Molecular Cloning And Expression Under The Different Conditions Of Peroxisome Proliferator Activated Receptors-α And Carnitine PalmitoyltransferaseⅠin Trachinotus Ovatus

The full-length c DNA of PPAR α and CPTⅠgene in T. ovatus were cloned, Tissues expression and the influence of the expression of them under three different conditions as well as discussed in this paper. The results follow: 1.cloned and analyzed of the full-length c DNA of PPAR α and CPTⅠgeneThe full-length c DNA of PPAR α and CPTⅠ in T. ovatus were cloned by RACE(Rapid Amplification of c DNA Ends), and the protein structures, physical,chemical properties and their functional domain structures which encoded by PPAR α and CPTⅠ gene were analyzed by bioinformatics method. The results showed that c DNA sequence of PPAR α in T. ovatus was 1930 bp, and the open reading frame was 1425 bp, encoding 474 amino acid(Gen Bank accession No. KP893147). The protein was unstable protein without signal peptide and trans-membrane. The secondary structure of PPAR α contains alpha helix, beta turn, and extended strand, random coil. At the same time, alpha helix was 45.15%. There were some typical structures in PPAR α, including a DNA-binding domain with zinc-finger structures and a ligand binding domain which can integrate ligands and activate PPAR α. The result showed that cDNA sequence of CPTⅠ in T.ovatus was 2841 bp, consisting of a 5′untranslated region(5′-UTR) of 142 bp, a 335 bp 3′-UTR of 335 bp, and open reading frame was 2 363 bp, encoding 787 amino acid(GenBank accession No. KP987456). CPTⅠ was a stable protein with hydrophilic and large molecular, and with aliphatic index 85.63, grand average of hydropathicity(GRAVY)-0.213. The protein contained two helical transmembrane regions, two N-glycosylation sites in 312 th and 367 th of the amino acid sequence, and there were possible phosphorylation sites in Ser, Thr and Tyr. the sequences of T. ovatus PPAR α were highly homologous with those cloned from Japanese sea perch, cobia, large yellow croaker and gilthead sea-bream(81%--89%), phylogenetic tree analysis of PPAR α protein showed that the nearest relationship existed between T. ovatus and cobia among all the species mentioned above, and the lowest homology was human.Phylogenetic tree analysis of CPTⅠ protein showed that the nearest relationship of T.ovatus was perch with 94%, and large yellow croaker and Gilthead seabream followed by 93%, the lowest homology were human and mouse 2. Tissues expression of PPAR α and CPTⅠmRNAUsing quantitative real-time RT-PCR, we observed that PPAR α and CPTⅠ mRNA were expressed predominantly in ten tissues likes brain-lipid, spleen, intestine and head-kidney tissues of T. ovatus.The results showed that PPAR α and CPT Ⅰhad expressed in these ten tissues, and have higher quantity in some fat metabolism active organizations. PPARα had rich expression in brian tissue, head kidney, intestine and spleen(P<0.05). CPTI gene expression in liver tissue is significantly higher than other groups(P<0.05).3. Effects of fenofibrate to the relatively expression of gene PPAR α and CPTⅠThe fenofibrate solution with concentration of 20, 50,100 mg/kg were injected into the abdomen of Trachinotus ovatus respectively, and the relatively expression of gene PPAR alpha and CPTI in livers and muscles were analyzed by RT-PCR, and the effects of the agonists on relatively expression of PPAR alpha and CPTI gene was studied. The result showed that the expression of PPAR α in group 50 mg/kg is significant higher than that in other groups(P<0.05) and CPTI had higher expression in group 20mg/kg(P<0.05). There had no significant difference in the expression of PPAR alpha and CPTI gene in PBS group(P>0.05), but the expression of PPAR α in group 50 mg/kg is growth before decrease and return to the initial level trend finally as time changes. Meantime, CPTI with time gone which fall after rise and stable rising last, following the highest expression at 30 h after injection in livers and highest expression at 18 h in muscles(P<0.05). In the mass, the relatively expression of gene PPAR alpha in livers and muscles with fenofibrate solution is higher than PBS. So we know that the fenofibrate can induce the expression of PPAR α, but, there isn’t obviously pertinence change trend between PPAR α and CPTI.4. The relatively expression analysis of gene PPAR α and CPTⅠ under different temperaturePut Trachinotus ovatus into the oval with 16℃,19℃,22℃,25℃ and 28℃ to stimulated. Then test the expression change trend of PPAR α and CPTⅠgene in livers and muscles by RT-PCR to discusse the influence of genen expression by temperature. The results showed that PPAR α and CPTⅠmRNA expression had rised over time which put T. ovatus into 16℃,19℃,22℃,25℃ water directly from keep water with about 28℃. Among the temperature gradient, those two genes had increased significantly in livers and muscles at 16℃and 19℃(P<0.05). And the expressionreached the maximum at 12 h, finally, it land back to the initial value. In contrast, at 22℃ and 25℃, the two genes also had raised trend, but not significant(P>0.05), which CPTI gene albeit slowly ups and downs at each time, and lower than the initial value at 48 h. 5. The expression analysis of PPARα and CPTI with hunger conditionThough short-term starvation for T. Ovatus get muscles and livers to test the expression of PPAR α and CPTI by RT-PCR which provides reference materials and theoretical basis for investigating the function for those two genes,the mechanism and regulation of fat deposition in fish study, as well as research in fish physiology hunger plays an important value in academic and applied.The results of this study showed that the expression of PPARα and CPTImRNA i n muscle tissue changes over time are to reduce first, and incrise then, and reach a maxi mum at 32 ~ 48 h and then reduce. While in the liver tissue, the two genes over time first increased and then decrease. In addition, expression of starvation CPTI gene is generally higher than the normal control group fed, while PPAR α gene expression only when the maximum value exceeds the control group, the other time points lower than the control group, which may have a great relalationship with the main function of two genes. CPTI gene is directly related to the fact that fat changes into energy. Under starvation conditions, it is expressed higher than normal beacsue there needs energry for fish metabolism and life activities.