Comparison Study On Regulation Of Δ6 Fatty Acyl Desaturase Among Rainbow Trout, Japanese Seabass And Large Yellow Croaker

The experiments were conducted to investigate the difference in regulation of Δ6 fatty acyl desaturase in freshwater, enryhaline and saltwater fishes. Rainbow trout. Japanese seabass and large yellow croaker, which are wildly cultured carnivorous species in China, were designed as representative of freshwater, enryhaline and saltwater fishes. The results are summarized as follows:1. Effects of replacement of fish oil by vegetable oil on growth performance, tissue accumulation of lipid and fatty acids and histology of rainbow trout, Japanese seabass and large yellow croaker.A 70-days feeding experiment were conducted to investigate the effect of replacement of vegetable oil by fish oil on growth performance, body composition, tissue accumulation of lipid, histology and anabolic LC-PUFA of rainbow trout (initial mean weight 11.24±0.07 g). Japanese seabass (18.60±0.36 g) and large yellow croaker (8.93±0.21 g) in freshwater indoor recirculation aquaculture system and seawater floating net cages, respectively. Three isoprotein (41% crude protein) and isolipidic (12% crude lipid) diets were formulated to contain graded levels of vegetable oil blend (0.50 and 100% dry weight) by supplementation of soybean oil and inseed oil. The 0% vegetable oil blend group was treated as the control group.The results showed that no significant differences were observed in final weight (FW). specific growth rate (SGR). feed intake (FI). feed efficiency ratio (FER) and survival rate (SR) among rainbow trout fed diets with graded levels of vegetable oil. while these parameters were significantly decreased with increasing dietary vegetable oil in Japanese seabass and large yellow croaker. The hepatosomatic index in three fish were not significantly different, but viscerosomatic Index (VSI) in three fish fed diets with vegetable oil were significantly higher than fish fed the diet with fish oil. The fatty acid composition of experimental fish reflected closely those of diets. The level of n-3 LC-PUFA was lower when vegetable oil was replaced by fish oil. but no significant difference was found between fish oil group and 50% vegetable oil group in muscle of rainbow trout. Increased lipid vacuoles in hepatocytes cytoplasm and serious intestinal inflammation was induced by increasing level of fish oil replacement in all three fish species. Among the three kinds of fish, liver and intestinal structure of large yellow croaker was damaged the most seriously by the vegetable oil.2. Cloning and bioinformatics analysis of Δ6FAD gene promoter in rainbow trout, Japanese seabass and large yellow croaker.The upstream sequences from the translation start codon of Δ6FAD in rainbow trout, Japanese seabass and large yellow croaker were cloned by the genome walking method,1479bp,2064bp and 996bp. Alignments of Δ6FAD promoter fragments of rainbow trout. Japanese seabass, large yellow croaker. European sea bass, and Atlantic salmon, including potential elements of transcription factors (specificity protein 1 (Spl), nuclear factor Y (NF-Y). and SREBPs (SRE), showed that the Δ6FAD promoter of rainbow trout and large yellow croaker possesses elements of NF-Y and SREBPs as Japanese seabass. European sea bass, and Atlantic salmon, but lacks Spl element which Atlantic salmon possesses.3. Cloning and bioinformatics analysis of SREBP-1 and PPAR-α in rainbow trout, Japanese seabass and large yellow croaker.The full length of SREBP-1 cDNA of rainbow trout and large yellow croaker were 4220bp and 3750bp, each of which contained an entire open reading frame for a protein with high identity to mammalian SREBP-1. The deduced protein sequence of SREBP-1s displayed typical structure of SREBP. a bHLH-zip domain. SREBP-1 with other previously characterized SREBPs from fish and human showed that the sequences in fish to be more similar to human SREBP-la than to SREBP-lc. The phylogenic tree of SREBP-1 showed that marine fish and freshwater fish species clustered together, respectively, and formed sister groups to the branch of mammal species, chicken and microorganism.The full-length sequence of PPAR-α2 cDNA from rainbow trout was 1401 bp, all which is an entire open reading frame. The sequence of PPAR-α cDNA from large yellow croaker was 2051bp. including a 5’-UTR of 149bp. a 3’-UTR of 468bp, and an open reading