内蒙古白绒山羊Hairless基因实时荧光定量PCR标准质粒和标准曲线的构建

Construction of Standard Plasmid and Standard Curve of Real-time PCR for Inner Mongolia Cashmere Goats Hairless Gene

根据GenBank中绵羊、牛的Hairless和β-actin基因编码区保守序列,设计特异性引物,提取皮肤组织总RNA,经反转录RT-PCR扩增,产物回收纯化后与pGM-T载体连接,转化大肠杆菌感受态细胞Top10,质粒提取后经酶切、PCR和测序鉴定,获得阳性Hairless、β-actin重组质粒;将阳性重组质粒按103～107拷贝/反应梯度稀释作为模板,进行实时荧光定量PCR,系统软件自动生成标准曲线及回归方程。结果显示,产物熔解曲线峰值单一,说明引物特异性高,且无引物二聚体;Hairless和β-actin基因标准曲线相关系数分别为r2=0.998、r2=0.999,说明线性关系好。重复性试验结果显示,所构建的重组质粒9次同条件扩增Ct值具有良好的重现性,且变异率小于6%(107拷贝/反应除外),说明标准曲线重复性好。试验成功构建了目的基因Hairless、内参基因β-actin的标准质粒和标准曲线,为实时荧光定量PCR检测绒山羊Hairless基因的mRNA表达水平奠定基础。

Pairs of specific primer were designed according to the conserved coding region of sheep,Bos taurus Hairless and β-actin genome from GenBank.We synthesized the first strand cDNA by reverse transcription PCR after isolated the RNA from the skin tissues.The amplification products were ligated into the pGEM-T vector after recycling and purification,and transformed into competent Top10 cells eventually.Plasmid DNA was purified and identified by restricted enzyme digestion,PCRamplification and sequencing.The positive plasmids enter into the real-time PCR steps after gradient dilution from 103 to 107.The standard curve and regressive curve were generated automatically by the DNA continuous fluorescence detection system.The results showed that no specific amplicons was found according to the derived melting curve of the PCR products, indicating the specification of the present primers.The correlation coefficients of the standard for the Hairless and β-actin genes were 0.998 and 0.999, respectively, suggesting the strong linear relationship between the two groups.The results of repeatability experiments showed that the Ct values hold well for the recombinant plasmids with nine times PCR amplification under the same condition,and the coefficient of variation（CV）was less than 6% among groups（except for 107 copies/reaction）,suggesting the great repeatability and stability of the standard curve.Therefore, the construction of the standard plasmid and standard curve established the foundation of the procedures focusing on the quantitation of Cashmere goats Hairless gene mRNA expression in skin tissues.