略阳乌鸡METTL21C基因克隆及超表达载体构建

旨在克隆略阳乌鸡 METTL21C基因,构建其超表达载体。采用实时荧光定量PCR(qPCR)技术检测 METTL21C基因在略阳乌鸡7种组织中的表达,通过PCR扩增技术克隆 METTL21C基因完整的CDS区序列,分析其核苷酸序列及不同物种序列进化关系,将 METTL21C基因片段连接至pCD513B载体,构建其超表达载体并转染至293T细胞。结果显示: METTL21C基因在略阳乌鸡心肌中表达量最高,骨骼肌次之;克隆获得略阳乌鸡 METTL21C基因完整编码区序列,共747 bp,编码249个氨基酸残基,与原鸡的相应序列具有很高的同源性;构建的 METTL21C超表达载体能转染293T细胞并成功表达,可直接用于后续 METTL21C基因功能探索的研究。

METTL21C Gene Cloning and Overexpression Vector Construction in Lueyang Black-bone Chicken

The aim of this study was to clone METTL21C gene of Lueyang black-bone chicken and construct its overexpression vector. The tissue expression patterns was performed by real time fluorescence quantitative PCR (qPCR). The whole coding sequence (CDS) of METTL21C gene was cloned by PCR, and analyzed the evolution relationship in different species. Lentiviral overexpression vector pCD513B was inserted in the METTL21C gene fragment, then was transfected into 293T cell. The results showed that the expression level of METTL21C gene mRNA was highest in myocardium, followed by skeletal muscle. Meanwhile, a complete coding region of METTL21C gene was cloned from Lueyang black-bone chicken, which contained 747 bp coding 249 amino acids, and had high homology with Gallus gallus.The overexpression vector of METTL21C was transfected into 293T cells, and METTL21C was successfully expressed. Our research could lay a foundation for further exploration of METTL21C gene function.