| Stearoyl-CoA desaturase 1(SCD1)is aΔ-9 desaturase that catalyzed carbon-14 to carbon-19 fatty acyl-CoAs to yield the corresponding monounsaturated fatty acids(MUFAs).It has recently been identified as one of the major enzymes in the control of lipid metabolism.SCD1 primarily converts palmitic acid and stearic acid to palmitoleic acid(16:1 n-7)and oleic acid(18:1 n-9),respectively.SCD1 is also responsible for the conversion of trans 11-C18:1(tll-C18:1)to cis9,trans11-conjugated linoleic acid(c9,t11-CLA).MUFAs and CLA have a range of positive health effects on reducing body fat,cardiovascular diseases,cancers and immune responses.As is known to all,milk and milk products contribute significantly to total SFAs in the human diet.If SCD1 activity was increased in the mammary gland,the total amount of unsaturated fatty acids(USFAs)could be increased,thus resulting in a decrease in the amount of saturated fatty acids(SFAs)in milk.The research of Scdl exclusively in transgenic mammary glands of dairy goats has not yet been reported.Thus,we selected Scdl as the research object.Firstly,the primary culture of dairy goat mammary epithelial cells(GMECs)were established and the enhanced green fluorescent protein(EGFP)reporter vector was construced to detect the function of milk synthesis in GMECs.Secondly,the dairy goat Scdl cDNA was amplified and the bioactivity of SCD1protein was evaluated in eukaryotic cells.Thirdly,the effect of Scdl on lipid metabolism related genes in GMECs was studied by overexpression and siRNA interference of Scdl.Finally,a mammary-specific expression vector of Scdl was constructed and the efficiency of this vector was proved in GMECs.The main results were as follows:1.Isolation of dairy goat mammary epithelial cells and development of a fluorescence reporter system Dairy goat mammary epithelial cells(GMECs)which retained their mammary gland specific function are useful model for understanding the development,differentiation and involution of the mammary gland and models of bioreactor.The aim of this study was to isolate and optimize culture conditions of GMECs and to develop an effective and convenient way to evaluate the secretory capacity of GMECs in primary culture.Firstly,the primary culture of GMECs were established by tissue culture.The effects of serum,ITS(Insuline-Transferrine-Selenium Solution)and insulin on cell proliferation,cell cycle synchronization,apoptosis and transfection efficiency were studied.Secondly,a reporter vector containing enhanced green fluorescence protein(EGFP),driven by the CSN2(Capra hircusβ-casein)promoter,was constructed,and introduced into GMECs by lipofectamine.Finally,to evaluate the homogeneity and secretory capacity of GMECs,we measured the expression of cytoskeletal proteins and theβ-casein protein by immunofluorescence and Western blot analysis,respectively.Taking the proliferation,cell cycle synchronization,apoptosis and transfection efficiency into consideration,the 10ITS group(10%FBS+1%ITS+5μg/mL Hydrocortisone +10 ng/mL EGF)was found to be the best suited for cell culture and was chosen for further experiments.The results of RT-PCR and EGFP fluorescence showed that the expression of EGFP gene can only be induced in GMECs.The immunofluorescence results showed that almost all of the epithelial cells revealed a strong positive fluorescence signal for cytokeratin 18,and only a few of GMECs were stained lightly by anti-vimentin antibody.GMECs reacted positively with an anti-β-casein antibody.The results showed that(1)GMECs retained their mammary gland specific function,which can be used as GMECs models;(2)The reporter system was an easy,convenient and efficient method to detect the secretory capacity of GMECs in primary culture.2.Research on Scdl clone and biological activity of the expressed products To botain the conding sequence and evaluate the biological activity of the expressed product of Scdl.In this study,the full length fragment of cDNA encoding Scdl was amplified from mammary gland tissue of a lactating dairy goat by RT-PCR.The fragment was inserted into the pMD(?)19-T vector,followed by sequencing.To study the localization of SCD1,the Scd1 sequence without termination codon was amplified and inserted into pNl-EGFP by XhoI and EcoRI sites.The