CDNA Clone, Sequence Analysis And Expression Research Of ATGL And HSL Gene In Xinong Saanen Dairy Goats

Adipose triglyceride lipase(ATGL) and hormone-sensitive lipase(HSL) are the key and rate-limiting enzymes in lipid lipolysis, and play a decisive role in the processes of energy metabolism and physical transformation. In this experiment, RT-PCR and RACE(Rapid Amplification cDNA Ends) methods were used for the isolation and cloning of the whole cDNA of ATGL and HSL gene in Xinong Saanen dairy goats, real-time fluorescence quantitative-PCR approach was used for detecting the expression levels of ATGL and HSL gene in the tissues of goats. In addition, we also did initial research in the regulation of ATGL and HSL gene in mammary epithelial cells of goat. The major results are as follows:(1) The full length of goat ATGL gene(GenBank accession No. GQ918145) cloned is 2 074 bp, which contains 141 bp of 5'UTR, 472 bp of 3'UTR, and 1 461 bp of entire coding sequence (CDS), encoding a protein of 486 amino acid residues(GenBank accession No. ADD25174).The full length cDNA of goat HSL gene (GenBank accession No. EU273879) is 2 877 bp, which includes 185 bp of 5'UTR, 421 bp of 3'UTR, and 2 271 bp entire coding sequence (CDS), encoding a 756-amino acid residues of goat HSL (GenBank accession No. ABX79556.1). The AA of goat ATGL are with high similarity, more than 85%, compared with bovine(Bos taurus), Rat(Rattus norvegicus), mouse(Mus musculus), pig(Sus scrofa), and human(Homo sapiens) in GeneBank, and the results of the amino acids of goat HSL aligned with that of cattle(Bos taurus), sheep (ovis aries), mouse (Mus musculus,), rat (Rattus norvegicus), and human (Homo sapiens) in GeneBank, are also with high level, above 83%, which were indicated by AA sequence alignment. The protein structure analysis showed that the estimated molecular weight of ATGL and HSL are 53 243 Da and 82 584.3 Da, and the isoelectric point(pI) are 7.4 and 6.2 the estimated molecular weight of 83 kD. Both of the two proteins contain theα/βhydrolase folds and GXSXG(Gly-X-Ser-X-Gly) structure, which are the typical characteristic of lipases. A patatin domain and a transmembrane helix were speculated in the N-terminal of ATGL gene, and no signal peptide found in the entire sequence of both ATGL and HSL.(2) We analyzed the tissue expression of ATGL and HSL gene. The results showed that the mRNAs of ATGL and HSL gene were existed in all the 12 detected organizations, of which the most abundant expression are in the adipose tissue, and much higher than other tissues, but the level of relative expression of HSL is more than 11 times much higher than ATGL in the adipose tissue.In addition, There is higher expression level of HSL gene but lower level of ATGL gene relatively in the small intestine, and the expression level of ATGL gene is above 4 times much higher than HSL in the liver. For other tissues, the trend of the relative expression of the two genes are consistent, and the ratio of ATGL and HSL gene is about 0.6~1.5. Further more the levels of the two genes in heart are in the minimum amount, relatively.(3) In mammary gland epithelial cells, adding dexamethasone and estradiol could significantly increased the mRNA levels of ATGL and HSL genes, the difference was significant (P <0.01); and adding NaF, insulin and oleate could obviously decreased the mRNA level of ATGL gene with significant difference(P <0.01). Besides, the mRNA levels of HSL gene in NaF, insulin and oleate treated groups also decreased obviously, and which were significant (P <0.05, P <0.001 and P <0.01). In addition, the analysis also found that prolactin could down regulation the mRNA level of ATGL gene, but the difference was not significant (P> 0.05); while the mRNA level of HSL gene showed upward trends, the difference was significant (P <0.001 ). The result of detecting the content of triglyceride and cholesterol in all the experimental groups, showed that NaF, oleate and insulin could significantly increased the content of cellular TG, and NaF and oleate also could significantly reduced the levels of celluar cholesterol, suggesting that ATGL and HSL gene were related with metabolism of triglyceride and cholesterol ester in mammary gland epithelial cells.