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| Cloning and Subcellular Distribution of Chicken (Gallus gallus) Perilipin1 Gene |
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Abstract Surrounding the membrane surface of lipid droplets, Perilipin1 plays important roles in the formation and maturation of lipid droplets, and regulation of fat mobilization (lipolysis). Considered as one of the important marker genes of adipocyte differentiation, many Perilipin1 studies in mammals have been carried out, while in chickens (Gallus gallus) relatively few. The objectives of the present study were to clone full length mRNA of chicken Perilipin1 gene, and analyze its subcellular location during the progression of chicken preadipocyte differentiation. We extracted total RNA of abdominal adipose tissue from chickens at 7 weeks of age. RT-PCR and rapid amplification of cDNA ends (RACE) methods were used to clone the 5'-untranslated region (5'-UTR), the coding domain sequence (CDS) and the 3'-untranslated region (3'-UTR) sequences of chicken Perilipin1 gene, and then the gene structure was analyzed. The subcellular localizations of Perilipin1 in chicken preadipocyte was examined by immunofluorescence confocal microscopy at different time points (12~120 h). Results showed that the whole mRNA length of chicken Perilipin1 gene was 2 379 bases (bp), consisted of nine exons and eight introns (with the start codon ATG located in the second exon). The CDS length of chicken Perilipin1 gene was 1 551 bp, encoding 517 amino acids, and lengths of 5'-UTR and 3' UTR were 94 and 733 bp, respectively. Sequences have been submitted to NCBI database, and assigned the serial number (Accession No. GU327532). In addition, we performed the multiple alignment for the chicken Perilipin1 CDS sequence with mouse (NM_175640) and human (NM_001145311) sequences deposited in NCBI database by the Clustalw software (http://www.genome.jp/tools/Clustalw/), and obtained the homology percentages, 38% and 41%, respectively. Therefore, sequence divergence at nucleotide level was quite large, suggesting that the role of chicken Perilipin1 in adipocye differentiation may not be fully consistent with mammals. We used immunofluorescence method combined with laser confocal microscopy to check the subcellular location of chicken Perilipin1 in the process of preadipocyte differentiation, which was induced by oleate at different time points (12, 24, 48, 72, 96 and 120 h), started on primary chicken preadipocytes cultured by the digestion method. At the first 12h of induction, few small lipid droplets were visualized in the preadipocytes, and Perilipin1 located on the surface of neonatal lipid droplets. As time passed on (24 to 48 h), more lipid droplets emerged, and Perilipin1 surrounded the lipid droplets. At late stage (72 to 120 h), several large lipid droplets formed through the fusion of small lipid droplets, and Perilipin1 convened and packaged on the surface of lipid droplets. Therefore, our results indicated that chicken Perilipin1 surrounded the membrane surface of lipid droplets during the whole process of adipocyte differentiation, and we speculated that basically, the function of chicken Perilipin1 might help in forming a protective barrier separating lipase from lipids stored in the lipid droplet. In conclusion, we have successfully cloned the full length mRNA of chicken Perilipin1 gene, and confirmed the subcellular position of Perilipin1 during chicken preadipocyte differentiation. The current study provided some molecular information for further investigating the function of chicken Perilipin1 gene.
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Received: 10 March 2016
Published: 06 August 2016
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