Functional Identification And Transcriptional Regulation Of Elovls And Fads Genes In Trachinotus Ovatus

Polyunsaturated fatty acids(PUFA)are essential for the growth and development of fish and juveniles.At present,invertebrates and freshwater fish have been shown to have the ability to synthesize PUFAs de novo,while marine fish have a lack or weak ability to synthesize PUFAs.In recent years,it has been found that marine fish such as Siganus canaliculatus and Solea senegalensis can synthesize linoleic acid and linolenic acid into long-chain polyunsaturated fatty acid.Trachitotus ovatus belongs to the Perciformes,Trachonotus.It is one of the important marine fish in the Asia-Pacific region.Its meat is delicious and has high nutritional value.It is a high quality edible marine fish.At present,there are few reports on the key enzymes in the LC-PUFA synthesis pathway,such as Elongases of very long chain fatty acids(Elovls)and Fatty acid desaturases(Fads).Therefore,the clarification of its function and transcriptional regulation mechanism in the PUFA synthesis pathway is of great significance for clarifying the network regulation pathway in the PUFA synthesis pathway,and will help to ascertain the reasons for the general low PUFA synthesis ability of marine fish.In order to study the regulation mechanism of anatomical metabolism of LC-PUFA in T.ovatus,this study cloned the c DNAs of key enzymes Elovl4 a,Elovl4b,Elovl4-like and Fads2 a,Fads6 genes in the LC-PUFA synthesis pathway,and analyzed its tissue expression profile by q PCR.The functional characteristics of yeast were studied by heterologous expression of yeast.The transcriptional regulation mechanisms of Elovl4 a and Fads6 were preliminarily elucidated by promoter activity analysis and point mutation technique.The transcriptional regulation mechanisms of Elovl4 a and Fads6 were preliminarily elucidated by promoter activity analysis and point mutation technique.The main results obtained are as follows:1.Elovls and Fads bioinformatics and tissue expression analysis.The full length of Elovl4 a,Elovl4b and Elovl4-like c DNA were 1606 bp,2430 bp and 2180 bp,respectively,and the open reading frame(ORF)encoded 320,305 and 263 amino acids,respectively.The amino acid sequences of the coding region thereof contain significant structural features of the Elovl4 family;Multiple sequence alignments show the homologous proteins were similar to those of other teleosts;Phylogenetic analysis showed that Elovl4 a,Elovl4b,Elovl4-like and other species of Elovl4 similar protein clustered into one large branch.The full-length c DNAs of Fads2 a and Fads6 are 2041 bp and 1594 bp,respectively,and the ORF encodes 442 and 380 amino acids,respectively.Multiple sequence alignments showed the homologous proteins were similar to those of other teleosts with significant structural features of the Fads family.Phylogenetic analysis indicated that Fads2 a and Fads6 were clustered with homologous proteins of other species,respectively.The expression profile revealed that Elovl4 a m RNA is highly expressed in brain tissue;The Elovl4 b m RNA is highly expressed in male gonads,female gonads and brain tissues;The Elovl4-like m RNA is underexpressed in male gonads,spleen and female gonads;Both Fads2 a and Fads6 m RNA are highly expressed in brain tissue.2.Functional studies of Elovls and Fads genes.Yeast heterologous expression was used to detect the function of the T.ovatus Elovls and Fads genes.Heterologous expression analysis of yeast showed that Elovl4 a can prolong C18:3n-6 to C20:3n-6with a conversion rate of 1.05%;Elovl4b can extend C18:2n-6,C18:3n-6 and C20:5n-3to C20:2n-6,C20:3n-6 and C22:5n-3,respectively,with conversion rates of 5.16%,9.35%and 5.42%,respectively.Elovl4-like can extend C18:2n-6 and C20:5n-3 to C20:2n-6and C22:5n-3,respectively,and the conversion rates are 0.83% and 0.82%,respectively;Both Fads2 a and Fads6 have the function of a Δ4/Δ5/Δ8 desaturase.Both Fads2 a and Fads6 can convert C20:2n-6,C20:3n-6,C22:5n-3,C22:4n-6 into C20:3n-6,C20:4n-6,C22:6n-3,C22: 5n-6,respectively.The conversion rates of Fads2 a were 0.18%,0.56%,43.82%,and 1.24%,respectively.The conversion rate of Fads6 was 0.17%,0.54%,39.29%,and 1.04%,respectively.According to the current research results of the laboratory Elovls and Fads on the T.ovatus,T.ovatus has a complete unsaturated fatty acid synthesis pathway,and there is a synthetic pathway of "Elovls-?8-?5" in the scorpion.3.The transcriptional regulation mechanisms of Elovl4 a and Fads6.By constructing a series of deletion and promoter activity assays,the results indicated that the core promoter region of Elovl4 a is located at(-148 bp ～ + 258 bp).And Elovl4 a transcription is positively regulated by the transcription factor peroxisome proliferator-activated receptors(PPARαb).Point mutation analysis indicated that the transcriptional activity of the Elovl4 a promoter was significantly reduced after targeting the M2 binding site of the mutant PPARαb(+ 209 bp ～ + 223 bp).In addition,the core promoter region of Fads6 is located at(-448 bp ～ +1 bp),and transcription is also positively regulated by PPARαb.Point mutation analysis indicated that the activity of the Fads6 promoter was significantly reduced after targeting the M2 binding site of the mutant PPARαb(-113 bp ～-87 bp).