Table 1.
Sequences of the primers used in the experiment.
Figure 1.
Nucleotide and deduced amino acid sequences of CfDDC.
The nucleotides and amino acids are numbered along the left margin. The asterisk (*) indicates the stop codon. Polyadenylation signal is bolded and italicized. The predicted PLP-binding motif and the predicted N-glycosylation sites are underlined and double-underlined respectively.
Figure 2.
Multiple alignment of the Pyridoxal_deC domain of CfDDC with other DDCs deposited in GenBank.
The black shadow region indicates positions where all sequences share the same amino acid residue. Similar amino acids are shaded in grey. Gaps are indicated by dashes to improve the alignment. The asterisk (*) indicates the conserved cysteine residue. The species and the GenBank accession numbers are as follows: H. sapiens (AAP35655), M. musculus (CAI23994), X. tropicalis (NP_001011289), D. rerio (AAH56292), B. mori (AAK48988), T. castaneum (ABU25222), P. dumerilii (CAJ38793).
Figure 3.
Consensus neighbour-joining tree based on the sequences of DDC from different animals.
The numbers at the forks indicate the bootstrap. The protein sequences used for phylogenetic analysis include: B. Taurus (ABG66984), S. scrofa (ABO15741), H. sapiens (AAP35655), M. musculus (CAI23994), R. norvegicus (AAA41087), X. tropicalis(NP_001011289), O. latipes (BAH37023), D. rerio (AAH56292), T. castaneum (ABU25222), A. aegypti (AAC31639), M. sexta (AAC46604), A. pernyi (AAR23825), B. mori (AAK48988) and P. dumerilii (CAJ38793).
Figure 4.
Tissues distribution of the CfDDC transcript detected by SYBR Green real-time RT-PCR.
CfDDC transcript level in hepatopancreas, kidney, adductor muscle, gonad, gill and mantle of six adult scallops is normalized to that in haemocytes. Vertical bars represent the mean ± SE (N = 6).
Figure 5.
Temporal expression of CfDDC mRNA detected by real-time RT-PCR in scallop haemocytes at 3, 6, 12, 24 and 48 h after LPS stimulation.
β-actin gene is used as an internal control to calibrate the cDNA template for all the samples. Each values are shown as mean ± SE (N = 6). Significant difference between challenged group and blank group is indicated by asterisks (P<0.05).
Figure 6.
SDS-PAGE analysis of rCfDDC and the western blotting of anti-rCfDDC.
Lane M: protein molecular standard (kDa). Lane 1: negative control for rCfDDC (without induction). Lane 2: IPTG induced rCfDDC. Lane 3: purified rCfDDC. Lane 4: the western blotting of anti-rCfDDC.
Figure 7.
Encapsulated beads (%) of scallop haemocytes to agarose beads coated with rCfDDC and BSA at 6 h and 24 h.
Vertical bars represent the mean ± SE (N = 3).
Figure 8.
The haemocyte encapsulation promoted by rCfDDC.
Agarose beads coated with rCfDDC (a, b), BSA (c, d, as a control protein), or anti-rCfDDC (e, f) were incubated with scallop haemocytes.
Figure 9.
ROS level in haemocytes of scallops at 6, 12 and 24 h after the injection of DDC inhibitor methyldopa.
Each values are shown as mean ± SE (N = 6). Significant difference between challenged group and blank group is indicated by asterisks (P<0.05).