鸡Elovl2基因组织表达谱及脂代谢功能研究

Construction of Elovl2 Gene Overexpression Recombinant Plasmid and the Related Functions in Chicken

为探究Elovl2基因在鸡体内的生物学功能,对鸡ELOVL2蛋白的理化性质进行分析,通过RT-qPCR技术检测该基因在鸡各组织中的表达情况。将Elovl2基因过表达重组质粒与最佳干扰片段分别转染鸡LMH细胞系,通过RT-qPCR技术检测Elovl2基因及脂代谢相关基因mRNA水平的变化,利用GC-MS技术检测细胞内相关脂肪酸含量的变化。结果显示:鸡ELOVL2蛋白属于稳定蛋白,该基因mRNA主要表达于脑和肝脏组织。转染pcDNA3.1-Elovl2后,基因相对表达水平显著上升,过表达重组质粒构建成功。三组siRNA均显著抑制Elovl2基因表达,抑制效果最佳的为siRNA-681。抑制Elovl2基因表达后,pck1基因的表达量较对照组相比上升2倍,但二者差异不显著。过表达Elovl2基因后,各种脂肪酸含量均有所增加,而经RNAi技术抑制Elovl2基因表达后,各种脂肪酸则有不同程度减少,受其影响最明显的为C22∶1N9。试验结果为进一步研究鸡Elovl2基因的功能奠定了基础。

In order to explore the biological functions of Elovl2 gene in chicken,the physical and chemical properties of chicken ELOVL2 protein were analyzed,and the expression of Elovl2 gene in chicken tissues were detected by RT-qPCR technology.The Elovl2 gene overexpression recombinant plasmid and the best interference fragment were respectively transfected into chicken LMH cell,and the changes of Elovl2 gene and lipid metabolism-related gene mRNA levels were detected by RT-qPCR technology,and the changes of related fatty acid content in the cells were detected by GC-MS technology.The results showed that chicken ELOVL2 protein was a stable protein,and the mRNA of Elovl2 gene was mainly expressed in brain and liver tissues.After transfection of pcDNA3.1-Elovl2,the relative gene expression level increased significantly,and the overexpression recombinant plasmid was successfully constructed.All three groups of siRNA significantly inhibited the expression of Elovl2 gene,and siRNA-681 had the best inhibitory effect.After inhibiting the expression of Elovl2 gene,the expression of pck1 gene was increased 2 times compared with the control group,but the difference was not significant.After overexpression of Elovl2 gene,the content of various fatty acids increased,and after the RNAi technology inhibited the expression of Elovl2 gene,the content of fatty acids were decreased to varying degrees,and the most obvious effect was for C22∶1 N9.The results laid the foundation for further research on the function of chicken Elovl2 gene.