山羊SREBP-1基因的超表达对脂肪酸代谢相关基因表达的影响

Effects of SREBP-1 over-expression on fatty acid metabolism related genes expression in goats

摘要:

本研究旨在通过构建西农萨能羊固醇调节元件结合蛋白-1 (SREBP-1) 基因的重组腺病毒超表达载体，获得有感染性的病毒颗粒，并检测该基因过表达后对乳腺上皮细胞中脂肪酸代谢相关基因的影响，为进一步研究该基因在脂肪酸代谢及泌乳调控中的功能奠定基础。根据GenBank中收录的西农萨能羊SREBP-1基因的序列设计引物，PCR扩增后进行测序。将测序正确的目的基因连接到穿梭载体pAdTrack-CMV后并进行线性化，转化含有腺病毒骨架载体pAdEasy-1的大肠杆菌Escherichia coli BJ5183感受态细胞中进行同源重组，将重组成功的质粒进行PacⅠ酶线性化，转染HEK 293细胞进行重组腺病毒的包装、扩繁及滴度测定。病毒液感染原代乳腺上皮细胞，实时荧光定量检测SREBP-1超表达效果及对脂肪酸代谢相关基因的影响。结果表明：重组腺病毒质粒构建成功，腺病毒滴度为109 U/mL。病毒液感染乳腺上皮细胞48 h后，SREBP-1基因表达量上升大约15倍，72 h后，表达量上调了30倍；对脂肪酸代谢相关基因的影响在72 h较为明显，其中，脂肪酸合酶 (FASN) 及酰基辅酶A羧化酶 (ACC) 均显著上调了大约两倍，过氧化物酶体增殖物激活受体 (PPARγ) 上调了大约1.5倍，肝素X受体 (LXRα) 及甘油三酯水解酶 (ATGL) 表达量升高了1.2倍；其中硬脂酰辅酶A去饱和酶 (SCD) 表达水平无明显变化。表明在西农萨能羊乳腺上皮细胞中，SREBP-1能够促进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。

Abstract:

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 PmeⅠ 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 PacⅠ 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 109 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 receptorγ (PPARγ) increased by 1.5 times. Liver X receptorα (LXRα) 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.