Study On Expression Of Fatty Acid Synthase Gene In Goat Mammary Gland By RNA Interference

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.This study uses different concentrations of 17β-estradiol and RNA interference technology to study the function of FAS gene, can provide the foundation for goat mammary gland fatty acid mechanism. Considering above-mentioned reasons, some studies were taken basing on the following aspects:(1) Healthy Xinong saanen goat were used as experimental animal to construct in vitro culture system of dairy goat mammary epithelial cells. Its growth characteristics, morphological features, cytoplasmic inheritance andβ-casein protein expression and other relevant characteristics were analysed and identified to provide foundation to study the function of genes in goat mammary gland.(2) Dairy goat mammary epithelial cells were treated with 17β-estradiol. Subsequently, the effects of 17β-estradiol on dairy goat mammary epithelial cells proliferation and differentiation were deteced by MTT, LDH activity analysis and RT-PCR.Finally, fatty acid composition were assyed by gas chromatograph. (3) The change of dairy goat mammary epithelial cells were checked by chemical synthesis,liposome transfection,MTT,LDH activity analysis,RT-PCR and western blotting while the silencing of FAS gene.The main results were as follows:(1) Primary culture of goat mammary gland cells could be derived by outgrowth of migrating cells from fragment of tissue. Fibroblast and epithelial cells could be pured according to their different sensibility to trypsin. Morphological observation revealed that the cultured cells possessed the typical character of epithelial cells. Karyotyping analysis showed the normal chromosome number in cultured cells. The tissue specific expression of cytokeratin 5 and 8 genes in mammary epithelial cells was identified by immunofluorescent cytochemical staining. The transcription of the beta-casein gene was detected by RT-PCR , when the purified cells were induced(2)17β-estradiol plays an important regulatory role in dairy goat mammary epithelial cells proliferation and differentiation. 25μmol/L, 250μmol/L and 500μmol/L 17β- the estradiol can promote cell proliferation and FAS gene expression, change the fatty acids composition in goat mammary gland,and change ultrastructure of the dairy goat mammary epithelial cells .100μmol/L 17β- the estradiol can inhibit FAS gene expression ,cause dairy goat mammary epithelial cells pathology Injury, and induce apoptosis of the dairy goat mammary epithelial cells.(3)Silenceing of FAS gene could inhibit dairy goat mammary epithelial cells proliferation, cause its pathology Injury, decrease the abundance of FAS mRNA , and change the fatty acids composition in goat mammary gland.There were extremely significant decrease on short-chain fatty acid and significant increase on medium-chain fatty acid concentrations. Silenceing of FAS gene could induce apoptosis of the dairy goat mammary epithelial cells, emerge apoptotic body. At the same time, the mitochondria also appears swelling, breakage phenomenon...