Macular Blue Fish HUFA synthesis involved three key Cloning and Characterization
|Keywords||Macular Blue Fish HUFA anabolic Δ4 fatty acid desaturase Δ6/Δ5 bifunctional fatty acid desaturase Carbon chain elongase Nutritional regulation|
Generally, fish fatty acids can be saturated and the carbon chain to the prolonged action of linoleic acid and linolenic acid 18C 20-22C into the highly unsaturated fatty acids (HUFA), and sea fish, or lack of capacity of the kind weak . Recently, however, our group in the macula Aiko fish (Siganus canaliculatus) was first discovered and proved saltwater fish have HUFA conversion of linoleic and linolenic as the ability, and the kind of transformation capacity in the low salinity waters (10 ppt ) than in high-salinity water (32 ppt) strong (Li et al., 2008). This suggests that the fish basket HUFA synthesis seems both freshwater and saltwater fish characteristics, clear blue Fish HUFA anabolic regulatory mechanisms will help to ascertain the ability of marine fish causes of low HUFA synthesis, research and development to improve Saltwater fish HUFA synthesis methods, thus improving the ability of marine fish use vegetable oil, marine fish feed to promote research and application and development of aquaculture. In order to study Blue Fish HUFA anabolic regulatory mechanism of its regulation of the thesis cloned HUFA synthesis of the two fatty acid desaturase (fatty acyl desaturases, fad1 and fad2) and carbon chain extending enzyme (Elovl5) gene (cDNA), and its functional characteristics and tissue expression, as well as feed fatty acid composition of its mRNA expression were studied. The main results are as follows: 1. Fad1 gene has been cloned before us but not yet functional characterization Blue Fish Δ6 fatty acid desaturase gene (accession number: EF424276). The functional identification display, not only has the Δ6 desaturase enzyme activity, but also has a Δ5 desaturase activity, i.e. Fad1 Δ6/Δ5 is a dual function with fatty acid desaturase activity, Δ6 desaturation activity, but greater than the Δ5 desaturase activity. Fad1 full length cDNA of 1846 bp (excluding Poly A tail), encoding 443 amino acids. The amino acid sequence of the cloned or Δ5 fish Δ6 fatty acid desaturase which has 70%? 81% similarity, and has a typical fatty acid desaturase structural characteristics. 2. Fad2 mainly as Δ4 desaturase activity, but also a small amount of Δ5 desaturase activity. Its full-length cDNA of 1831 bp, encoding 445 amino acids (accession number: GU594278). The amino acid sequence of the cloned or Δ5 fish Δ6 fatty acid desaturase having 67%? 79% similarity, and has a typical fatty acid desaturase structural characteristics. 3 carbon chain extending enzyme gene (accession number: GU597350) with a typical extension enzyme Elovl5 activity with full-length cDNA of 1254 bp, encoding 291 amino acids. The amino acid sequence of the cloned enzyme Elovl5 fish carbon chain extension of the amino acid sequence having 69%? 83% similarity, and has a typical carbon chain extension of the structural properties of the enzyme. 4 tissue-specific expression studies have shown, Fad2 and Elovl5 in the brain, liver, intestine tissue mRNA expression highest, followed by ocular tissues, in the spleen, muscle, heart and gill tissues of the lowest level or difficult to detect. This shows that the liver, brain, and intestinal tissue may be synthesized Aiko HUFA main parts of fish. 5 Use the Soviet oil and triolein fat source, preparation linolenic acid: linoleic acid ratio was 2.5:1 but 0:1,0.5:1,1:1,1.5:1,2:1 and HUFA-free feed for the test group to add fish oil feed as the control group, feeding the fish basket analyzed after 12 weeks of the three enzymes in liver tissue mRNA expression levels. The results showed that: Su oil and triolein group mRNA levels than fish oil group, but linolenic acid and linoleic acid on gene expression role in promoting and feed on these two fatty acid ratio. Linolenic acid and linoleic acid ratio greater than or less than 1:1, the expression of the three genes have a more significant role in promoting the promotion ratio of 1:1 weakest; ratio of 2:1, the fad2 desaturase enzymes and enzyme expression in the carbon chain elongation promoted significantly; ratio of 2.5:1, the right fad1 desaturase expression role in promoting the strongest. 6 main innovation of this study include: ① the first to get from one marine fish has Δ6/Δ5 bifunctional fatty acid desaturase activity, but also for the first time in seawater fish proved Δ5 desaturase activity existence; ② first reported in vertebrates function with Δ4 fatty acid desaturase. The results will help to reveal the saltwater fish HUFA biosynthesis molecular mechanism, improve fish HUFA synthesis pathway; while vegetable substitute for fish depth study provides a theoretical basis; For rich fish nutrition, especially Molecular Nutrition learning content has important theoretical significance and academic value.