Identification of Fatty Acid Desaturase 6 in Golden Pompano Trachinotus Ovatus (Linnaeus 1758) and Its Regulation by the PPARαb Transcription Factor
Abstract
:1. Introduction
2. Results
2.1. Molecular Cloning and Phylogenetics of T. ovatus Fads6
2.2. Heterologous Expression of the Desaturase ORF in Saccharomyces cerevisiae
2.3. Tissue Distribution of ToFads6
2.4. Promoter Analysis of PPARαb Regulation
2.5. Transcriptional Regulation of ToFads6 by PPARαb
2.6. Binding of PPARαb to Fads6 Promoters
3. Discussion
4. Materials and Methods
4.1. Ethics Statement
4.2. Animals and Tissue Collection
4.3. Gene Cloning and Bioinformatics of T. ovatus Fads6
4.4. Functional Characterization of the ToFads6 Desaturase
4.5. Real-Time Quantitative PCR Analysis
4.6. Preparation of a Fads6 Polyclonal Antibody and Western Blotting Analysis
4.7. Cloning of the Fads6 Promoter and Construction of Deletion Mutants
4.8. Construction of Truncated Mutants for the Identification of Predicted Transcription Factor (TF) Binding Sites in the Fads6 Promoter
4.9. Cell Culture, Transfection, and Luciferase Assay
4.10. Expression Analysis of ToFads6 with ToPPARαb
4.11. Electrophoretic Mobility Shift Assay (EMSA)
4.12. Statistical Analysis
Supplementary Materials
Author Contributions
Acknowledgments
Conflicts of Interest
Abbreviations:
Fads | Fatty acid desaturases |
EMSA | Electrophoretic mobile shift assays |
LC-PUFA | Long-chain polyunsaturated fatty acids |
PPARαb | Transcription factor peroxisome proliferator-activated receptor alpha b |
Elovl | Elongation of very long-chain fatty acids |
HNF4 | Hepatocyte nuclear factor-4 |
NR1H3 | Nuclear receptor subfamily 1 group H member 3 |
SREBP | Sterol-regulatory element binding protein |
NF-κB | Nuclear factor-kappa B |
PPREs | PPAR response elements |
FAMEs | Fatty acid methyl esters |
TLC | Thin-layer chromatography |
TOCF | T. ovatus caudal fin |
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FA Substrate | Product | Conversion (%) | Activity |
---|---|---|---|
C18:2n-6 | C18:3n-6 | 0 | Δ6 |
C18:3n-3 | C18:4n-3 | 0 | Δ6 |
C20:2n-6 | C20:3n-6 | 0.17 ± 0.02 | Δ8 |
C20:3n-6 | C20:4n-6 | 0.54 ± 0.06 | Δ5 |
C22:5n-3 | C22:6n-3 | 39.29 ± 4.4 | Δ4 |
C22:4n-6 | C22:5n-6 | 1.04 ± 0.11 | Δ4 |
Putative Binding Sites | Nucleotide Sequence | Mutation |
---|---|---|
M1 | 5′ CTGGACTGCATCAAGAGTCA 3′ | deletion |
M2 | 5′ ATGCTAACTTTGACCTGTGGACTGTGC 3′ | deletion |
M3 | 5′ AGCAGCTGCAGTTTTGTGTG 3′ | deletion |
M4 | 5′ GGGGGGAACAGAGGAGAGG 3′ | deletion |
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Zhu, K.-C.; Song, L.; Guo, H.-Y.; Guo, L.; Zhang, N.; Liu, B.-S.; Jiang, S.-G.; Zhang, D.-C. Identification of Fatty Acid Desaturase 6 in Golden Pompano Trachinotus Ovatus (Linnaeus 1758) and Its Regulation by the PPARαb Transcription Factor. Int. J. Mol. Sci. 2019, 20, 23. https://doi.org/10.3390/ijms20010023
Zhu K-C, Song L, Guo H-Y, Guo L, Zhang N, Liu B-S, Jiang S-G, Zhang D-C. Identification of Fatty Acid Desaturase 6 in Golden Pompano Trachinotus Ovatus (Linnaeus 1758) and Its Regulation by the PPARαb Transcription Factor. International Journal of Molecular Sciences. 2019; 20(1):23. https://doi.org/10.3390/ijms20010023
Chicago/Turabian StyleZhu, Ke-Cheng, Ling Song, Hua-Yang Guo, Liang Guo, Nan Zhang, Bao-Suo Liu, Shi-Gui Jiang, and Dian-Chang Zhang. 2019. "Identification of Fatty Acid Desaturase 6 in Golden Pompano Trachinotus Ovatus (Linnaeus 1758) and Its Regulation by the PPARαb Transcription Factor" International Journal of Molecular Sciences 20, no. 1: 23. https://doi.org/10.3390/ijms20010023