Characterization And Function Analysis Of Fads2 Desaturase And Elovl5 Elongase Gene In Trachinotus Ovatus

It is generally believed that the Long chain polyunsaturated fatty acid(LC-PUFA)synthesis capacity of marine fish is lacking or weak,so it is necessary to add fish oil in its feed to meet the needs of its normal physiological function.In recent years,the global fish oil was shortage and expensive,and looking for other alternative oil to replace the fish oil was imminent.Vegetable oil is rich in C18 PUFA,which has become the most potential alternative oil.However,Vegetable oil almost has no LC-PUFA,which is difficult to become the replacement.Studies have shown that marine fish such as Siganus canaliculatus,Solea senegalensis and Channa striata has the ability to convert linoleic acid and linolenic acid to LC-PUFA.However,the conversion capacity of different marine fish is different.Trachinotus ovatus is one of the most important marine aquaculture fish in southern China.It has delicious meat,highly fat and protein content which become a high quality seawater edible fish.However,the research on the mechanism of molecular regulation of LC-PUFA in Trachinotus ovatus was rarely reported.Clarified the mechanism of LC-PUFA synthesis is helpful to improve its ability to utilize vegetable oil,to promote the research of Trachinotus ovatus feeds.It can promote the study of fish oil substitutes in the aquatic feed of Trachinotus ovatus and cut the costs.In this study,we cloned the full-length cDNA sequence of the fatty acid desaturase 2 and Elongation of very long chain fatty acids protein 5 gene in LC-PUFA synthesis in Trachinotus ovatus.The expression characteristics in different tissues and the expression of nutritional regulation were both analyzed by RT-PCR.Fads2 and Elovl5 gene functional validation was studied by yeast heterologous expression.The results were shown as follows:1.Effects of different diets on muscle fatty acid composition in Trachinotus ovatus.A feeding trial was conducted to investigate the effects of feeds type on growth and fatty acid composition of muscle in healthy Trachinotus ovatus(Initial body weight: 146.22 ± 3.26 g).In the experiment,three different feeds were used to feed the Trachinotus ovatus,which were pelleted feed,frozen squid(Loligo chinensis),forzen fish(Trachurus japonicus).Results showed that pelleted feed can significantly promote the growth of Trachinotus ovatus and reduce the feed conversion rate in the case of the same amount of feeding.The content of LC-PUFA in frozen squid was highest among the three kinds of feed.The pelleted feed had high LA and ALA level.The highest LC-PUFA content was detected in the muscle of the frozen squid group(P <0.05),while the content of MUFA(mainly C18: 1n9)in the muscle of the pelleted feed group was higher.The content of unsaturated fatty acids in the muscle of the frozen fish group was in the middle level.In conclution,the muscle LC-PUFA content is affected by the level of feed fatty acid.2.Gene structure and expression regulation of Fads2 and Elovl5.Fads2 can specifically insert double bonds at specific positions of the carbon chain to produce desaturation.In this study,a full-length of Fads2 cDNA sequence was identified from Trachinotus ovatus.Fads2 cDNA is 2215 bp in length,and ORF coding for 442 amino acids.It has the characteristic structures of Fads2 family.The homology analysis showed that the Fads2 protein of Trachinotus ovatus has a high homology with the ?4Fad of teleost(75%-80%).Phylogenetic analysis showed that there was no obvious clustering phenomenon between Fads2 and ?4Fad of other species.Elovl5 plays an important role in the biosynthesis of LC-PUFA,especially in the C18 extension.A full-length of Elovl5 cDNA sequence was identified from Trachinotus ovatus.Elovl5 cDNA is 3770 bp in length,and ORF coding for 294 amino acids.It has the characteristic structures of Elovl family.The phylogenetic analysis showed Trachinotus ovatus Elovl5 is clustered together with Elovl5 of other species.Multiple sequence alignment found that the Trachinotus ovatus Elovl5 polypeptide had 77–95% aa sequence identity to Elovl5 elongases of other teleost fish.The difference of tissue expression showed that Fads2 and Elovl5 mRNA both has higher expression in the brain,eye and liver.The expression of Fads2 and Elovl5 gene at T0 and T6 of the three experimental groups showed that,Fads2 and Elovl5 in the intestinal tissue was lower expression in the pelleted feed group at T0,and the expression level of the two genes in the frozen squid group was significantly decreased at T6.In the liver,Fads2 was lower expression in the frozen fish group and no significant difference in Elovl5 between three experimental groups at T0.The expression of Fads2 and Elovl5 was significantly down-regulated in the frozen squid group and the frozen fish group at T6.The result showed that the expression levels of Fads2 and Elovl5 are regulated by dietary fatty acid precursor and products.3.The functional of Fads2 and Elovl5 gene.Yeast heterologous expression was used to verify the functional of Fads2 and Elovl5.The recombinant expression vector containing the target gene was transformed into Saccharomyces cerevisiae,and the possible PUFA substrate of the target gene was added to the induction medium.The composition of PUFA in recombinant cells of Saccharomyces cerevisiae was analyzed by gas chromatography.The structure of the fatty acids production was determined by GCEI-MS.The result showed that Fads2 had the function of ?4 desaturase,Elovl5 had a function of prolonging C18 and C20 carbon chains,and preferred 20:5n-3 than 20:4n-6 as substrate.