Identification And Function Analysis Of SREBP-1A And SREBP-1Cgene In Dairy Goat

Sterol regulatory element binding protein(Sterol Regulatory Element Binding Protein,SREBPs) is an important transcriptional factor family which have key regulatory functions on lipid and cholesterol biosynthesis in mammals. SREBP-1 is one of the key regulators of lipid synthesis, and recent studies also demonstrate that SREBP-1 is the central regulator of milk lipid synthesis in bovine mammary gland during lactation period, it has important regulatory function on milk fatty acid biosynthesis, transportation, desaturation, elongation,triacylglycerol biosynthesis, lipid droplet formation and secretion. It has been found that SREBP-1 transcriptional factor family have two isoforms: SREBP-1a and SREBP-1c, studies on human and mouse have characterized the sequence structure difference and the function difference of the two isoforms, however, there is no report about the sequence information of the two isoforms in ruminant animals such as bovine and dairy goat.The present study identified the promoter sequence of SREBF1 two isoforms: SREBP-1a and SREBP-1c in Xinong Sannen dairy goat, and then amplified the functional nuclear form of the two isoforms, and constructed them into pc DNA3.1 overexpression vector. After 48 h transfection of the recombinant overexpression vector into goat mammary epithelial cells, we measured the overexpression effects of SREBP-1a and SREBP-1c, and also measured the effects of their overexpression on the m RNA level changes of fatty acid synthesis related genes and the changes of cellular total cholesterol and triacylglycerol. Our results provide experimental basis for further investigation of goat SREBP-1 gene family on fatty acid metabolism and milk lipid synthesis. The main results are as follows:(1) By comparing the goat genome sequence and the c DNA sequence of goat SREBP-1gene, we cloned the promoter sequence of goat SREBP-1a and SREBP-1c, contained 1 947 bp and 2 012 bp fragment upstream of transcriptional stat site(TSS), respectively. By constructing the promoter luciferase reporter vector(p GL3-basic), we transfected it along with p RL-TK into goat mammary epithelial cells, and then collected the cells for promoter activity assay after 48 h transfection. Results showed that, the obtained fragment all have gothigh promoter activity, indicating that the cloned fragment are all promoter sequence.(2) By comparing the promoter sequence of two SREBF1 isoforms with SREBP-1 gene c DNA sequence, we designed primers to amplify the nuclear form sequence of goat SREBP-1a and SREBP-1c. Results showed that, a 1 209 bp fragment sequence of SREBP-1a and 1 137 bp fragment sequence of SREBP-1c were amplified. The sequence difference of the two isoforms are mainly in the region of N-terminal, SREBP-1a have a longer sequence of 72 bp than SREBP-1c gene sequence, the remaining sequence are exactly the same.(3) By constructing the nuclear form of goat SREBP-1a and SREBP-1c isoforms into pc DNA3.1 overexpression vector and transfecting them into goat mammary epithelial cells.48 h later, RT-q PCR results showed that pc DNA3.1-SREBP-1a and pc DNA3.1-SREBP-1c transfected group resulted in a 10 times increase and more than 30 times increase of SREBP-1a and SREBF1(P<0.05), respectively, Western blot also showed that the nuclear form of SREBP-1 protein increased significantly in pc DNA3.1-SREBP-1a and pc DNA3.1-SREBP-1c transfected group than in pc DNA3.1 transfected group.Overexpression of SREBP-1a and SREBP-1c lead to a significant increase of fatty acid synthesis related genes: fatty acid synthetase(FASN), stearoyl-coenzyme A desaturase 1(SCD1), Acetyl Co A carboxylase α(ACACA), and elongase of very long chain fatty acids 6(ELOVL6) and Lipin(LPIN1)(P<0.05). Overexpression of SREBP-1a and SREBP-1c lead to increase of cellular total cholesterol content, but the effect is not statistically significant(P>0.05).In summary, our present study cloned the promoter sequence of the two isoforms of goat SREBF1 gene family, and then identified the nuclear nucleotide sequence of the two isoforms.By constructing and transfecting the pc DNA3.1-SREBP-1a and pc DNA3.1-SREBP-1c vector into goat mammary epithelial cells for 48 h, a significant m RNA increase of fatty acid synthesis related genes are observed, and the cellular total cholesterol content were increased by SREBP-1a and SREBP-1c overexpression. Our results provide theoretical and experimental basis for further definition of the two isoforms of SREBF1 gene family on fatty acid metabolism and validation of their exact functions in mammary gland.