Molecular characterization of histone gene in golden pompano (Trachinotus ovatus) and antimicrobial activity of its derived peptides.

In addition to controlling gene expression, mediating DNA folding into chromatin, and responding to immunological stimuli, histones are also thought to have antimicrobial effects. This study identified the molecular characteristics of core Histone MacroH2A2 (TOMacroH2A2) and Histone H2B 1/2 (TOH2B) from Trachinotus ovatus, and the antimicrobial potential of their derived peptides (To.mh2a and To. h2b). The open reading frames (ORFs) of TOMacroH2A2 and TOH2B from T. ovatus were 1010 bp and 375 bp, encoding polypeptides of 369 and 124 amino acids, respectively. The TOMacroH2A2 included an H2A domain and an A1pp domain, while TOH2B included an H2B domain. The amino acid sequences of TOMacroH2A2 and TOH2B demonstrated high homology with other teleost's sequences of histone macroh2a2 and histone h2b, with homologies exceeding 90 %. Expression analysis showed high expression of TOMacroH2A2 in brain, stomach, heart, and skin tissues and TOH2B in gill, brain, and skin tissues. In addition, the histone-derived peptides To. mh2a and To. h2b, synthesized based on two histone sequences from T. ovatus, exhibited typical physical characteristics of antimicrobial peptides, including positive charges, amphipathicity, hydrophobicity, and rich α-helix structure. Crucially, the vitro antibacterial results demonstrated that To. mh2a and To. h2b can inhibit the growth of various aquatic pathogens including Streptococcus agalactiae, Staphylococcus aureus, Bacillus subtilis, Acinetobacter baumannii, Aeromonas hydrophila, and Escherichia coli to varying degrees. Specifically, To. mh2a and To. h2b were capable of disrupting the cell surface structures of S. aureus and penetrating the cell membrane, leading to the leakage of cellular contents, thereby exerting their antibacterial effects. Furthermore, gel electrophoresis migration assays showed that To. mh2a and To. h2b participated in antimicrobial activity by binding to bacterial genomic DNA and reducing the migration rate of gDNA in a dose-dependent manner. The minimum effective concentration for binding to DNA was approximately 50 µM. In conclusion, our study suggested that To. mh2a and To. h2b can act as antimicrobial peptides, providing a potential strategy for controlling bacterial diseases in T. ovatus.

Molecular characterization of TNF-ß and IFN-γ in yellowfin seabream (Acanthopagrus latus, Hottuyn, 1782) and their immune responses to density stress during transport.

The inflammatory cytokines TNF-ß and IFN-γ are important mediators of the vertebrate inflammatory response and coordinators of the immune system in regard to NF-κB signalling pathways. In this study, the TNF-ß and IFN-γ genes of yellowfin seabream, Acanthopagrus latus were identified, and the multiple sequence alignments, evolutionary relationships and gene expressions of the two genes were also determined. AlTNF-ß contained a 762 bp open reading frame (ORF) encoding 253 amino acids, while AlIFN-γ contained a 582 bp ORF encoding 193 amino acids. An amino-acid sequence alignment analysis showed that these proteins have highly conserved transmembrane structural domains among teleosts. Moreover, AlTNF-ß has a close affinity with TNF-ß of yellowfin seabream while AlIFN-γ has a high evolutionary correlation with A. regius and Sparus aurata. In addition, the mRNAs of AlTNF-ß and AlIFN-γ are widely expressed in various tissues. AlTNF-ß is highly expressed in gill and intestinal tissues, and the mRNA levels of AlIFN-γ are higher in spleen, skin, and gill tissues than in other tissues. Under transportation density stress, the mRNA level of AlTNF-ß was significantly elevated in the intestine of the high-density group, while AlTNF-ß transcription in the gills did not vary significantly among the density groups. Furthermore, AlIFN-γ expression was increased in liver, intestinal, and gill tissues under high transportation density. The results of this study show that TNF-ß and IFN-γ expression in yellowfin seabream is greatly affected by density stress. The density of 125 per bag for 4-5 cm fry or 1200 per bag for 1-2 cm fry is most suitable for the transportation of live fish. These results might provide a reference for further studies on the immunomodulatory response process and auxiliary function of immune stress of TNF and IFN genes in fish under density stress.

Characterization of the MMP9 Gene and Its Association with Cryptocaryon irritans Resistance Traits in Trachinotus ovatus (Linnaeus, 1758).

