Characterization and expression analysis of chymotrypsin after bacterial challenge in the mud crab, Scylla paramamosain

Abstract

Chymotrypsin is one of the serine proteases families that have various biological functions. A chymotrypsin gene was isolated from hepatopancreas of the mud crab, Scylla paramamosain (designated SpCHY) in this study. The full-length cDNA of SpCHY contained 942 nucleotides with a polyadenylation sequence and encoded a peptide of 270 amino acids with a signal peptide of 17 amino acids. The SpCHY gene contains seven exons, six introns, a TATA box and several transcription factor binding sites that were found in 5'-promoter region which is 1221 bp in length. Real-time quantitative PCR analysis indicated that the expression level of SpCHY mRNA in hepatopancreas was significantly higher than that in other tissues. Immunocytochemistry and in situ hybridization exhibited the CHY-like reactivity presented in resorptive cells of the hepatopancreas. After bacterial challenge with Vibrio alginolyticus, the expression level of SpCHY mRNA was extremely up-regulated at 3 h in hepatopancreas. Our results suggest that SpCHY might play an important role in the mud crab's immune response.

Keywords: Scylla paramamosain; chymotrypsin; immune response; immunocytochemistry; in situ hybridization.

Figure 1

Nucleotide and deduced amino acid sequences of the SpCHY gene (GenBank accession no. JF831535.1). The nucleotides are numbered on the right, and the amino acids on the left. The putative signal peptide is underlined. The trypsin-like SP domain is wave underlined. The catalytic triad (H, D, and S) is gray shadowed. The boxed letters are the polyadenylation signal. The asterisk (*) indicates the stop codon and arrows indicate the location of introns.

Figure 2

Organization of the SpCHY gene. The positions of the exons (open boxes 1–7), introns (A–F), and CA repeat sequence (filled box) are denoted.

Figure 3

Phylogenetic analysis of SpCHY with other chymotrypsins. A NJ tree was produced with Mega3.1 software. One thousand bootstraps were carried out to check the repeatability of the result. L. vannamei-CHYA (CAA71672), L. vannamei-CHYB (CAA71673), F. chinensis-CHY (ACC68669), P. humanus corporis-CHY (AAV68346), P. cochleariae-CHY (CAA76928), T. molitor-CHY (DQ356031.1), M. sexta-CHY (2120321A), A. grandis-CHY (AAT09847.1), A. gambiae-CHY1 (CAA79325), A. gambiae-CHY2 (CAA79326), B. Taurus - CHYB (P00767), G. morhua-CHYB (P80646), S. aurata-CHYB (AAT45258), M. musculus-CHY (AAL11034), H. sapiens-CHY (CAA74031.1), B. taurus-CHYC(AAI51507.1), X. laevis-CHYC(NP_001085458), B. taurus-CHYB (NP_001098800.1), D. rerio-CHYB1 (NP_997783.1), and O. dioica-CHYB (AAT47850).

Figure 4

The results of quantitative real-time PCR analysis of SpCHY expression in various tissues. Expression of a β-actin gene was used as control. Values were shown as means ±S.E. (N = 3). Abbreviations: Br, brain; TG, thoracic ganglion; Ht, heart; Gi, gill; Hp, hepatopancreas; St, stomach; Mu, muscle; Ov, ovary.

Figure 5

Location of SpCHY by immunocytochemistry and in situ hybridization in hepatopancreas of S. paramamosain. (A) histological observation; R resorptive cells, B blister cells, E embryonic cells, F fibrillar cells, Nu nucleus. (B) immunocytochemistry results; the arrows point to immunocytochemical positive signals. (C) in situ hybridization results; arrows indicate the specific SpCHY mRNA hybridization signal with the antisense riboprobe. (D) The negative control with the sense riboprobe showed no specific signal. Scale bars: 50 μm.

Figure 6

Transcript profiles of SpCHY in hepatopancreas of S. paramamosain following challenge with V. alginolyticus,. The relative SpCHY transcript levels in crabs challenged with V. alginolyticus were compared to those of saline injected animals. The expression of a β-actin gene was used as endogenous control. Significant differences of SpCHY expression between the challenged and the control group are indicated with asterisks. **p < 0.01.