Functional Study On HUFA Synthesis Related Genes In Large Yellow Croaker(Larimichthys Crocea)

Highly unsaturated fatty acids(HUFA)are a kind of linear fatty acids with at least three double bonds and twenty carbon atoms.They are essential fatty acids for human.They are not only play a pivotal role in maintaining the function and integrity of bio film structure,but also play an essential role in brain development,retinal formation and vision protection.In addition,HUFA are also involved in the anti-stress response and immune response of human body,and play an important role in the prevention and treatment of some diseases,such as nephropathy,diabetes,alzheimer disease and cardiovascular disease.In particular,EPA(C20:5n-3)and DHA(C22:6n-3)are known as heart gold and brain gold.Fish,especially marine fish,are human’s favorite food and the main source to obtain HUFA.However,studies have shown that most marine fish lack HUFA synthesis ability or have weak synthesis ability.In order to investigate the HUFA anabolism mechanism of large yellow croaker(Larmichthys crocea),the structure and function of Elovl4a gene were cloned in this study,and the regulatory effects of transcription factors(LXR,HNF4,PPAR)on the activity of Elovl4a promoter were also investigated.In addition,the expression patterns of related genes(Elovl4,Elovl4a,Elovl5 and Fads2)in the HUFA synthesis pathway of fish were compared and analyzed in eleven different tissues and five brain regions of large yellow croaker.All this study enriching and improving the HUFA biosynthesis pathway of large yellow croaker and laying a foundation for elucidation the HUFA anabolism mechanism.The main findings are as follows:1)The full-length cDNA of Elovl4a gene is 3,230 bp(NCBI:MK887188),including a 404 bp 5’UTR,a 1,863 bp 3’UTR and a 963 bp open reading frame coding a polypeptide of 320 amino-acid.Large yellow croaker ELOVL4a is a membrane protein with seven α-helical transmembrane regions and has the ELO superfamily typical conserved structural domains as well as KxxExxDT;QxxFLHxYHH;NxxIHxxMYxYY;YLxxxQxxQ four characteristic motifs of the fatty acid elongase enzymes.Phylogenetic analysis showed that fish ELOVL4 with ELOVL4b was a small cluster and ELOVL4a was a small cluster,and the ELOVL4a of large yellow croaker had higher homology with the amino acid sequence of other fish’s ELOVL4a.2)Quantitative Real-time PCR results showed that the Elovl4 gene was primarily expressed in the eyes,brain and gonads,with the highest expression in the eyes(P<0.05).Elovl4a gene was widely expressed in multiple tissues of large yellow croaker,and the expression in the brain was significantly higher than in other tissues(p<0.05).Elovl5 gene was also widely distributed in tissues;the expression level was highest in eyes but can be barely detected in gonad,muscle,kidney and head kidney.Fads2 gene was mainly expressed in the brain,gonad,muscle and eyes,and the expression level in brain was the highest(p<0.05).Besides,all the four genes were highly expressed in eyes,brain and gonad of large yellow croaker,which may be related to the growth and development of the brain,eyes and gonad required a great amount of HUFA.What’s more,the four genes were widespread in brain,cerebellum,thalamus,medulla oblongata and optic lobe,but the expression of each gene was different without obvious regularity.3)Saccharomyces cerevisiae eukaryotic expression system was used to verify that the large yellow croaker Elovl4a gene can extend C18 and C20 fatty acid substrates to C22 fatty acid,but it does not have the function of extending C22 fatty acid substrates to C24 or even C36.In addition,the affinity of large yellow croaker ELOVL4a to n-6 series fatty acids is poor and cannot catalyze 18:2n-6 and 18:3n-6.4)A 1,730 bp(-1249 bp～+481 bp)candidate Elovl4a gene promoter region was cloned by RACE in large yellow croaker.Predicting transcription factor binding sites have SP1(Specificity protein 1),SRE(Sterol regulatory element protein),YY1(YIN-YANG 1),NF-Y(Nuclear factor Y),HNF4α(Hepatocyte nuclear factor 4α),PPARγ-RXRα,etc.Dual-Luciferase assay showed that the transcription factors HNF4,LXR and PPARa had a positive activation effect on the Elovl4a gene promoter,and the degree of regulation had no difference.