Measurements of pectenolone and pectenoxanthin contents
As shown in Figure 2, precursor ions [M + H]+ at 581.4 and 565.38 m/z were observed in the MS spectrum, indicating the presence of pectenolone and pectenoxanthin, respectively, as judged by their molecular weights and product ions [3]. As shown in Figure 3, the pectenolone content in adductor muscles of the group without RNAi was significantly higher than that of the group treated with RNAi (P < 0.05, Figure 3a), whereas no significant difference in pectenoxanthin content was observed between the two groups (P > 0.05, Figure 3b).
Transcriptomic analyses and Real-time PCR
Six libraries from the two groups were sequenced and 358,313,800 raw reads (GEO accession number: GSE138590), with 349,906,798 clean reads were obtained. The Q20 values were obtained at 97.6%–97.82% and 97.56%–97.77% in the group with and without RNAi, respectively. More than 7.0 Gb clean data were obtained from each sample (Table 2). In total, 90.89%–92.11% and 90.96%–91.83% of gene-mapped reads from group with and with RNAi were mapped onto the bay scallop genome, respectively (Table 3).
A total of 534 DEGs, including 246 upregulated and 288 downregulated genes, were identified between the two groups (Figure 4). Among these DEGs, 3 downregulated genes, including LDLR, CYP 450 and NPC1L1, and 1 upregulated gene, ABC transporter, were found to be related to carotenoid metabolism.
The expression levels of these genes related to carotenoid accumulation, as well as VPS29 were further evaluated by Real-time PCR in the adductor muscles of the two groups. Notably, the expression levels of VPS29 were significantly decreased in the group treated with RNAi of VPS29. Among the other genes, only the mRNA level of ABC transporter was significantly higher whereas those of NPC1L1, LDLR, and CYP450 were lower in the group treated with RNAi of VPS29 (Figure 5).
The GO functional analyses revealed that most of the DEGs were assigned to DNA integration. Among the biological processes, the DEGs were mainly assigned to the small-molecule, single-organism, and organic-acid biosynthetic processes. Among the cellular components, the DEGs were focused on the proton-transporting two-sector ATPase complex, dynein complex, and plasma membrane. Among the molecular functions, most of the DEGs were assigned to the peptidase inhibitor, peptidase regulator, and enzyme inhibitor activities (Figure 6). In addition, gene enrichment was also observed in the binding of tetrapyrrole, a non-carotenoid pigment.
All the DEGs were assigned to 50 KEGG pathways. Among the KEGG pathways, lysosome, one carbon pool by folate, biosynthesis of amino acids, citrate cycle, and 2-oxocarboxylic acid metabolism were the top five pathways. In addition, porphyrin metabolism was also found in the KEGG pathways.
As the most significantly enriched pathway, lysosome pathway was enriched with 7 upregulated genes, included NPC1L1, phosphatidic acid phosphatase, papain family cysteine protease, saposin-like type B, acid phosphat A, V-type ATPase 116kDa subunit, and cation-independent mannose-6-phosphate receptor repeat.