miR-200a对奶山羊乳腺上皮细胞乳脂合成相关基因mRNA表达的影响

摘要

旨在构建pAd-pri-miR-200a重组腺病毒,使其在奶山羊乳腺上皮细胞中稳定表达miR-200a,研究miR-200a对乳脂合成相关基因mRNA表达的影响.以西农萨能羊DNA为模板扩增pri-miR-200a.采用Ad-Easy系统构建重组腺病毒载体pAd-pri-miR-200a,并于HEK 293细胞株中进行病毒的包装、扩繁.TCI50法测定病毒滴度.qRT-PCR检测miR-200a及16个乳脂合成相关基因mRNA表达水平.序列分析结果显示,pri-miR-200a包括pre-miR-200a 86 bp和侧翼序列共297 bp.酶切结果证实pAd-pri-miR-200a构建成功.Ad-pri-miR-200a滴度达8×109 PFU· mL-1.实时定量结果表明,Ad-pri-miR-200a(MOI为200)感染奶山羊乳腺上皮细胞72 h,miR-200a的表达量较对照高出2.4倍.同时miR-200a的过表达引起10个基因mRNA表达量下调,6个基因mRNA表达量上调.其中脂肪酸从头合成相关基因FASN、脂滴生成相关基因TIP47及脂肪酸运输相关基因FABP4降幅较大,分别下降了0.47、0.89及0.65倍.而TAG生成相关基因DGAT1和脂解相关基因HSL较对照分别增加了0.52和1.49倍.本研究获得的重组腺病毒Ad-pri-miR-200a能在山羊乳腺上皮细胞中稳定表达miR-200a,同时miR-200a过表达影响乳脂合成相关基因mRNA表达水平.%To study the effect of miR-200a on mRNA expression of genes related to milk fat synthesis, recombinant adenovirus pAd-pri-miR-200a was constructed and miR-200a was stably expressed in goat mammary gland epithelial cell. Pri-miR-200a was amplified from DNA from Xinong Saanen Goat. Recombinant plasmid pAd-pri-miR-200a was constructed using Ad-Easy System. The virus was packaged and amplified in HEK 293 cells. Virus titer was indentified by TCI50 assay. The expression of miR-200a and 16 genes related to milk fat synthesis in dairy goat mammary gland epithelial cells were analyzed by qRT-PCR. Sequencing analysis showed that the length of pri-miR-200a was 297 bp, including 86 bp pre-miR-200a and flank sequences. Enzyme digestion analysis demonstrated that the recombinant adenouvirus was constructed successfully and the virus titer reached T = 8× 109 PFU · mL-1. The results of qRT-PCR showed that the expression of miR-200a in cell infected by Ad-pri-miR-200a (MOI was 200) was 2. 4 times more than that of the control. Expression of 10 genes mRNA were down regulated and 6 genes mRNA were up regulated. There was remarkable decrease in expression level of FASN (involved in de novo fatty acid synthesis) , TIP47(involved in lipid drop formation) and FABPA (involved in fatty acid transportation). These genes mRNA were 0. 47, 0. 89 and 0. 65 times less compared with that of control, respectively. The expression of DGAT1 (involved in TAG synthesis) and HSL (involved in lipolysis) mRNA were 0. 52 and 1. 49 times more than that of the control. In conclusion, miR-200a over-expression by Ad-pri-miR-200a could affect gene mRNA level related to milk fat synthesis in goat mammary gland epithelial cells.