京海黄鸡钠氢离子交换泵1基因实时荧光定量PCR标准曲线的建立

Establishment of Standard Curves for Detection of Na^+/H^+ Exchanger 1 Gene in Jinghai Yellow Chicken Using Real-time PCR

为阐明钠氢离子交换泵1(Na+/H+exchanger 1,NHE1)基因在京海黄鸡组织表达和生理功能,依据GenBank登录的NHE1基因的核苷酸序列(登录号:DQ256198)设计特异性引物,经RT-PCR技术从京海黄鸡组织样中扩增和克隆了上述因子的204bp核苷酸片段。测序鉴定后,将重组质粒10倍系列稀释后作为标准模板,通过实时荧光定量PCR方法,建立了它们各自的标准曲线及直线回归方程,得到了相关系数R2=0.9903、回归方程为y=-3.4643x+35.9000的标准曲线,成功构建了NHE1基因的标准质粒和标准曲线,为京海黄鸡组织中NHE1mRNA水平的定量检测提供了必要的技术手段。

The purpose of this study was to elucidate the Na＋/H＋ exchanger 1 （NHE1） gene expression and its normal physiological functions. The primers were designed and synthesized based on the nucleotide sequences of NHE1 gene available in GenBank. The 204 bp fragments were amplified by RT-PCR from mRNA of Jinghai Yellow chickens, and then the frag- ments were cloned and sequenced. The recombinant plasmids were diluted by 10-fold serial and used as the Real-time PCR standard templates. The constructed standard curve had a relative coefficient R2= 0. 9903 and the regressing equation： y =- 3. 4643x＋35. 9000 suggested it was successful, and they provided powerful tools for quantification of mRNA of NHE1 gene in Jinghai Yellow chickens.