Figure 1.
Overview of the three SSH libraries.
(A) Schematic of SSH library construction. The D–T library was constructed using D-shape stage larval cDNA as the tester and trochophore stage larval cDNA as the driver. The U–D library was constructed using umbonal stage larval cDNA as the tester and D-shape stage larval cDNA as the driver. The J–U library was constructed using juvenile stage larval cDNA as the tester and umbonal stage larval cDNA as the driver. Bar = 15 µm in oosperm, 15 µm in trochophore and D-shaped stages, 40 µm in umbonal and juvenile stages, and 4 cm in the adult stage. (B) Length distribution of unigenes in the three SSH libraries. The white columns represent unigenes in the D–T library, grey columns represent unigenes in the U–D library, and the dark grey columns represent unigenes in the J–U library. Unigene-length frequencies for each library are plotted in 100-bp bins.
Table 1.
Summary of the three SSH libraries.
Figure 2.
Secreted proteins predicted in the three SSH libraries.
Unigenes with a coding region >150 base pairs, processing a signal sequence, predicted to be secreted proteins using TargetP [88] on TMHMM Server v. 2.0 (http://www.cbs.dtu.dk/services/TMHMM/), and GPI modification predictor [89] together [90]. The unigenes coding for secreted proteins were searched by BLASTX using the Genbank nr database with a cutoff e-value of 10e–05. (A) Unigenes in the D–T library. (B) Unigenes in the U–D library. (C) Unigenes in the J–U library.
Figure 3.
Schematic of unigenes coding for proteins with tandem-arranged repeat units.
XSTREAM [73] was used to isolate unigenes coding for proteins with tandem-arranged repeat units. Unigenes in the D–T library (A), U–D library (B), and J–U library (C), with a coding region longer than 150 base pairs that were subjected to this analysis. As a control, shematrin family genes found in P.fucata were analyzed using XSTREAM (D). The identities of tandem-arranged repeat units are marked by numbers on the schematic at the top left of each motif (red font). The copy number of repeat units is shown on the top right of the motif (black font). On the top of each motif there is a label of identity*copy number. Bars = 50 amino acids (in A, B, C, D).
Figure 4.
Gene expression patterns for selected genes in tissues of P.fucata.
(A) Tissue-specific gene expression of selected unigenes by RT-PCR analysis. Total RNA was extracted from mantle edge (ME), mantle pallial (MP), foot, viscus (Vis), gonad (Gon), adductor muscle (Mus), hemocytes (Hem), and gill. RT-PCR was used without a template as a negative control (Control). The housekeeping gene GAPDH was used as a positive control. (B) A section of oyster mantle was hybridized with GFP anti-sense RNA probes as a negative control, and no hybridization signals (dark purple) were detected in this section. DT_Cluster236 (C) and DT_Cluster524.seq.Contig1 (E) were expressed in all mantle epithelial cells (indicated by arrowheads), except the cells at the bottom of the periostracal groove (indicated by arrows). (D) Expression of DT_cluster252 was localized to mantle epithelial cells (indicated by arrowheads) with weaker signals in the inner epithelial cells of the outer fold, and the outer epithelial cells of the middle fold at the periostracal groove (indicated by arrows). (F) Expression of UD_Cluster94.seq.Singlet1 was localized to the epithelial cells of the inner fold, the inner epithelial cells of the middle fold, and the epithelial cells at the top of the outer fold (indicated by arrowheads). (G) Expression of IU_Cluster32 was localized to nearly all the mantle epithelial cells (indicated by arrowheads), but at the bottom of the periostracal groove we detected stronger signals in epithelial cells (indicated by arrows). OF, outer fold; MF, middle fold; IF, inner fold; PG, periostracal groove. Scale bar, 0.5 mm.
Figure 5.
Expression levels of the selected genes knocked down by RNAi.
The expression levels of the selected genes were measured using real-time quantitative PCR. The expression levels were analyzed six days after dsRNA injection and five individuals were tested in each group. For the control, the expression levels of the PBS injected group were measured to a relative value of 1.0. The asterisk represents a significant (p<0.005) difference compared with the PBS-injected groups. A 160 µg dosage was only used in the krmp-injected group, because the expression did not decrease significantly in the 40 µg dosage-injected group.
Figure 6.
Effect of selected genes on shell growth.
Expression levels of the selected genes were decreased by RNAi (see Materials and Methods for SEM image details). (A1) SEM image of the internal nacreous layer surface of the normal shell, showing stair-like growth pattern. (B–K2) SEM images of the internal nacreous layer surface of the shells in the dsRNA injected groups, which shows that the growth of the tablets was disrupted. (L1) SEM image of the ‘aragonitic line’ shows the growth of nacreous tablets on the left side and the growth of calcitic prisms on the right side. (M1–N2) SEM image of the ‘aragonitic line’ of the UD_Cluster94.seq.Singlet1-injected group. (O) SEM image of the normal prismatic layer of the shell. (P–Q) SEM image of the krmp-injected groups. Low dosage (80 µg for krmp, 40 µg for the other genes) injection of nacrein (B), DT_Cluster236 (D1), DT_Cluster252 (F1), DT_Cluster524.seq.Contig1 (H1), IU_Cluster32 (J1) UD_Cluster94.seq.Singlet1 (M1), and krmp (P). High dosage (160 µg for krmp, 80 µg for the other genes) injection of nacrein (C), DT_Cluster236 (E1), DT_Cluster252 (G1), DT_Cluster524.seq.Contig1 (I1), IU_Cluster32 (K1), UD_Cluster94.seq.Singlet1 (N1), and krmp (O). (A2, D2, E2, F2, G2, H2, I2, J2, K2, L2, M2, and N2) enlargement of the box in (A1, D1, E1, F1, G1, H1, I1, J1, K1, L1, M1, and N1). Bar = 10 µm in A2, B, C, F2, G2, H2, J2, K2, L2, D2, E2, I2, M2; Bar = 50 µm in A1, F1, G1, H1, J1, N2, P, Q; Bar = 100 µm in D1, E1, I1, K1, M1, O, L1, N1.