Adenovirus-Mediated Over-Expression Of Stearoyl-CoA Desaturase (SCD) Gene In Goat Mammary Gland

Stearoyl-CoA desaturase (SCD) is the rate-limiting enzyme catalyzing the synthesis of unsaturatedfatty acids in milk, and also a key enzyme in the endogenous synthesis of conjugated linoleic acid (CLA) of ruminant meat and milk. Changing the fatty acid composition of goat milk by reasonable regulation of SCD gene expression is of great importance to the exploration of milk formation mechanism. In this research, SCD gene in Xinong Saanen goat mammary gland was amplified by RT-PCR and sub-cloned into shuttle vector pAdTrack-CMV after T/A cloning. The recombinant shuttle vector was linearized by Pme I digestion, and then translated into E.coli BJ5183, which had already contained adenoviral backbone vector pAdEasy-1, to attain homologous recombined adenovirus plasmid Ad-SCD. The reconstructed plasmid, after linearization using enzyme Pac I, was used to transfect HEK 293 cells, so that the virus be packed and amplified to obtain the high-titer adenovirus which was used to infect dairy goat mammary epithelial cells (MOI = 200) to investigate changes of fatty acid composition and several fatty acid metabolism-related genes after over-expression of SCD. The results of this study provide preliminary understanding of the molecular mechanisms for goat mammary gland fatty acid mechanism.The results of this research are as follows:(1)Goat mammary gland SCD gene had an open reading frame of 1080 nucleotides encoding 359 amino acids, the GenBank No. was GU947654. The homologies of nucleotide and peptide sequence of this SCD with sheep(GQ904712), bovine (AF188710), human (NM₀05063), and mouse (BC055453) were 98%, 95%, 87%, 82% and 100%, 94%, 86%, 83%. In our bioinformatic analysis, we found five transmembrane areas on the peptide that the SCD gene encodes, locating at amino acid residue 77⁹4, 101¹18, 217²37, 246²71 and 317³34; and a fatty acid desaturases family I signature appears at site 294-308. Besides, four casein kinaseâ…¡phosphorylation sites, five PKC phosphorylation sites, two N-myristoylation sites and two N-glycosylation sites were predicted in the peptide. The physical property analysis indicated that the molecular weights of goat SCD were 41.6 kDa and its isoelectric point ( pI ) was 9.19. No signal peptide sequence was found. In the end, it is a non secretory protein.(2)Quantitative Real-time PCR results showed that, goat SCD gene generally expressed in Liver, Lungs, Spleen, Heart, Kidneys, Mammary Gland, Small Intestine, Sebum, Rumen, Ovarian and Pituitary tissues, and highly expressed in Mammary Gland, Lung and Sebum, lowly expressed in Heart, Rumen and Ovary.(3)The recombinant adenovirus vector Ad-SCD was constructed through homologous recombination, and was packed in the HEK 293 cell to obtain mature adenovirus granule. The virus titer reached 5.82×108 PFU/mL after amplifying. The optimal MOI value of dairy goat mammary epithelial cells was obtained at 72h post-infection by adenovirus(MOI=200).(4)The results from Quantitative Real-time PCR and Western blotting showed that the expression of the SCD gene in goat mammary epithelial cells after the infection was nearly 16.62 folds higher than the control group. The GC-MS results showed that the content of saturated fatty acids C16:0 and C18:0 in the cells decreased by about 4.68% and 12.47% respectively after SCD gene over-expression, and content of the unsaturated fatty acids 9z-C16:1, 9z-C18:1, 11e-C18:1 and 9z,12z-C18:2 increased by about 9.35%, 4.38%, 2.59% and 0.83% respectively. Also, results from qRT-PCR showed that the mRNA expression level of FAS, A-FABP, LEPR and LXRαgene decreased by 49%, 60%, 54% and 22% respectively, in the meantime expression of PPAR-γand LPL gene were 7.30 and 2.82 times greater of those in the control group respectively.The recombinant adenovirus vector of goat SCD gene was successfully constructed, and high-titer recombinant virus preservation solution was obtained. There was a significant effect of SCD gene over-expression in dairy goat mammary epithelial cells which was post-infection by adenovirus(MOI=200), the total fatty acids content and the proportion of unsaturated fatty acids were increased. The study shows that SCD gene plays an important role in the regulation in unsaturated fatty acid synthesis.