Cloning, Sequencing And Analysis Of Fatty Acid Synthase (FAS) Gene And Its Promoter In Mammary Gland Of Xinong Saanen Goat

Fatty acid synthase(FAS) plays an important role in de novo fatty acids synthesis in mammals. FAS helps to catalyze all the reaction steps in the conversion of acety1-CoA, malonyl-CoA and NADPH to saturated fatty acids. Short- and medium- chain fatty acids in goat mammary synthesis are functioned by a transferase reaction in the region of Acetyl-CoA and malonyl-CoA transacylases(AT/MT). It′s one of the important means to solve goat milk odor from genetics and breeding aspect by regulating FAS gene expression in order to change fatty acids composition in goat mammary gland. Study of goat milk short- and medium-chain fatty acids metabolism can provide the foundation for goat milk odor formation mechanism. At the same time, it is of important practical significance for making rational use of the character of goat milk fatty acids and raising goat milk fatty acids content which is beneficial to human bodies.The study uses premature, normal delivery and healthy Xinong Saanen goat in Xinong Saanen Goat Breeding Farm of Northwest A&F University and Goat Seed Farm in Qianyang of Shaanxi Province as experimental materials, using RT-PCR combining with Rapid Amplification of cDNA Ends(RACE) method to clone and analyze the sequence of Xinong Saanen goat mammary FAS gene full-length cDNA and partial promoter region. It adopts real time PCR method to identify FAS gene mRNA expression abundance of mammary gland of Xinong Saanen goat during different lactation stages(peak period, middle period, late period, end period, dry period), aiming to establish foundation for FAS gene function in mammary gland of Xinong Saanen goat. By mastering FAS gene expression, it can provide experimental foundation for mammary fatty acids metabolism, FAS gene function and regulation mechanism and genetics of goaty milk flavor. The experimental results are as follows:1. The cloning sequence of FAS encoding region consists of KS domain (exon2-exon9), AT/MT domain (exon8-exon16), DH-ACP domain (exon16-exon39) and TE domain (exon39-exon42), and the length of each domain is 1052 bp, 1449 bp, 4356 bp and 787 bp respectively. The Genbank number of each sequence is EF042286, DQ915966, EF068247, EF059988 respectively(These four data have been published). There are 1250 bp in 3′end and 360 bp in 5′end. The full-length cDNA sequences of FAS gene have been formed by joint for the first time, whose size is 8217 bp and encoding for 2514 amino acids.2. The length of FAS promoter gene fragment is 719 bp, and the Genbank number is EF556550, which is still unpublished. Sequence analysis shows that TATA box is located in -41 bp, and a similar CAAT box is located in -74 bp. Both are the typical eukaryotic promoter elements. TATA box upstream domain contains 76% G/C and four GC boxes(GGGCGG and CCGCCC), and potential binding sites for nuclear transcription factor Sp1 at positions -99 bp,-174 bp,-255 bp and-320 bp.3. FAS gene mRNA has the highest expression rate in the late lactation period and the lowest in dry period. mRNA expression level of FAS gene in mammary gland of Xinong Saanen goat expresses differently and its content change is probably related to the change of milk yield and milk fat content.