The MMPs are endogenous proteolytic enzymes that require zinc and calcium as cofactors. MMP9 is one of the most complex matrix metalloproteinases in the gelatinase family and has many biological functions. In mammals, mmp9 is thought to be closely associated with cancer. However, studies in fish have rarely been reported. In this study, to understand the expression pattern of the ToMMP9 gene and its association with the resistance of Trachinotus ovatus to Cryptocaryon irritans, the sequence of the MMP9 gene was obtained from the genome database. The expression profiles were measured by qRT-PCR, the SNPs were screened by direct sequencing, and genotyping was performed. The ToMMP9 gene contained a 2058 bp ORF encoding a putative amino acid sequence of 685 residues. The homology of the ToMMP9 in teleosts was more than 85%, and the genome structure of ToMMP9 was conserved in chordates. The ToMMP9 gene was expressed in different tissues of healthy individuals and was highly expressed in the fin, the gill, the liver and the skin tissues. The ToMMP9 expression in the skin of the infected site and its adjacent sites increased significantly after C. irritans infection. Two SNPs were identified in the ToMMP9 gene, and the SNP (+400A/G) located in the first intron was found to be significantly associated with the susceptibility/resistance to C. irritans. These findings suggest that ToMMP9 may play an important role in the immune response of T. ovatus against C. irritans.

Effects of cysteine addition to low-fishmeal diets on the growth, anti-oxidative stress, intestine immunity, and Streptococcus agalactiae resistance in juvenile golden pompano (Trachinotus ovatus).

As the precursor of taurine, cysteine serves physiological functions, such as anti-oxidative stress and immune improvement. Investigation of cysteine and its derivatives has made positive progress in avian and mammalian species, yet the study and application of cysteine in aquatic animals are relatively rare. Therefore, we evaluated the effects of supplementing a low-fishmeal diet with various levels of cysteine on the growth, antioxidant capacity, intestine immunity, and resistance against Streptococcus agalactiae of the juvenile golden pompano (Trachinotus ovatus). According to our study, exogenous supplementation with 0.6-1.2% cysteine greatly increased the final body weight (FBW) and specific growth rate (SGR) of golden pompano compared to the control group. Under the present conditions, the optimum dietary cysteine supplementation level for golden pompano was 0.91% based on the polynomial regression analysis of SGR. Meanwhile, we found that the Nrf2/Keap1/HO-1 signaling pathway was notably upregulated with the increase of exogenous cysteine, which increased antioxidant enzyme activity in serum and gene expression in the intestine and reduced the level of reactive oxygen species (ROS) in the serum of golden pompano. In addition, morphological analysis of the midgut demonstrated that exogenous cysteine improved muscle thickness and villi length, which suggested that the physical barrier of the intestine was greatly strengthened by cysteine. Moreover, cysteine increased the diversity and relative abundance of the intestinal flora of golden pompano. Cysteine suppressed intestinal NF-κB/IKK/IκB signaling and pro-inflammatory cytokine mRNA levels. Conversely, intestinal anti-inflammatory cytokine gene expression and serum immune parameters were upregulated with the supplementary volume of cysteine and improved intestine immunity. Further, exogenous cysteine supplementation greatly reduced the mortality rate of golden pompano challenged with S. agalactiae. In general, our findings provide more valuable information and new insights into the rational use of cysteine in the culture of healthy aquatic animals.

Effects of exogenous taurine supplementation on the growth, antioxidant capacity, intestine immunity, and resistance against Streptococcus agalactiae in juvenile golden pompano (Trachinotus ovatus) fed with a low-fishmeal diet.

Taurine has various biological functions in fish, playing an essential role in growth, resistance to oxidative stress, and intestine immunity. Here, we evaluated the effects of exogenous taurine added to low-fishmeal diets on the growth, anti-oxidative stress, intestine immunity, and Streptococcus agalactiae resistance in juvenile golden pompano (Trachinotus ovatus). Our study showed that exogenous taurine supplementation of 1.2% (T3 group) greatly enhanced the weight gain rate and specific growth rate (SGR) of juvenile golden pompano, significantly upregulating growth-related factor expression in the brain and liver, as well as the levels of growth-related parameters in the serum. Polynomial regression analysis using SGR estimated the optimal dietary taurine level for golden pompano at 1.18%. Moderate exogenous taurine also increased the muscular thickness and villus length within the intestine, maintained intestinal physical barrier stability, activated the Nrf2/Keap-1/HO-1 signaling pathway, increased intestinal antioxidant enzyme gene expression and antioxidant enzyme activity in the serum, and upregulated immunoglobulin and complement levels in parallel with declining reactive oxygen species (ROS) levels in the serum. Antioxidant factor expression was also upregulated in the intestine. Furthermore, supplementation suppressed NF-κB signaling and intestinal pro-inflammatory cytokine gene expression, increased anti-inflammatory cytokine gene expression, and improved intestine immunity. Finally, taurine supplementation improved the survival rate of golden pompano challenged with S. agalactiae. Overall, our findings provide additional information and support for the rational use of taurine in healthy aquatic animal farming.

