapid amplification of cDNA ends (RACE) approach. The full-length cDNA of EsHc was 2573 bp, consisting of a 5' untranslated region of 51 bp, a 3' untranslated region of 458 bp, and an open reading frame of 2064 bp. The deduced protein had 688 amino acid residues with molecular mass of 77,997.31 Da. Quantitative real-time RT-PCR analysis showed that the EsHc gene was expressed in haemocytes, hepatopancreas, muscles, gills, and intestines with the highest level of expression in the hepatopancreas and the lowest in the muscles. After Aeromonas hydrophila challenge, the relative expression level of EsHc in hemolymph was up-regulated at 3 h post-injection of bacteria followed by a gradual recovery from 12 to 24 h. In the second set of transcriptional studies, the mRNA expression patterns of EsHc in haemocytes and hepatopancreas were measured by quantitative real-time RT-PCR after the Chinese mitten crab were fed six diets containing different levels of copper (0, 10, 20, 40, 80 and 400 mg kg(-1)) for 8 weeks, respectively. The feeding trial showed that the expression levels of EsHc mRNA significantly increased at the copper levels of 20-40 mg kg(-1). This study implies that the expression levels of EsHc could be affected by dietary copper in the hepatopancreas and haemocytes, and hemocyanin may be potentially involved in the immune responses of the Chinese mitten crab.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aeromonas hydrophila / immunology*
  • Analysis of Variance
  • Animals
  • Base Sequence
  • Brachyura / genetics*
  • Brachyura / immunology*
  • Brachyura / microbiology
  • Cloning, Molecular
  • Copper / administration & dosage
  • Copper / metabolism
  • Copper / pharmacology
  • DNA Primers / genetics
  • DNA, Complementary / genetics
  • Expressed Sequence Tags
  • Gene Expression Profiling
  • Gene Expression Regulation / drug effects
  • Gene Expression Regulation / immunology*
  • Hemocyanins / genetics*
  • Hemocyanins / metabolism
  • Hemocytes / metabolism
  • Hepatopancreas / metabolism
  • Molecular Sequence Data
  • Protein Subunits / genetics*
  • Protein Subunits / metabolism
  • RNA, Messenger / metabolism*
  • Real-Time Polymerase Chain Reaction
  • Reverse Transcriptase Polymerase Chain Reaction
  • Sequence Analysis, DNA

Substances

  • DNA Primers
  • DNA, Complementary
  • Protein Subunits
  • RNA, Messenger
  • Copper
  • Hemocyanins