广西三黄鸡PPARγ基因克隆及实时荧光定量PCR的建立

Cloning of peroxisome proliferator-activated receptors gama (PPARγ) gene in Guangxi Sanhuang chicken and its establish ment of real-time quantitative PCR

【目的】克隆广西三黄鸡过氧化物酶体增殖物激活受体γ(PPARγ)基因,建立该基因的实时荧光定量PCR,为后续开展广西三黄鸡PPARγ基因组织表达谱及品种间表达差异研究奠定基础。【方法】根据GenBank已公布的鸡PPARγ基因序列保守区域,用Oligo 6.0软件设计合成1对引物,以广西三黄鸡脂肪总RNA为模板,用RT-PCR从广西三黄鸡克隆PPARγ基因。以携带PPARγ基因片段的重组质粒为标准品,建立基于SYBR Green I染料法的实时荧光定量PCR标准曲线。【结果】广西三黄鸡PPARγ基因的编码区序列(CDS)长1428 bp,编码475个氨基酸;与GenBank已公布的鸡PPARγ基因(AF163811)参考序列比对,其同源性为99.7%,存在4个位点碱基突变,但均为无义突变。广西三黄鸡PPARγ基因的实时荧光定量PCR标准曲线方程为:y=-3.568x+28.50,扩增效率为1.907,实时荧光定量PCR扩增产物的溶解曲线峰单一。【结论】鸡PPARγ基因在进化中比较保守;建立的广西三黄鸡PPARγ基因实时荧光定量PCR是可行的。

[Objective ]The aim of the present study was to clone the peroxisome proliferator-activated receptor γ （PPARγ） gene and establish its real-time fluorescence quantification PCR in order to provide references for further research on tissue expression profile of PPARγ and expression differences among different varieties. [Method]In this experiment, according to the conservative region of PPARγ gene in Gallus gallus registered in GeneBank, a pair of primers was designed and composed by Oligo 6.0. With the total RNA from Guangxi Sanhuang chicken as the template, PPARγ gene was amplified using RT-PCR. The plasmids carrying partial sequence of PPARγ gene were used to establish the standard curve of real-time fluorescence quantitative PCR using the method of SYBR Green I. [Result]The overall length of cDNA sequence of PPARγ gene, cloned from Guangxi Sanhuang chicken, was 1428 bp with 475 amino acids. It had 99.7% homology with the reference sequence. There were 4 site nonsense mutations. The standard curve with only one peak was y= -3.568x＋28.50. The amplification efficiency was 1.907. [Conclusion]PPARγ gene was rather conservative during its evolution. It was feasible to establish the qRT-PCR method for the PPARγ, gene of Guangxi Sanhuang chicken.