Gill oxidative damage caused by acute ammonia stress was reduced through the HIF-1α/NF-κb signaling pathway in golden pompano (Trachinotus ovatus).

This study aimed to investigate the intoxication mechanism of golden pompano (Trachinotus ovatus) exposed to high ammonia levels and the effects on the immune and antioxidant mechanisms of gills. Juvenile golden pompano was exposed to ammonia (total ammonia: 26.9 mg/L) to induce 96 h of ammonia stress, and a 96 h recovery experiment was performed after poisoning. Then, we evaluated hematological parameters, the histological structure and the expression of related genes. In this experiment, continuous exposure to high levels of ammonia led to a significant increase in plasma alkaline phosphatase (ALP), acid phosphatase (ACP) and lactate dehydrogenase (LDH) levels (P < 0.05), and the levels of triiodothyronine (T3) and tetraiodothyronine (T4) were significantly reduced (P < 0.05). Moreover, the expression of antioxidant genes such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX) and inflammatory cytokines such as tumor necrosis factor α (TNF-α) and interleukin 1ß (IL-1ß) increased (P < 0.05). These results indicate that ammonia activates the active osmotic regulatory mechanism of fish gills and participates in defense and immune responses. However, with prolonged exposure to ammonia, the balance of the defense system is disrupted, leading to oxidative damage and inflammation of the gill tissue. This research not only helps elucidate the intoxication mechanism of golden pompano by ammonia at the molecular level but also provides a theoretical basis for further research on detoxification mechanisms.

Transcription factor pparαb activates fads2s to promote LC-PUFA biosynthesis in the golden pompano Trachinotus ovatus (Linnaeus 1758).

Fatty acyl desaturase 2 (fads2) is a rate-limiting enzyme in long chain polyunsaturated fatty acids (LC-PUFAs) biosynthesis. In mammals, the lipid metabolism is modulated by a transcription factor, peroxisome proliferator-activated receptor alpha ß (pparαß); however, the detailed mechanism via pparαß regulates fads2 remains unclear in fish. In the present study, we identified the sequence features of Trachinotus ovatus fatty acyl desaturase 2a (Tofads2a) and fatty acyl desaturase 2b (Tofads2b), which both encoded 442 amino acid polypeptides containing cytochrome-b5-like domains and three representative histidine-rich domains. The Phylogenetic and genome organization analyses revealed characteristic phylogeny: the majority of fads2s exhibited a highly conserved exon/intron architecture. Tissue expression patterns by quantitative real-time PCR (qRT-PCR) showed that the two Tofads2s were prominently expressed in the brain. A nutritional study indicated that the transcription of the two Tofads2s was significantly implicated by treatment with a 1: 1 ratio of fish oil: soybean oil (FO:SO) in the liver and brain. Furthermore, functional characterization in yeast demonstrated that both Tofads2a and Tofads2b possessed Δ4/Δ5/Δ8 desaturation activity. Furthermore, promoter activity assays showed that the expressions of the two Tofads2s were actively regulated by pparαß. Moreover, mutation analyses showed that the M1 and M4/M5 binding sites of pparαß were functionally vital for binding to Tofads2a and Tofads2b promoters, respectively. Transcriptional activities of the two Tofads2s promoters were significantly reduced after targeted mutation of M1 or M4/M5. Electrophoretic mobile shift assays (EMSAs) verified that pparαß interacted with the M1 binding site in Tofads2a promoter to accommodate Tofads2a transcription. Briefly, pparαß plays an important role in Tofads2 expression and may promote the LC-PUFAs biosynthesis by regulating the expression of two Tofads2s.

