Xu Huifen, Luo Jun, Li Fang, Yu Kang, Shi Hengbo, Li Jun, Lin Xianzi, Zhu Jiangjiang
College of Animal Science and Technology, Shaanxi Provincial Key Laboratory of Molecular Biology for Agriculture, Northwest A&F University, Yangling 712100, Shaanxi, China.
Sheng Wu Gong Cheng Xue Bao. 2012 Nov;28(11):1306-16.
The aim of the study was to construct a recombinant adenovirus overexpression vector for Sterol Regulatory Element Binding Protein-1 (SREBP-1) of Xinong Saanen dairy goat, and to detect its effect on genes related to fatty acid metabolism in goat mammary epithelial cells, to establish foundation for further study of its roles in metabolism of fatty acid synthesis and lactation. First, we designed primers based on the SREBP-1 gene sequence in GenBank for PCR amplification and inserted the sequence into shuttle vector pAdTrack-CMV. The recombinant plasmid pAdTrack-CMV-SREBP-1 linearized by Pme I was transformed into E. coli BJ5183 competence cell containing the backbone vector pAdEasy-1 to obtain recombinant vector pAd-SREBP-1 by homologous recombination. pAd-SREBP-1 was linearized by Pac I and transfected into HEK 293 cell. Then we infected goat mammary epithelial cells with recombinant adenovirus which was packaged in HEK 293 cell line. The results showed that the recombinant adenovirus vector containing SREBP-1 was successfully constructed, and the titer of virus was 10(9) U/mL. Compared with the control group, mRNA level of SREBP-1 increased by about 15 times after infected for 48 h and 30 times after infected for 72 h. Fatty acid synthase (FASN) and Acetyl-CoA carboxylase (ACC) was upregulated by almost 2 times. The expression level of Peroxisome proliferator activated receptorgamma (PPARgamma) increased by 1.5 times. Liver X receptoralpha (LXRalpha) and Adipose triglyceride lipase (ATGL) upregulated by 1.2 times compared with that of control. But Stearoyl-coenzyme A desaturase (SCD) had no obvious change. In conclusion, SREBP-1 can activate the expression of genes related to fatty acid synthesis in mammary epithelial cells of Xinong Saanen dairy goat, demonstrated a regulatory function on the fatty acid metabolism in goat mammary gland.
本研究旨在构建西农萨能奶山羊固醇调节元件结合蛋白-1（SREBP-1）重组腺病毒过表达载体，并检测其对山羊乳腺上皮细胞脂肪酸代谢相关基因的影响，为进一步研究其在脂肪酸合成代谢及泌乳中的作用奠定基础。首先，根据GenBank中SREBP-1基因序列设计引物进行PCR扩增，并将该序列插入穿梭载体pAdTrack-CMV。用Pme I线性化的重组质粒pAdTrack-CMV-SREBP-1转化至含有骨架载体pAdEasy-1的大肠杆菌BJ5183感受态细胞中，通过同源重组获得重组载体pAd-SREBP-1。用Pac I将pAd-SREBP-1线性化并转染至HEK 293细胞。然后用在HEK 293细胞系中包装的重组腺病毒感染山羊乳腺上皮细胞。结果表明，成功构建了含SREBP-1的重组腺病毒载体，病毒滴度为10(9) U/mL。与对照组相比，感染48 h后SREBP-1的mRNA水平增加约15倍，感染72 h后增加30倍。脂肪酸合酶（FASN）和乙酰辅酶A羧化酶（ACC）上调近2倍。过氧化物酶体增殖物激活受体γ（PPARγ）的表达水平增加1.5倍。肝X受体α（LXRα）和脂肪甘油三酯脂肪酶（ATGL）与对照组相比上调1.2倍。但硬脂酰辅酶A去饱和酶（SCD）无明显变化。综上所述，SREBP-1可激活西农萨能奶山羊乳腺上皮细胞中脂肪酸合成相关基因的表达，对山羊乳腺脂肪酸代谢具有调控作用。
