ACTA VETERINARIA ET ZOOTECHNICA SINICA ›› 2012, Vol. 43 ›› Issue (7): 1028-1036.doi:

• 遗传繁育 • Previous Articles     Next Articles

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. 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

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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.