Characterization and functional analysis of liver-expressed antimicrobial peptide-2 (LEAP-2) from golden pompano Trachinotus ovatus (Linnaeus 1758).

The liver-expressed antimicrobial peptide-2 (LEAP-2) is an important component of the innate immune defense system and plays an important role in resisting the invasion of pathogenic microorganisms. In this study, LEAP-2 from golden pompano (Trachinotus ovatus) was characterized and its expression in response to Photobacterium damselae was investigated. The full-length LEAP-2 cDNA was 1758 bp, which comprised a 5'-UTR of 250 bp, an ORF of 321 bp, and a 3'-UTR of 1187 bp, encoding 106 amino acids. LEAP-2 consisted of a conserved saposin B domain and four conserved cysteines that formed two pairs of disulphide bonds. The genomic organization of LEAP-2 was also determined and shown to consisted of three introns and two exons. The predicted promoter region of ToLEAP-2 contained several putative transcription factor binding sites. Quantitative real-time (qRT-PCR) analysis indicated that LEAP-2 was ubiquitously expressed in all examined tissues, with higher mRNA levels observed in the muscle, liver, spleen, and kidney. After P. damselae stimulation, the expression level of LEAP-2 mRNA was significantly upregulated in various tissues of golden pompano. In addition, SDS-PAGE showed that the molecular mass of recombinant LEAP-2 expressed in pET-32a was approximately 23 kDa. The purified recombinant protein showed antibacterial activity against Gram-positive and Gram-negative bacteria. Luciferase reporters were constructed for five deletion fragments of different lengths from the promoter region (-1575 bp to +251 bp), and the results showed that L3 (-659 bp to +251 bp) presented the highest activity, and it was therefore defined as the core region of the LEAP-2 promoter. The seven predicted transcription factor binding sites were deleted by using PCR technology, and the results showed that the mutation of the USF transcription factor binding site caused the activity to significantly decrease. The results indicate that golden pompano LEAP-2 potentially exhibits antimicrobial effects in fish innate immunity.

Functional Analysis of IRF1 Reveals its Role in the Activation of the Type I IFN Pathway in Golden Pompano, Trachinotus ovatus (Linnaeus 1758).

Interferon (IFN) regulatory factor 1 (IRF1), a transcription factor with a novel helix-turn-helix DNA-binding domain, plays a crucial role in innate immunity by regulating the type I IFN signaling pathway. However, the regulatory mechanism through which IRF1 regulates type I IFN in fish is not yet elucidated. In the present study, IRF1 was characterized from golden pompano, Trachinotus ovatus (designated ToIRF1), and its immune function was identified to elucidate the transcriptional regulatory mechanism of ToIFNa3. The full-length complementary DNA (cDNA) of IRF1 is 1763 bp, including a 900-bp open reading frame (ORF) encoding a 299-amino-acid polypeptide. The putative protein sequence has 42.7-71.7% identity to fish IRF1 and possesses a representative conserved domain (a DNA-binding domain (DBD) at the N-terminus). The genomic DNA sequence of ToIRF1 consists of eight exons and seven introns. Moreover, ToIRF1 is constitutively expressed in all examined tissues, with higher levels being observed in immune-relevant tissues (whole blood, gill, and skin). Additionally, Cryptocaryon irritans challenge in vivo increases ToIRF1 expression in the skin as determined by Western blotting (WB); however, protein levels of ToIRF1 in the gill did not change significantly. The subcellular localization indicates that ToIRF1 is localized in the nucleus and cytoplasm with or without polyinosinic/polycytidylic acid (poly (I:C)) induction. Furthermore, overexpression of ToIRF1 or ToIFNa3 shows that ToIRF1 can notably activate ToIFNa3 and interferon signaling molecule expression. Promoter sequence analysis finds that several interferon stimulating response element (ISRE) binding sites are present in the promoter of ToIFNa3. Additionally, truncation, point mutation, and electrophoretic mobile shift (EMSA) assays confirmed that ToIRF1 M5 ISRE binding sites are functionally important for ToIFNa3 transcription. These results may help to illuminate the roles of teleost IRF1 in the transcriptional mechanisms of type I IFN in the immune process.

Functional characterization of NK-lysin in golden pompano Trachinotus ovatus (Linnaeus 1758).

