Table 1.
Sequences of the PCR primers used in this study.
Fig 1.
Nucleotide and deduced AA sequences for the elovl5 gene.
Uppercase letters indicate the translated region, and lowercase letters indicate the untranslated region. The start codon (ATG) and the stop codon (TAG) are in bold. Double-underlined letters indicate the polyadenylation signal (AATAA).
Fig 2.
Comparison of the deduced AA sequence of Elovl5 from orange spotted grouper with that from other fish, mice and humans.
The AA sequences were aligned using ClustalX, and identity/similarity shading was based on a 75% identity threshold. Identical residues are shaded black, and similar residues are shaded gray. The conserved HXXHH histidine box motif, five (I–V) putative membrane-spanning domains and the ER retrieval signal predicted by Tvrdik et al. [50] are also indicated.
Fig 3.
Phylogenetic tree comparing grouper Elovl5 with elongase proteins from other organisms.
The phylogenetic tree was constructed using the neighbor joining method [42] in MEGA4. The horizontal branch length is proportional to the AA substitution rate per site. The numbers represent the frequencies (%) with which the tree topology presented was replicated after 1000 iterations.
Fig 4.
Functional characterization of the putative grouper Elovl5 in transgenic S. cerevisiae grown in the presence of the substrate fatty acid 18:4n-3 (Panel A), 18:3n-6 (B), 18:3n-3 (C), 18:2n-6 (D), 20:5n-3 (E) or 20:6n-3 (F).
Fatty acids were extracted from yeast transformed with the pYES2 vector containing the ORF of the putative Elovl5 as an insert. Peaks 1–4 represent the primary endogenous fatty acids of S. cerevisiae, specifically 16:0 (1), 16:1n-7 (2), 18:0 (3) and 18:1n-9 (4). The remaining major peaks correspond to the exogenously added fatty acid and the products of its elongation (18:1n-7 (5), 20:1n-9 (6) and 20:1n-7 (7) resulted from the elongation of 16:1n-7, 18:1n-9 and 18:1n-7, respectively). Vertical axis, flame ionization detector (FID) response; horizontal axis, retention time.
Table 2.
Activity of the putative grouper fatty acyl elongase in yeast.
The proportion of substrate fatty acids that were converted to elongated fatty acid product(s) was calculated as follows: [areas of first product and longer-chain products/(areas of all products with longer chain than substrate + substrate area)].
Fig 5.
Tissue expression of Elovl5 in the orange-spotted grouper.
The results are expressed as the means±standard error (n = 3). Different letters above the bars denote significant (P<0.05) differences between tissues.
Fig 6.
Relative mRNA expression of elovl5 (A), lxrα (B) and srebp-1 (C) in visceral mass from grouper larvae (29DAH) fed a diet containing various n-3 LC-PUFA concentrations for 4 weeks.
Relative mRNA expression was evaluated via RT-qPCR. The values are presented as the means ± S.E.M. (n = 3). Bars for the same gene bearing different letters are significantly different based on Duncan’s test (P<0.05).
Fig 7.
Results of the dual-luciferase reporter assays.
The type of transfection plasmid applied to each group was as follows: NIC: PGL3-basic+PCS2+PRL-CMV; E: PGL-Elo5 promoter+PCS2+PRL-CMV; E-L: PGL-Elo5 promoter+PCS2-LXRα+PRL-CMV; and E-S: PGL-Elo5 promoter +PCS2-SREBP-1+PRL-CMV.