fusion expression vector pN1-SCD1 was constructed and introduced into GMECs.A eukaryotic expression vector for dairy goat Scdl was constructed and introduced into dairy goat ear skin-derived fibroblast cells(GEFCs)to evaluate the activity of SCD1 by lipid analysis.The results showed that the cloned sequence was 100%homologous with the Scdl sequence available in Gen Bank.The fusion protein of SCD1-EGFP was localized to the cytoplasm.qRT-PCR analysis confirmed that the relative expression level of Scdl was 16.5-fold higher than that in control cells.Fatty acid analysis showed that Scd1-overexpression resulted in an increase in levels of palmitoleic acid(16:1 n-7,1.73±0.02%vs.2.54±0.02%,P<0.01)and oleic acid(18:1 n-9,27.25±0.13%vs.30.37±0.04%,P<0.01).In this study,the conding sequence of Scdl was obtained and the biological activity of SCD1 protein was confirmed in vitro.The research lays a foundation for studying the role of Scdl in fatty acid metabolism and the construction of Scdl mammary-specific vector.3.The effect of Scdl on lipid metabolism related genes There were numerous genes involved in the milk fat synthesis,decomposition and regulation in mammary gland,which formed a complex network.However,few studies about the effect of Scdl on genes related to lipid metabolism were reported.It aims to study the effect of Scdl on genes related to lipid metabolism in GMECs by overexpression and siRNA interference of Scd1.Firstly,the pCAG-SCD1 vector was constructed on the basis of pCAGGS vector and was identified by PCR,restriction enzyme digestion and sequencing.Secondly,the Scd1siRNA sequence was designed,synthesized and the highest interference sequence was selected.Finally,the pCAG-SCD1 vector and Scdl siRNA sequence were introduced into GMECs,and the mRNA expression levels of Scdl and lipid metabolism related genes were detected by qRT-PCR.The qRT-PCR results showed that the expression levels of triglyceride synthesis related genes(Agpat6,Dgat1)and the lipid droplet formation related genes(Adrp,Tip47)were significantly upregulated while Scd1overexpressed.However,the interference of Scd1,the expression levels of triglyceride synthesis related genes(Gpam)and the lipid droplet formation related genes(Adrp,Tip47)were significantly downregulated along with the interference of Scdl.These results indicated that the Scdl might involve in the regulation of triglyceride synthesis and lipid droplet formation related genes and have effect on the triglyceride synthesis and lipid droplet formation.The research lays a foundation for further studying the central role of Scdl in fatty acid metabolism.4.Construction and expression of Scd1 mammary specific-vector in dairy goat mammary epithelial cells If SCD1 activity was increased in the mammary gland,the total amount of unsaturated fatty acids(USFAs)could be increased,thus resulting in a decrease in the amount of saturated fatty acids(SFAs)in milk.The aim of this study was to develop an effective Scd1vector that was specifically expressed in dairy goat mammary glands.Firstly,the Scd1cDNA was inserted into pBC1 which contained goat β-casein promoter,and the obtained vector was named pBCl-SCD1.The selectable marker genes(Neo and EGFP)contained two Loxp sites with the same orientation for the deletion of marker genes from the genome on their ends,and they were cloned into pBCl-SCD1.The obtained vector was named as pBC1-SCD1-LINE and the vector was confirmed by enzymatic digestion and partial DNA sequencing.Secondly,GMECs were transiently transfected with pBC1-SCD1-LNIE and the expression of Scdl was analyzed by real-time PCR and Western blot analysis.The mammary gland-specific plasmid pBC1-SCD1-LNIE was successfully confirmed by PCR,restriction enzyme digestion and sequencing.qRT-PCR analysis showed a 14.1-fold increase in the expression of Scdl in T-GMECs compared with cells transfected with the pBCl-LINE control plasmid.Western blot analysis showed a 7-fold increase in the expression of SCD1 protein in T-GMECs In this study,the mammary-specific vector was constructed and Scdl could be effectively expressed in GMECs.Therefore,this work lays a foundation for both the preparation of Scdl donor cells and the generation of transgenic dairy goats which could produce more USFAs. |