NK-lysin is an important part of the innate immune defence system and plays an important role in resisting the invasion of pathogenic microorganisms. In this study, NK-lysin from golden pompano (Trachinotus ovatus) was characterized and its expression in response to Photobacterium damselae was investigated. The full-length NK-lysin cDNA was 731 bp, which comprised a 5'-UTR of 63 bp, an ORF of 444 bp, and a 3'-UTR of 224 bp, and encoded 147 amino acids; NK-lysin consisted of a conserved saposin B domain and six conserved cysteines that formed three pairs of disulfide bonds. The genomic organization of NK-lysin was also determined and the gene consisted of four introns and five exons. The predicted promoter region of ToNK-lysin contained several putative transcription factor binding sites. Quantitative real-time (qRT-PCR) analysis indicated that ToNK-lysin was ubiquitously expressed in all examined tissues; the highest mRNA levels were observed in the skin, kidney and intestine, while the lowest expression level was detected in the stomach. After P. damselae stimulation, the expression level of NK-lysin mRNA was significantly upregulated in various tissues of golden pompano. In addition, SDS-PAGE showed that the molecular mass of recombinant NK-lysin expressed in pGEX-6P-1 was approximately 37 kDa. The purified recombinant protein showed antibacterial activity against gram-positive and gram-negative bacteria. The results indicate that golden pompano NK-lysin has potential antimicrobial roles in fish innate immunity.

Structural and expression analysis of golden pompano Trachinotus ovatus IRF5 and its role in regulation of type I IFN.

The interferon regulatory factor 5 (IRF5) is a mediator of the type I IFN signalling pathways, thereby playing a key role in innate immunity. However, the detailed mechanism through which IRF5 regulates type I IFN in fish remains unclearly. In the present study, we first describe the identification of IRF5 (ToIRF5) from golden pompano (Trachinotus ovatus) and its features at the genomic sequence and expression level. The genomic DNA sequence consists of eight exons and seven introns. The full-length ToIRF5 cDNA is composed of 2, 059 bp and encodes for 499 amino acid polypeptides. The putative protein sequence shares 66.3%-82.9% identity to fish IRF5 and possesses three representative conserved domains (a DNA-binding domain (DBD) at the N-terminus, an IRF-associated domain (IAD), and a virus-activated domain (VAD) at the C-terminus) and one highly variable domain (middle region (MR)). Furthermore, the ToIRF5 transcript is constitutively expressed in all examined tissues, with higher levels observed in the immune relevant tissues. The mRNA levels of ToIRF5 are increased by polyinosinic: polycytidylic acid [poly (I: C)], lipopolysaccharide (LPS) and flagellin stimulation in the immune- and nonimmune-related tissues. The subcellular localization indicates that ToIRF5 is mainly localized in the cytoplasm with or without poly (I: C) induction. In addition, to explore whether ToIRF5 is a modulator of ToIFNa3, promoter analysis is performed. The region from -200 bp to +1 bp is identified as the core promoter by different truncated mutants of ToIFNa3. Mutation analyse declares that the activity of the ToIFNa3-5 promoter significantly decreases after targeted mutation of M2 binding sites. Moreover, overexpression of ToIRF5 in vitro memorably aggrandizes the expression of some IFN/IRF-based signalling pathway genes. These results provide new insights into the roles of teleost IRF5 in transcriptional mechanisms of type I IFN in the immunity process.

Functional characterization of IRF8 regulation of type II IFN in golden pompano (Trachinotus ovatus).

Interferon regulatory factor 8 (IRF8) increases type I IFN transcription levels by binding to IFN promoters, thereby playing a role in innate immunity. Nevertheless, the detailed mechanism through which IRF8 regulates type II IFN in fish remains ambiguous. In the present study, two genes from the golden pompano (Trachinotus ovatus), IRF8 (ToIRF8) and IFN gamma (ToIFNγ), were identified in the IFN/IRF-based signalling pathway. The full-length ToIRF8 cDNA was composed of 2,141 bp and encoded a 421 amino acid polypeptide; the genomic DNA was 2,917 bp in length and consisted of 8 exons and 7 introns. The putative protein showed the highest sequence identity (90-92%) with fish IRF8 and possessed a DNA-binding domain (DBD), an IRF-association domain (IAD) and a nuclear localization signal (NLS) motif consistent with those of IRF8 in other vertebrates. Furthermore, the ToIRF8 transcripts were expressed in all examined tissues of healthy fish, with higher levels observed in the central nervous and immune relevant tissues. They were upregulated by polyinosinic acid: polycytidylic acid [poly (I: C)], lipopolysaccharide (LPS) and flagellin treatments in the blood, liver, intestine and kidney. The results from assays of subcellular localization showed that ToIRF8 was localized to the cytoplasm. Moreover, to investigate whether ToIRF8 was a regulator of ToIFNγ, a promoter analysis was performed using progressive deletion mutations of ToIFNγ. The results indicated that the region from -601 bp to -468 bp includes the core promoter. Mutation analyses indicated that the activity of the ToIFNγ promoter significantly decreased after the targeted mutation of the M1-M3 binding sites. Additionally, overexpressed ToIRF8 in vitro notably increased the expression of several IFN/IRF-based signalling pathway genes. These results suggest that IRF8 is vital in the defence of T. ovatus against bacterial infection and contributes to a better understanding of the transcriptional mechanisms of ToIRF8 on type II IFN in fish.

