Genetic Variants, Transcriptional Regulation And Functional Studies On Bovine Cidec Gene

As people are Seeking after beef quality, the improvement of the content of intramuscular fat has became an important goal of the beef cattle breeding. The research in rat and other model animals show that cell death-inducing DFFA-like effector c (CIDEC/Fsp27) is required for unilocular lipid droplet formation and optimal energy storage. And it has emerged as an important regulator of metabolism associated with lipodystrophy, insulin-resistant diabetes, and hepatic steatosis. The research of genetic variats, transcriptional regulation and regulatory mechanism for the deposition of fat in bovine primary adipocytes in bovine CIDEC gene is important for marker-assisted selection and transgenic breeding of beef cattle. DNA pool sequecing, forced-PCR-RFLP, gene clone, prokaryotic expression, eukaryotic expression, packaging of the adenovirus and cell culture were used in this research to verify the function of bovine CIDEC gene. The expression profiles of CIDEC in different growth period of Qinchuan cattle, the verification of relationship between CIDEC gene genetic variation and growth traits, subcellular localization of CIDEC protein, screening of the core promoter region and PPRE reaction elements in CIDEC gene were studied, at last the function of bovine CIDEC in apoptosis and glucolipids metabolism were confirmed. The main results of this research including:1. CIDEC gene has a higher expression level in bovine adipose tissueReal-time RT-PCR was used to analyse the expression of CIDEC gene in different tissues of Qinchuan newborn and adult cattles, the results revealed that the expression of this gene in these two stages was similar, the expression level was highest in adipose tissue, then in lung and gut, in other tissues its expression was very low. These suggesting that bovine CIDEC gene has an important role in lipid metabolism.2. Variats in the promoter of CIDEC gene could affect its transcription thereby affect the growth traitsTen novel SNPs were discovered in5’regulation region of CIDEC gene by DNA pool sequencing in five Chinese cattle breeds, SNP2,3,4,6and9linked completed, SNP8and10linked completed. The association analysis revealed that different heplotypes was associated with Nanyang cattle growth traits. By detection of the transcriptional activity of different heplotypes, we found that the haplotype with less variats show higher transcriptional activity, the individuals with this haplotype also show better growth traits. Combined with the analysis of the cis-acting elements, we can speculate that these10SNPs could changed the cis-acting elements, then haplotypes of the cis-acting elements may affect transcriptional activity, thus affecting the growth traits of livestock. These10SNPs loci could be used as candidate loci in bovine molcular breeding.3. CIDEC and PPARg have different subcellular localizationThe recombinant eukaryotic expression vector pEGFP-C1-CIDEC and pEGFP-C1-PPARg were transfected in3T3-L1cell, CIDEC protein located in cytoplasm and PPARg protein located in nucleus, suggesting that these two protein can’t interacted with each other by directed protein-protein contact. According to the research in other species, we speculated that bovine PPARg gene may regulate the transcription activity of CIDEC gene by binding to its PPRE reaction element, which located in the5’regulation region of CIDEC gene.4. Bovine PPARg could regulate the transcription activity of CIDEC geneBy searching the Genomatix software, there are2promoter region (promoter1:-1936to-1336, promoter2:+3552to+4154) and3PPRE response elements (-1691to-1669,-1684to-1662,-1677to-1655) in bovine CIDEC gene. The results of dual luciferase activity analysis shown that the promoter1was the core promoter, and its active region was PPRE response elements, the activity of the PPRE response elements elevated by adding the agonist of PPARg gene:rosiglitazone. So this result confirmed our inference, bovine PPARg gene regulates the transcription activity of CIDEC gene by binding to PPRE reaction element, which located in the5’regulation region of CIDEC gene.5. Cloning, sequence analysis and prokaryotic expression of bovine CIDECThe ORF region of bovine CIDEC contain696bp, encodes peptide with222amino acids, the deduced molecular weight of CIDEC protein was25KDa, the isoelectric point was7.97. The main secondary structure of this protein was spiral tube. Phylogenetic analysis revealed that cattle exhibit greater sequence similarity with dogs, chimpanzees, humans, monkeys, pigs and mice than chicken or fish. The optimal condition for the expression of the recombinant plasmid pET-28a(+)-CIDEC were:37℃,1.0mmol/L IPTG and induced6h. Western-blot confirmed the products were the target protein.6. Overexpression of CIDEC inducing apoptosis of the bovine primary adipocytesAdenovirus vector of bovine CIDEC gene was constructed, after packaging and amplification successfully, high-titer of Ad-CIDEC was infected with bovine primary adipocytes. According to the expression of the Fas gene we found that overexpression of CIDEC gene inducing apoptosis of bovine primary adipocytes, which was consistent with the research in human and mouse.7. CIDEC gene regulate the glucolipid metabolism of bovine primary adipocytes We obtained one shRNA sequence for bovine CIDEC gene which could cause obvious interference effect (pENTR/CMV-GFP/U6-CIDEC-shRNAl). High-titer of Ad-siCIDEC was obtained after packaging and amplification (3×108PFU/mL). In bovine primary adipocytes, the RNA interference efficiency of Ad-siCIDEC was up to72%. The reduction of CIDEC gene can result in the up-regulation of PPARg, PLIN, HSL (lipoprotein metabolism), GLUT4(glucose metabolism), UCP1(mitochondrial activity). Bovine CIDEC gene participated in the glucolipids metabolism, when the expression of CIDEC was decreased, the level of lipolysis was up regulated, the balance of glucose was affected, the number and activity of mitochondrial was increased, in brief, the bovine adipocytes was changed from energy storage to energy consumption.