Table 1.
Sequences of the primers used in the experiment.
Figure 1.
Nucleotide and deduced amino acid sequences of CfAChE.
The amino acid residues in the mature protein were assigned positive numbers, and those in signal peptide were assigned negative numbers. The stop codon was labeled with box. The nucleotides and amino acids were numbered along the left margin. The predicted N-glycosylation sites were bold and underlined. The conserved cysteine residues were marked in italic and grey. The three residues forming the catalytic triad, S220, E354, and H479, were grey and labeled with box.
Figure 2.
Multiple sequences alignment of CfAChE with other AChEs deposited in GenBank.
The CfAChE amino acid sequence was aligned with AChEs from Hydra vulgaris (CAA06981), Loligo opalescens (AAD15886), Aedes aegypti (NP_476953), Danio rerio (AAC14022) and Homo sapiens (NP_000656). The amino acid numbers for each sequence were indicated at right. The AChE catalytic triad residues were indicated by filled triangle. The PAS site was labeled by hollow triangle. The choline binding site was labeled by filled circles. And the intramolecular disulfide bonds were indicated by number and open circles.
Figure 3.
Consensus neighbor-joining tree based on the sequences of CfAChE and other AChEs from different animals.
The protein sequences used for phylogenetic analysis include: Hydra vulgaris (CAA06981), Ascaris suum (ADY44392), Ciona intestinalis (NP_001122349), Caenorhabditis elegans (AAC14022), Drosophila melanogaster (NP_476953), Apis mellifera (NP 001035320), Loligo opalescens (AAD15886), Aedes aegypti (NP_476953), Danio rerio (AAC14022), Torpedo Californica (P04058), Mus musculus (NP 033729), Bos taurus (NP 001069688) and Homo sapiens (NP_000656).
Figure 4.
SDS-PAGE analysis of recombinant CfAChE.
After electrophoresis, the gel was visualized by Coomassie brilliant blue R250 staining. Lane 1: purified recombinant CfAChE; Lane 2: protein molecular standard; Lane 3: recombinant CfAChE after deglycosylation by PNGase F; Lane 4: PNGase F as control.
Figure 5.
The activity of CfAChE in serum after DDVP stimulation for 0, 3, 6, 12, 24, 48 and 96 h.
The CfAChE activities were determined in each group and the unstimulated scallops were used as the blank groups. Values were shown as mean ± S.E., N = 6.
Figure 6.
Tissue distribution of the CfAChE transcripts detected by SYBR Green RT-PCR.
CfAChE transcript level in adductor muscle, mantle, gill, hepatopancreas, kidney and gonad of six adult scallops was normalized to that of haemocytes. Vertical bars represented the mean ± S.E. (N = 6), and bars with * are significantly different (P<0.05).
Figure 7.
The temporal expression of CfAChE mRNA in haemocytes after LPS stimulation for 3, 6, 12, 24, 48 and 96 h.
Data was expressed as the ratio of the CfAChE mRNA to the β-actin mRNA. The relative CfAChE expression level was determined for each group, and the unstimulated scallops (0 h) were used as the blank group, and scallops injected with PBS were used as the control group. Values were shown as mean ± S.E. (N = 6), and bars with * are significantly different (P<0.05).
Figure 8.
The temporal expression of CfAChE mRNA in haemocytes after TNF-α stimulation for 1, 3, 6, 9 and 12 h.
Data was expressed as the ratio of the CfAChE mRNA to the β-actin mRNA. The relative CfAChE expression level was determined for each group, and the unstimulated scallops (0 h) were used as the blank group, and scallops injected with PBS were used as the control group. Values were shown as mean ± S.E. (N = 6), and bars with * are significantly different (P<0.05).
Figure 9.
The temporal expression of lysozyme mRNA in haemocytes of C. farreri after DDVP treatment for 0, 3, 6, 12, 24, 48 and 96 h.
Data was expressed as the ratio of the CfAChE mRNA to the β-actin mRNA. The relative CfAChE expression level was determined for each group and the unstimulated scallops were used as the control group. Values were shown as mean ± S.E. (N = 6), and bars with * are significantly different (P<0.05).