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

畜牧兽医学报 ›› 2012, Vol. 43 ›› Issue (7): 1028-1036.doi:

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

林先滋1，罗军1*，张犁苹1，朱江江1，石恒波1，苟德明2

1. 西北农林科技大学动物科技学院陕西省农业分子生物学重点实验室，杨凌 712100；2. 深圳大学生命科学学院，深圳 518060

收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2012-07-27 发布日期:2012-07-27
通讯作者: 罗军

The Effect of miR200a on Gene mRNA Expression Related to Milk Fat Synthesis in Dairy Goat Mammary Gland Epithelial Cells

LIN Xianzi1, LUO Jun1*, ZHANG Liping1, ZHU Jiangjiang1,SHI Hengbo1,GOU Deming2

1. Shanxi Provincial Key Laboratory of Agricultural Molecular Biology, College of Animal Science and Technology, Northwest A＆F University, Yangling 712100, China; 2. College of Life Sciences, Shenzhen University, Shenzhen 518060, China

Received:1900-01-01 Revised:1900-01-01 Online:2012-07-27 Published:2012-07-27
Contact: LUO Jun

摘要/Abstract

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。

Abstract:

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 FABP4 (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.

林先滋;罗军;张犁苹;朱江江;石恒波;苟德明. 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[J]. 畜牧兽医学报, 2012, 43(7): 1028-1036.

LIN Xianzi;LUO Jun;ZHANG Liping;ZHU Jiangjiang;SHI Hengbo;GOU Deming. The Effect of miR200a on Gene mRNA Expression Related to Milk Fat Synthesis in Dairy Goat Mammary Gland Epithelial Cells[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2012, 43(7): 1028-1036.

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