Research Of Δ5Fatty Acyl Desaturase In Abalone, Haliotis Discus Hannai Ino

This study was conducted to investiguate the Δ5fatty acyl desaturase (Fad),crucial enzyme in the pathway of LC-PUFA biosynthesis, in abalone Haliotis discushannai Ino, a good resource of LC-PUFA for human beings. This paper includes thecontents of cloning, sequence analysis, functional characterization and tissuedistribution of Δ5Fad in abalone. Moreover, the effects of dietary fatty acids, indifferent type or different concentration, on survival, growth, fatty acid compositionand expression of Δ5Fad were further studied in hepatopancreas and muscle ofabalone.1. Long chain polyunsaturated fatty acid (LC-PUFA) biosynthesis is neglectablein most marine species, normally due to lack of Δ5fatty acyl desaturase (Fad) activity.Among exceptions, abalone possesses considerable LC-PUFA conversion ability fromC18precursors. However, its relevant enzymes and regulatory mechanism are notwell characterized. Here we successfully cloned and characterized Δ5Fad in abalone(Haliotis discus hannai Ino). Two Δ5Fad mRNA transcripts, Hdhfad1(GQ470626)and Hdhfad2(GQ466197), were found in abalone sharing96.82%similarity forcDNA sequence and96.58%similarity for amino acid sequence. Hdhfad1cDNA is1530bp in length, consisting of a99bp5’ untranslated region (UTR), an114bp3’UTR and an ORF coding for438amino acids. Hdhfad2cDNA is1525bp in length,composed of91bp5’ UTR,114bp3’ UTR and1320bp ORF encoding for439aminoacids. Both of them have characteristic features of front-end desaturase, includingthree histidine boxes, an N-terminal cytochrome b5domain with heme-binding motif(HPGG) and transmembrane regions. They have the closest relationship with Fad ofoctopus. HdhFad1possessed higher Δ5desaturase activity but expressed at a lowerlevel than HdhFad2. Both isoforms preferred20:4n-3than20:3n-6as substrate. Theseresults should provide valuable information on the molecular evolution of LC-PUFAbiosynthesis and better understanding of the nutritional regulation of LC-PUFAbiosynthesis in abalone. 2. A120-day feeding trial was conducted to investigate the effects of dietaryfatty acids on survival, growth, fatty acid composition and expressions of Δ5fattyacid desaturases (Fads, Hdhfad1and Hdhfad2) in juvenile abalone (Initial bodyweight:0.38±0.01g; Initial shell length:15.06±0.21mm). Six purified diets wereformulated to contain3.5%tripalmitin (TP), olive oil (OO, rich in18:1n-9), grapeseed oil (GO, rich in18:2n-6), linseed oil (LO, rich in18:3n-3), ARA oil (AO, rich inARA) or EPA oil (EO, rich in EPA and DHA) as dietary lipid. Each diet wasrandomly fed to triplicate aquaria and each aquarium was stocked with50abalones.Results showed that no significance was observed in survival rate of abalone fed withdifferent diets. Compared with those of control (TP) group, growth parameters suchas weight gain rate (WGR), specific growth rate of weight (SGRW), shell lengthincrease rate (SIR), daily growth rate of shell length (DGRSL) were significantlyincreased in abalone fed OO (rich in18:1n-9), AO (rich in ARA) and EO diets (richin EPA and DHA)(P<0.05). GO diet has no different effect on growth parameters ofabalone im comparison with TP group (P>0.05). The lowest growth was found inabalone fed LO diet containing high level of18:3n-3(P<0.05).20:1n-9and22:1n-9were significantly higher than those of other groups (P<0.05). N-6polyunsaturatedfatty acid (PUFA) in particular ARA in hepatopancreas and muscle of abalone fed GOdiet, was significantly higher than those of TP, OO, LO and EO groups (P<0.05). N-3PUFA, especially for EPA, in abalone fed LO diet was significantly higher than thoseof TP, OO, GO and AO groups (P<0.05). Expression levels of Δ5Fads in abalonewere stimulated by dietary LO (rich in18:3n-3) and suppressed by dietary AO andEO (rich in ARA, EPA or DHA), but unresponsive to dietary high level of18:1n-9or18:2n-6. In conclution, abalone has the capability to synthesis LC-PUFA fromC18PUFA. The expression levels of Δ5Fads are regulated by dietary C18precursor,ALA, as well as products, such as ARA, EPA and DHA. These results providedvaluable information on better understanding of the nutritional regulation of LC-PUFA biosynthesis in abalone.3. A120-day feeding trial was conducted to investigate the effects of dietaryincreasing levels of grape seed oil (GO) and linseed oil (LO), rich in linoleic acid (LA)and-linolenic acid (ALA) respectively, on growth, fatty acid composition andexpression levels of Δ5fatty acyl desaturases (Fads) in juvenile abalone (Haliotisdiscus hannai Ino). Seven experimental diets were formulated to contain increasing amounts (0%,0.875%,1.75%and3.5%) of GO or LO as dietary lipids. Tripalmitin(TP), rich in16:0, was supplemented to reach3.5%(dry weight) total lipid. Thesediets were named as0%GO/LO (TP),25%GO,50%GO,100%GO,25%LO,50%LOand100%LO. With the increase of dietary GO or LO inclusion, growth parameters(such as specific growth rate of weight) first increased and then decreased (R2>0.72,P=0.000). The survival rate of abalone was not significantly correlated with dietaryGO (R2=0.04, P=0.815) or LO inclusion (R2=0.50, P=0.046). With the increase of LAin GO diets, LA,20:2n-6and22:2n-6were increased in hepatopancreas and muscleof abalone; ARA in hepatopancreas first increased and thereafter reached a plateauwhile ARA in muscle was increased. With the increase of ALA in LO diets, ALA and20:3n-3were increased in hepatopancreas and muscle; EPA were first increased andthereafter reached a plateau while DHA were first increased and then decreased. DPAwas first increased and then decreased in hepatopancreas while it was first increasedand then reached a plateau in muscle tissue. Compared with those of TP-fed abalone,the mRNA levels of Δ5Fads were significantly increased in groups fed with50%GO,50%LO and100%LO diets (P<0.05), but no significant differences were observed inother experimental groups. These results indicated that biosynthesis of some longchain polyunsaturated fatty acid in abalone could be increased in response toincreasing levels of dietary LA or ALA through increased expressions of Δ5Fads.High intakes of dietary LA or/and ALA inhibited the biosynthesis of DHA and hadbad effects on growth performance.