Study On Gene Expression Rules Of Adipocyte Determination Factors In Pig Development

PPARγ, CCAAT/enhancer binding protein a (C/EBP α), ADDl have been implicated as key regulators of adipocyte differentiation. The patterns of PPARy, C/EBPa and ADDl gene expression in different growth stages and the relation with adipose deposition are studied. LPL, AP2 and adipsin are the markers of adipocyte differentiation. The pattern of PPARy, C/EBPa and ADDl gene expression in different growth stages are studied. Duroc× Landrace × Yorkshire pigs weighed 1, 30, 50, 70 and 90 kg were used to study developmental gene expression of PPARy, C/EBPa, ADDl, LPL, AP2 and adipsin in subcutaneous adipose tissue by means of semi-quantitative RT-PCR..The special primers for PPARγ, C/EBPα, ADDl, LPL, AP2, adipsin and P-actin were designed respectively according to the reported gene sequences. Then genome RNA was extracted from the subcutaneous adipose tissue of pigs and the target gene mRNA amplified through RT-PCR with special primers. The DNA fragments of PPARγ, C/EBPα, ADDl, LPL, AP2, adipsin and P-actin were obtained. The PCR product was cloned into PGEM-T vector and sequenced. Sequence analysis suggested that the gene homology of fragments obtained in this study were 99.3%, 98.9%, 100%, 99.6%, 99.6%, 100% å’Œ99.4% compared with that of the reported cDNA sequence (AF103946, U34070, AY307771, AY559454, AF264083, AF012821 and AJ312193) in Genebank. Based on the gene clone, the suitable anneal tempreture, MgCl₂ concentration, and cycles in PCR system were discussed and the optimal semi-quantitative RT-PCR methods were constructed to detect the amounts of all genes mRNA expression in pigs.In the experiment, total fifteen female Duroc X Landrace X Yorkshire pigs of five groups, each group of three pigs weighed at 1, 30, 50, 70 and 90 kg were slaughtered for sampling. Subcutaneous adipose tissues were collected, weighed, and rapidly frozen in liquid nitrogen, then stored at -70℃ until RNA analysis, for determination of developmental gene expression, so as to disscuss the pattern of gene expression in different growth stages. Adiposity was determined for the relation of leptin geneexpression and adipose deposition.The results showed that the PPARy, C/EBPa and ADDl mRNA levels of porcine adipose tissue continuously increased as pigs grew and deposited fat from 1 to 90 kg growth stages (P<0.05). The present data showed a close positive correlation between the levels of PPARy, C/EBPa and ADDl gene expression and the fat deposition rate in pigs (P<0.05). This implicated PPARy, C/EBPa and ADDl gene can effect the adipose deposition.The results showed that the LPL, AP2 and adipsin mRNA levels of porcine adipose tissues presented a biological developmental pattern, LPL, AP2 and adipsin mRNA levels continuously increased from 1 to 70 kg growth stages (P<0.05). slowly increased from 70 to 90 kg growth stages. This indicates that porcine adipocyte are differentiating from 1 to 90 kg growth stages.