| It is generally thought that freshwater fish can bioconvert linoleic andα-linolenic into HUFA. In contrast, marine fish are generally presumed to have a low capability or inability to convert linoleic and linolenic acids into HUFA. However, our recent study proved for the first time in marine teleost that Siganus canaliculatus had the ability to biocovert linoleic and linolenic acids into HUFA, and such ability was higher in low salinity (10 ppt) than that in high salinity (32 ppt) (Li et al., 2008). This indicates that the HUFA biosynthetic capability in S. canaliculatus possesses the features of both marine and freshwater fish. And thus the clarification of HUFA biosynthetic mechanism in S. canaliculatus will be helpful for revealing the reasons of low ability of HUFA biosynthesis in marine fish, and for the development of method in enhancing such ability. The latter will increase the utilization ability of marine fish to dietary vegetable oils, and thus accelerate the development and application of formulated feeds, as well as the development of aquiculture.In this study, for fully understanding the regulatory mechanisms of HUFA biosynthesis in S. canaliculatus, three genes (cDNAs) encoding the key enzymes involving in HUFA biosynthesis of S. canaliculatus were cloned, including two fatty acyl desaturase (fad1 and fad2) and a elongse (Elovl5) genes. Their functional characteristics, tissue specific distribution as well as the nutritional regulation by dietary fatty acids composition were also investigated. The results were shown as follows:1. The cDNA for fad1 had previously been cloned but not functionally characterized (Genebank accession no. EF424276). This time the functional characterization results showed that fad1 had both ?6 and ?5 desaturation activity, and that ofΔ6 desaturation was higher than that ofΔ5 desaturation. The fad1 cDNA was 1846 bp in length (excluding polyA tail) and encodes a peptide with 443 amino acids. The protein sequence possessed all the characteristics of microsomal fatty acid desaturases and shared 70-81% sequence identity to the previously cloned fish'sΔ6 orΔ5 desaturases.2. Fad2 mainly had theΔ4 desaturation activity and a littleΔ5 desaturation activity. The full length cDNA had 1831 bp specifying protein of 445 amino acids (accession no. GU594278). The protein sequence also had all the characteristics of microsomal fatty acid desaturases and showed 67-79% sequence identity with other fish'sΔ6 orΔ5 desaturases.3. The cDNA for fatty acid elongase (Elovl5) was 1254 bp in full length and encodes a peptide with 291 amino acids (accession no. GU597350). The protein sequence had characteristics of microsomal fatty acid elongases and shared 69-83% sequence identity with other fish's Elovl5.4. The mRNA expression of both fad2 and Elovl5 was mainly detected in brain, liver and intestine, and then in eye. Whereas their expression levels in spleen, white muscle, heart and gill was low or undetectable. These results suggested that brain, liver and intestine may be the main places of HUFA biosynthesis in S. canaliculatus.5. With perilla oil and glycerol trioleate as lipid source, six diets were formulated in a 0:1, 0.5:1, 1:1, 1.5:1, 2:1 and 2.5:1 ratio of linoleic toα-linolenic acids but without HUFA. Besides, the diet with fish oil was used as control. After experimentation with the diets for 12 weeks, the mRNA expression levels of above three genes were analyzed in livers of fish. The results showed that the mRNA expression levels were generally higher in fish fed the diets with perilla oil and glycerol trioleate compared to fish fed the fish oil diet. However, the effect of increment was related to the dietary ratio of linoleic toα-linolenic acids. The enhancing effects of linoleic andα-linolenic acids to gene expression was obvious when the ratio was <1:1 or >1:1, and was lowest in 1:1 ratio, among all the groups. Obvious enhancing effects were obtained to the expression ofΔ4 desaturase and elongase genes in a 2:1 ratio, and to that ofΔ6/Δ5 desaturase gene in a 2.5:1 ratio.6. This is for the first time to obtain a bifunctional desaturase with bothΔ6 andΔ5 desaturation activity in a marine fish species, and also the first detection ofΔ5 desaturation activity in marine fish. Besides, this is also the first report of a fad withΔ4 activity in any vertebrate species. The results will be helpful for clarifying the molecular mechanisms of HUFA biosynthesis of marine fish, and perfecting fish's HUFA biosynthesis pathway. Furthermore, the results will be of practical significance for directing the replacement of dietary fish oil with vegetable oils for the aquiculture, as well as important theoretic and academic significance for enriching fish nutrition physiology, especially molecular nutriology.
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