从江香猪FoxO 4基因真核表达载体的构建及其在皮下脂肪前体细胞中的表达

Construction of Recombinant Eukaryotic Expression Vector of FoxO 4 Gene from Congjiang Xiang Pig and Its Expression in Subcutaneous Preadipocytes

本研究旨在克隆从江香猪FoxO 4基因编码区序列,对FoxO 4基因功能进行初步探究。采用qRT-PCR技术对从江香猪(6月龄)不同组织中FoxO 4基因的相对表达量进行检测;PCR扩增FoxO 4基因CDS区,构建真核表达载体pEGFP-N3-FoxO 4;利用脂质体法将重组质粒pEGFP-N3-FoxO 4瞬时转染从江香猪皮下脂肪前体细胞,通过qRT-PCR方法,检测FoxO 4、PPARγ、LPL、FAS、ACC、ATGL、HSL的表达水平。结果显示:FoxO 4基因在从江香猪脂肪组织中表达量最高;构建的重组真核表达载体pEGFP-N3-FoxO 4在从江香猪皮下脂肪前体细胞中成功表达,与对照组相比,LPL、FAS、ACC、ATGL、HSL基因的表达均显著上调。综上表明,FoxO 4对脂质代谢具有一定的调控作用,可能是影响从江香猪猪肉品质的重要候选基因,为进一步了解从江香猪脂肪沉积机制提供理论基础。

function of FoxO 4 gene. The relative expression level of FoxO 4 gene was detected by qRT-PCR in different tissues of Congjiang Xiang pig at the age of 6 month.The recombinant plasmid pEGFP-N3-FoxO4 was constructed by inserting the FoxO 4 gene coding sequence into pEGFP-N3, and then transiently transfected into the subcutaneous preadipocytes of Congjiang Xiang pig by liposome-mediated transfection.The expression levels of FoxO 4, PPARγ, LPL, FAS, ACC, ATGL, HSL in transfected cells were detected by qRT-PCR. The results showed that FoxO 4 gene had the highest expression level in adipose tissue. The constructed plasmid pEGFP-N3-FoxO4 was successfully expressed in subcutaneous preadipocytes. The expression level of LPL, FAS, ACC, ATGL and HSL genes were significantly up-regulated in the subcutaneous preadipocytes when compared to the control group.These results revealed that FoxO 4 gene had certain regulation on fat deposition and might be a key candidate gene for the pork quality, which will provide theoretical basis for further studying the mechanism of fat deposition.