frame of 1434bp. The deduced protein sequences of PPAR-α2 from rainbow trout and PPAR-α from large yellow croaker were displayed a typical structure of PPAR. which contains a DNA binding domain (C4-type zinc finger) and ligand-binding domain. The sequences of PPAR-al and PPAR-α2 in Japanese seabass have been published, and the PPAR-a in rainbow trout has been reported previously (named PPAR-α1 here) was different from the one cloned in the persent study (named PPAR-α2 here). The phylogenetic analysis revealed that PPAR-α1 and PPAR-α2 of fish but rainbow trout were on different branches. Gene duplication might occur in the evolution of PPAR-α, resulting in two PPAR-α genes with diverged functions in some fishes.4. Effects of replacement of fish oil by vegetable oil on mRNA expression of Δ6FAD, SREBP-1 and PPAR-α of rainbow trout, Japanese seabass and large yellow croaker.The expression levels of Δ6FAD transcript were significantly up-regulated in the liver of three kinds of fish fed the diet with vegetable oil compared with that of fish fed diets with fish oil, but there are different results in intestinal, muscle and adipose tissues. The degree of tissues on vegetable oil response and difference synthesis of LC-PUFA in tissues might have remarkable difference in one kind of fish. The expression levels of SREBP-1 transcript were significantly up-regulated in the three kinds of fish fed the diet with vegetable oil compared with that of fish fed diets with fish oil, also, but the level of SREBP-1 expression were significantly decreased firstly then increased with increasing dirtary vegetable oil in adipose and intestinal of rainbow trout and liver and musle of Japanese seabass. The expression levels of PPAR-a were significantly up-regulated in the liver of rainbow trout and large yellow croaker fed the diet with vegetable oil compared with that of fish fed diets with fish oil. however. the level of PPAR-α gene were significantly down-regulated in the liver and musle of Japanese seabass. The results showed that the PPAR-α gene in different fish have varying effects on vegetable oil response.SREBP-1 mRNA levle was in great accordance with the regulation of Δ6Fad gene expression in the intestine of Japanese seabass by these diets. The correlation coefficient (r) between SREBP-1 and Δ6Fad gene expression regulated by these diets was 0.999, P<0.05. A significantly positive correlation was also observed between the large yellow croaker PPAR-a gene expression level and the Δ6Fad transcript level in the liver and muscle.5. Dual regulation of fish Δ 6-desaturase gene expression by SREBP-1 and PPAR-αFor functional analysis, cDNAs encoding the SREBP-1 and PPAR-α open reading frame from rainbow trout. Japanese seabass and large yellow croaker were subcloned into the PCS2+　expression vector. The Δ6FAD promters form three fish were directly inserted into the PGL3-Basic. which is the promoterless reporter plasmid and contains a luciferase gene. To determine whether the genominc 5’ flanking regions of Δ6FAD in three fish contain a functional promoter, respectively, and the role of SREBP-1 and PPAR-α in regulating A6FAD promoter activity in three fish. Hek293T cells were cotransfected with the Δ6FAD promoter-luciferase reporter plasmid and the SREBP-1 or PPAR-a expression plasmid. The PGL3-Basic and PCS2+ as the control plasmid in dual-luciferase reporter assay. In Hek293T cells, the fragment of the A6FAD in rainbow trout showed significantly higher activity than the fragment in Japanese seabass and large yellow croaker. All transcription factor of SREBP-1 up-regulated the promoter activity of Δ6FAD in three fish. In rainbow trout, only PPAR-α2 can be up-regulated the promoter activity of A6FAD. However, no effect of PPAR-α on promoter activity of Δ6FAD in large yellow croaker.Inhibition of mRNA expression in liver of rainbow trout injected with SREBP-1 or PPAR-α2 small interfering RNAs (siRNAs). Knockdown efficiency was quantitatively measured by real-time PCR analysis. The result showed that siRNAs of gene inhibited the expression of corresponding gene. The expresstion level of SREBP-1 or PPAR-α2 have reached the lowest point at 4 or 7 hour after injection, and the Δ6FAD expression levels were the lowest after the previous phenomenon appeared 2 or 6 hour, respectively. These results once again confirm that dual regulation of Δ6FAD gene expression by SREBP-land PPAR-α2 in rainbow trout.