Table 1.
Nucleotide sequences of the PCR primers used in this study.
Figure 1.
The cDNA nucleotide and deduced protein amino acid sequences of PmVRP15 (GenBank accession code KF683338).
The putative start codon (ATG) is in bold, the asterisk indicates the stop codon (TAA, in bold italics), the potential transmembrane domain is boxed and the proposed polyadenylation site is underlined.
Figure 2.
PmVRP15 transcript expression analysis in various P. monodon tissues by RT-PCR.
The tissues examined were antennal gland (AN), epipodite (EP), eye stalk (ES), gill (G), heart (H), hemocyte (HC), hepatopancreas (HP), intestine (I) and lymphoid (L). EF1-α was used as the internal reference and PCR control.
Figure 3.
Up-regulation of PmVRP15 transcripts in response to WSSV infection.
Relative expression ratios, as determined by qRT-PCR, of PmVRP15 transcript levels in the hemocytes of WSSV-infected P. monodon were compared to those of the control (non-infected) shrimps and standardized against β-actin as the internal reference, at 24, 48 and 72 hpi with WSSV. The data represent the mean ±1 SD relative expression of PmVRP15 post-infection (solid bar, right) and the control (open bar, left), derived from three independent experiments. Means with an asterisk are significantly different (P<0.05, paired samples t-test). A relative expression ratio of <1, 1 and >1 mean that the gene expression level is down-regulated, the same or up-regulated, respectively, in the hemocytes of WSSV-infected shrimps compared to the uninfected control.
Figure 4.
Western blot analysis of PmVRP15 native protein in control (NaCl) and WSSV- injected (WSSV) P. monodon hemocytes.
Hemocytes were collected at 48(HLS) was prepared and 70 μg of total HLS protein per track was subjected to duplicate SDS-PAGE resolution. Gels were then either stained with coomassie blue for total protein detection or subject to Western-blot analysis to detect PmVRP15 and β-actin using specific antibodies. M is the protein size markers.
Figure 5.
CFLM-derived images of the uninfected (control) and WSSV-infected hemocytes at 48 hpi with WSSV.
Rabbit anti-rPmVRP15 and mouse anti-VP28 primary antibodies were detected with corresponding Alexa488 and Alexa568 secondary antibodies revealing PmVRP15 (green color) and VP28 (red color), respectively. Scale bars represent (A) 5 μm and (B) 2 μm. Nucleus was stained with TO-PRO-3 iodide and color was adjusted to blue. The bright field image showed hyaline cell (HC), semigranular cell (SGC) and granular cell (GC).
Figure 6.
The PmVRP15 gene silencing in P. monodon hemocytes.
(A) Transcriptional level of PmVRP15 transcripts after 24 h post-WSSV infection and -PmVRP15 gene knockdown in the P. monodon hemocytes was determined by RT-PCR using gene specific primers. The control was shrimp that was injected with GFP dsRNA. Three individuals were used for each group and each experiment was performed in triplicate. (B) Protein expression level of PmVRP15 was detected in both groups to confirm the success of PmVRP15 knockdown. Hemocytes were collected at 24 h after PmVRP15 gene knockdown in WSSV-infected shrimp, 70 μg of total HLS protein was analyzed by SDS-PAGE and western blot analysis using antibody specific to PmVRP15 and β-actin protein, an internal control.
Figure 7.
The effect of PmVRP15 gene silencing on WSSV propagation in P. monodon hemocytes.
Transcript expression level of the WSSV genes: ie-1, wsv477 and vp28, in PmVRP15 gene-silenced P. monodon hemocytes were determined by qRT-PCR. Data are shown as the mean ±1 SD of three replicates and as the fold change of ie-1, wsv477 and vp28 after normalization to the EF-1α transcript levels (grey bar). The control group (GFP-dsRNA injected) are shown in the black bars.
Figure 8.
The involvement of knockdown PmVRP15 gene in WSSV infection in shrimp.
(A) Cumulative mortality of WSSV-infected PmVRP15 gene knockdown shrimp (black line) was compared with that of the control, WSSV-infected GFP gene knockdown shrimp (Grey line). Data are shown as the mean ±1 S.D. and are derived from three independent repeats. (B) After knockdown PmVRP15 gene in WSSV-infected shrimp, PmVRP15 gene recovery was observed after WSSV infection at 24, 36, 48 and 60 hpi.