Gene cloning and their relevances with polyunsaturated fatty acid of peroxisome proliferator-activated receptor two α and one γ form in Cobia（Rachycentron canadum）

• Main Authors: Lee-Jen Fan, 范力仁
• Other Authors: Ronshan Cheng
• Format: Others
• Language: zh-TW
• Published: 2009
• Online Access: http://ndltd.ncl.edu.tw/handle/84480225676821872048

碩士 === 國立臺灣海洋大學 === 水產養殖學系 === 97 === Food restriction period is critical for future compensatory growth in living species and the mechanism is largely unclear. During fasting stage, the lipid is acclimated to lipolysis and mobilize to provide as energy source or metabolic cofactors. Among them, the polyunsaturated fatty acids (n-3 and n-6 series) are endogenous ligands for peroxisome proliferators activated receptors (PPARs) genes, which function as nutrient sensing nuclear receptor and cellular surviving factors. In this experiment, degenerate polymerase chain reaction (PCR) and rapid amplification of cDNA end (RACE) techniques were used to clone cobia (Rachycentron canadum) PPARs. Two PPAR α (α-1 and α-2) and one PPAR γ were isolated with full cDNA length of 3,674 bp (α-1 form), 3,818 bp (α-2 form), and 3,146 bp (γ form). Two PPAR α form share identical coding region with deduced 470 amino acids and 3’ untranslated region (UTR) of 2,074 bp, but differs in 5’ UTR length with 190 bp in α-1 form and 334 bp in α-2 form. The two α form has identical 42 bp DNA immediately upstream of translational initiation site, but differs in length and DNA contexts of rest sequence. The cobia PPAR α deduced amino acid of coding region were ranging from 73% to 97% identity to known fish species in GeneBank. The PPAR γ form contains a coding region with deduced 532 amino acids, 3’ untranslated region (UTR) of 1,246 bp, and 5’ UTR of 304 bp. The cobia PPAR γ deduced amino acids blasting ranging from 68% to 92% identity to known fish species. Residues blasting indicate that a wide range of amino acid variation of these two genes in evolution. The cobia tissue fatty acid mobilization rate during fasting was analyzed by gas chromotography. Among the tissue analyzed, the muscle fatty acid mobilization rate has the most significant changes at 3-fasting-day, while brain and liver with much slower rate. The relative percentage of fatty acid mobilization rate in muscle is following: α-linolenic acid (53.4%), eicosapentaenoic acid (57.1%), docosapentaenoic acid (46.3%), docosahexaenoic acid (50.1%), linoleic acid (48.2%), arachidonic acid (40.3%), docosadienoic acid (23.5%), palmitic acid (45.3%), stearic acid (47.9%). The relative ratio of n-3 and n-6 series polyunsaturated fatty acids (n-3/n-6) were decreasing in muscle and liver, while there was no significant changes in brain, indicating a quicker mobilization rate of n-3 series than n-6 series during fasting in such tissues. Real-time PCR was used to evaluate fasting tissue PPAR gene expression level. The PPAR α gene in brain was significant increased by 4.7-fold and 9.6-fold in 3- and 6-fasting-day, respectively. The PPAR γ was most significant increased to 45-fold at 9-fasting-day in kidney tissue. The polyunsaturated fatty acids mobilization rate and its role in PPAR α and PPAR γ activation is still not clear and might play certain important role in fasting stages.