Elovl4a participates in LC-PUFA biosynthesis and is regulated by PPARαß in golden pompano Trachinotus ovatus (Linnaeus 1758).

The elongases of very long-chain fatty acids (Elovls) are responsible for the rate-limiting elongation process in long-chain polyunsaturated fatty acid (LC-PUFA) biosynthesis. The transcription factor, PPARα, regulates lipid metabolism in mammals; however, the detailed mechanism whereby PPARαb regulates Elovls remains largely unknown in fish. In the present study, we report the full length cDNA sequence of Trachinotus ovatus Elovl4a (ToElovl4a), which encodes a 320 amino acid polypeptide that possesses five putative membrane-spanning domains, a conserved HXXHH histidine motif and an ER retrieval signal. Phylogenetic analysis revealed that the deduced protein of ToElovl4a is highly conserved with the Oreochromis niloticus corresponding homologue. Moreover, functional characterization by heterologous expression in yeast indicated that ToElovl4a can elongate C18 up to C20 polyunsaturated fatty acids. A nutritional study showed that the protein expressions of ToElovl4a in the brain and liver were not significantly affected among the different treatments. The region from PGL3-basic-Elovl4a-5 (-148 bp to +258 bp) is defined as the core promoter via a progressive deletion mutation of ToElovl4a. The results from promoter activity assays suggest that ToElovl4a transcription is positively regulated by PPARαb. Mutation analyses indicated that the M2 binding site of PPARαb is functionally important for protein binding, and transcriptional activity of the ToElovl4a promoter significantly decreased after targeted mutation. Furthermore, PPARαb RNA interference reduced ToPPARαb and ToElovl4a expression at the protein levels in a time-dependent manner. In summary, PPARαb may promote the biosynthesis of LC-PUFA by regulating ToElovl4a expression in fish.

Identification of Fatty Acid Desaturase 6 in Golden Pompano Trachinotus Ovatus (Linnaeus 1758) and Its Regulation by the PPARαb Transcription Factor.

Fatty acid desaturases are rate-limiting enzymes in long-chain polyunsaturated fatty acid biosynthesis. The transcription factor peroxisome proliferator-activated receptor alpha b (PPARαb) regulates lipid metabolism in mammals, however, the mechanism whereby PPARαb regulates fatty acid desaturases is largely unknown in fish. In this study, we report the full length cDNA sequence of Trachinotus ovatus fatty acid desaturase, which encodes a 380 amino acid polypeptide, possessing three characteristic histidine domains. Phylogenetic and gene exon/intron structure analyses showed typical phylogeny: the T. ovatus fatty acid desaturase contained a highly conserved exon/intron architecture. Moreover, functional characterization by heterologous expression in yeast indicated that T. ovatus desaturase was a fatty acid desaturase, with Δ4/Δ5/Δ8 Fad activity. Promoter activity assays indicated that ToFads6 desaturase transcription was positively regulated by PPARαb. Similarly, PPARαb RNA interference decreased ToPPARαb and ToFads6 expression at the mRNA and protein levels in a time-dependent manner. Mutation analyses showed that the M2 binding site of PPARαb was functionally important for protein binding, and transcriptional activity of the ToFads6 promoter was significantly decreased after targeted mutation of M2. Electrophoretic mobile shift assays confirmed that PPARαb interacted with the binding site of the ToFads6 promoter region, to regulate ToFads6 transcription. In summary, PPARαb played a vital role in ToFads6 regulation and may promote the biosynthesis of long-chain polyunsaturated fatty acids by regulating ToFads6 expression.