花鲈PPARα基因实时荧光定量PCR质粒标准品的构建

Construction of plasmid standard for detecting Lateolabrax japonicus PPARα gene by real-time fluorescence quantitative PCR

利用SYBR荧光定量PCR技术，制备用于花鲈（Lateolabrax japonicus）PPARα基因实时荧光定量PCR检测的质粒标准品．通过PCR扩增出目的片断344-bp，纯化后连接至pMDI8-T载体，转化宿主菌E．coli DH5α．通过PCR鉴定、双酶切鉴定和测序分析后抽提重组质粒，测定其拷贝浓度，10倍梯度稀释成标准品模板10^8～10^4 copy／mm^3，进行荧光定量PCR分析．结果显示，循环阈值（Ct）与起始模板量的对数值之间有着良好的线性关系．

This experiment constructs a standard of recombinant plasmid for Lateolabrax japonicus PPARα gene detected with real- time quantitative PCR based on SYBR-Green fluorescence. A 344-bp DNA fragment was amplified by PCR, cloned into pMD18-T vector and transformed into bacterium DH5α. After PCR identification, double enzyme digestion and DNA sequencing, the recombinant plasmid was extracted. The concentration of purified plasmid DNA solution was determined and diluted into standard of 10^8 to 10^4 copy/mm^3 by ten-fold serially. As results standard curves constructed by real-time PCR detection with a series of plasmid standards show a high linear relationship between cycle threshold （Ct） and template concentration.