Study Of Gene Clone,Expression,Polymorphism Of Very Long Chain Acyl-coa Dehydrogenase In Cyprinus Carpio Var Jian

Very long chain acyl-CoA dehydrogenase(VLCAD)catalyzes the first step in the?-oxidation pathway of fatty acids.In this paper,we found two VLCAD gences(termed jlVLCAD-a and jlVLCAD-b)existed in the Cyprinus Carpio var.jian,and they all containd 20 exons and 19 introns.By utilizing RT-PCR and RACE methods,we get the full-length of jlVLCAD-a cDNA sequence.The length was 2543bp,containing 293 bp 5'-untranslated region,270 bp 3'-untranslated region and 1980bp open reading frame encoding 659 amino acids.For amplifying the complete DNA sequences,we designed the premers by the known cDNA sequences.Except intron 8 and inron 13 in jlVLCAD-a,partial sequences in the 5'-un translated region and 3'-untranslated region of jlVLCAD-b,all introns and exons were cloned.The analysis in the amino acids sequences of jlVLCAD-a revealed that the feature structure of jlVLCAD-a were N-terminal,middle domain and C-terminal which included conserved sites(Flavin Adenine Dinucletide binding site,substrate binding pocket and catalytic base).The sequence analysis showed that the amino acid homology of jlVLCAD-a shared more than 80%with other fish,such as Danio rerio,Oreochromis niloticus,Poecilia reticulate and so on,but only above 70%with mammals,respectively.The phylogenteic tree shown that jlVLCAD-a belonged to the Fish branch,and be closest to the VLCAD of Danio rerio.By using the Semi-quantitative RT-PCR method,the jlVLCAD mRNA was transcribed ubiquitously in all eight adult tissues(Liver,Musle,Heart,Foregut,Brain,Kidney,Spleen,Gill),with higher expression in heart,musle,Brain and lowest in Spleen.The expression of jlVLCAD in liver,kidney,gill and foregut were in the middle level.In general,?-oxidation pathway of fatty acids happened in the eight tissues.The discrepancy in expression levels indicated that energy demand in tissue was diffirent and that the heart needed most.VLCAD is the rate-limiting enzyme in ?-oxidation pathway of fatty acid,and have sepecial selection on the catalytic substrate.In order to investigate the effects of diffirent different dietary fatty acids on muscle and liver expression levels of jlVLCAD,the diets were supplemented with fish oil(rich in C22:6n-3)and linseed oil(rich in C18:3n-3).But there was no significant difference between the expressions of jlVLCAD(P>0.05).That meant C18 and C22 did not affect the expression of jlVLCAD in liver or muscle.After lasting a week starvation stress,the expression of jlVLCAD significantly increased in muscle(P<0.05).The same condition also happened in the low temperature stress experiment(P<0.01).It indicated that starvation stress and low temperature stress both enhanced the fatty acid oxidation enhanced in Cyprinus Carpio var.jian muscle.Fat content was an important criterion for fish breeding.In the paper,by using Bioelectrical Impedance analysis(BIA),we got the fat content of musle in Cyprinus carpio var.jian as the associated trait for SNPs detection.Because of the close association in fatty acid metabolism,four SNPs were identified in jlVLCAD-a.Except G1031T located at intron,the other(G44T,T81A,T57C)all was found in extron.G44T was selence mutations which did not cause akteration in amino acid sequence.By using PCR-RFLP methods,we found the association between the G44T polymorphism and fat content traits was significant(P<0.05).The GG type fish fat content significantly higher than TG type(P<0.01).So it meant G44T might have effective on the regulation of fat content.