实时定量PCR构建法夫酵母内参基因β-actin、gpd、18S rRNA标准品质粒和标准曲线

Establishment of Standard Curves and Standard Plasmids for β-actin,gpd and 18S rRNA Genes of Phaffia rhodozyma Using Real-time PCR

为建立检测法夫酵母JMU-MVP14中虾青素合成相关基因在不同生长时期表达水平的实时定量PCR方法,构建法夫酵母JMU-MVP14的管家基因β-actin、gpd、18S rRNA的标准质粒,进行实时定量PCR,制作标准曲线及回归方程。β-actin基因标准曲线相关系数(R2)=0.9956,扩增效率(E)=96.93%;gpd基因标准曲线相关系数(R2)=0.9901,扩增效率(E)=93.78%;18S rRNA基因标准曲线相关系数(R2)=0.9981,扩增效率(E)=98.76%。3个基因片段的熔解曲线均呈单峰;扩增曲线呈典型的S型动力学曲线,指数期和平台期明显,为理想的熔解曲线和扩增曲线。用geNorm软件对三个管家基因的稳定性进行分析,三个基因的稳定性排序为β-actin>18S rRNA>gpd,故β-actin和18S rRNA较适合作为研究法夫酵母JMU-MVP14定量实验的内参基因。

To establish a method for detecting the expression levels of astaxanthin biosynthesis genes of Phaffia rhodozy- ma by Real-time quantitative PCR under the different growth phases, we constructed standard plasmids of housekeeping genes （β-actin,gpd, 18S rRNA）, and Constructed standard curves and regression equation through Real-time PCR. The standard curve correlation coefficient （ R2 ） of β-actin gene was 0. 9956, amplification efficiency （E） was 96.93 % ; the standard curve：correlation coefficient （ R2 ） of gpd gene was 0. 9901, amplification efficiency （E） was 93.78 % , and the standard curve coerelaron coefficient （R2） of 18S rRNA was 0. 9981 ,amplification efficiency （E） was 98.76 %. The linearity and amplification efficiency of the three genes were good. The dissociation curves of the three genes showed uni- modal respectively, and the three amplification curves were all presented as typical kinetics curves of ＂S＂, with clear exponential phases and plateau phases. Therefore, the melting curves and amplification curves of the three genes were i- deal. We used the geNorm software to analyze the stability of the reference genes. The order of the stability is ： ^-actin 〉 18S rRNA 〉gpd. Therefore, β-actin and 18S rRNA genes are more suitable as a reference gene for Real-time PCR in Phaffia rhodozyma strain JMU-MVP14.