中国美利奴羊(新疆型)皮肤组织GAPDH基因实时荧光定量PCR方法的建立

Development of Real - time PCR for GAPDH Gene in the Skin Tissues of Merino Sheep

【目的】建立GAPDH内参基因实时荧光定量PCR方法,为进一步研究中国美利奴羊(新疆型)相关基因的定量表达奠定基础。【方法】采用中国美利奴羊(新疆型)皮肤组织为试材,根据Genebank中绵羊GAPDH基因序列,设计并合成一对引物,建立基于SYBR Green I染料技术的Real-Time PCR检测体系,绘制出标准曲线并对其熔解曲线分析。【结果】以cDNA为模板建立的标准曲线循环阈值(Ct)与标准cDNA模板在一定浓度范围内呈良好的线性关系,GAPDH基因标准曲线中模板拷贝数(X)与Ct值的关系为Ct=一3.679 51g/X+35.648,相关系数R=0.999 8;试验重复性好,批内和批间变异系数(CV%)分别为0.22%～3.45%和1.30%～4.89%。【结论】所建立的方法具有快速、线性范围广、重复性强等特点,GAPDH可作为内参基因用于绵羊相关基因的定量表达研究。

[ Objectlve]The purpose of this project was to develop SYBR Green Ⅰ real -time PCR assays as an effective tools for the detection and quantification of expression of genes associated with important traits of Merino sheep. [ Method] In this study, a real -time PCR system was developed for GAPDH gene of Merino Sheep, in which the skin tissues were used as the experimental materials. They were designed and synthesized with the primer according to the sequence of GAPDH gene in Gene bank, and then the standard curve and melting curve were analyzed. [ Result ] The result suggested that the linear relationship between threshold cycle and eDNA was favourable in certain range, the standard curve was Ct = - 3. 679 51gX ＋ 35. 648, whose coefficient correlation was 0. 9998; they were highly reproducible, and had a coefficient of variation from 0.22% to 3.45% for intra - assay and from 1.30% to 4.89% for inter - assay. [ Conclusion]The high sensitivity, specificity and good reproducibility of the method indicated that GAPDH gene could be used as a reference gene for studying the expression of candidate genes in Merino Sheep.