珠子参实时荧光定量PCR体系的建立与优化

Establishment and optimization of real-time fluorescence quantitative PCR system for Panax japonicus var. major

实时荧光定量PCR是一种快速用于鉴定基因表达量的方法,广泛用于微生物、植物和动物基因表达分析中。通过选取合适的内参基因β-actin构建珠子参实时荧光定量PCR体系,为珠子参及其近缘种的基因表达分析奠定基础。主要从两个最基本的要素进行体系优化,即引物浓度和模板浓度。优化先从模板浓度开始,设置模板浓度梯度分别为120 ng/μL、24 ng/μL、4. 8 ng/μL、0. 96 ng/μL、0. 192 ng/μL,基于扩增曲线和扩增效率的计算,得出扩增效率最高的一组,即为最优模板浓度。然后进行引物浓度优化,在最优模板浓度基础上设计一系列的引物浓度分别为10μmol/L、5μmol/L、2. 5μmol/L、1. 25μmol/L、0. 625μmol/L,基于扩增曲线和扩增效率,确定最佳的引物浓度。实验结果表明,当模板浓度为120 ng/μL,引物浓度为10μmol/L时PCR的扩增效率最高。在珠子参中首次建立和优化了以β-actin基因作为内参基因的实时荧光定量PCR体系。

Real-time fluorescence quantitative PCR is a rapid method for the identification of gene expression,which is widely used in the analysis of gene expression in microorganisms,plants and animals.In this study,we selected the appropriate internal reference geneβ-actin to construct the real-time fluorescent quantitative PCR system of the rhizome of Panax japonicus var.major,which laid the foundation for the analysis of the gene expression of Panax japonicus var.major.This experiment was performed from two aspects of the PCR condition,primer concentration and template concentration.The optimization process was firstly conducted from the template concentration.The template concentration gradient was set as 120 ng/μL,24 ng/μL,4.8 ng/μL,0.96 ng/μL and 0.192 ng/μL,respectively.Based on the calculation of the amplification curve and amplification efficiency,the group with the highest amplification efficiency was the optimal template concentration.Then,primer concentration was optimized.Based on the optimal template concentration,a series of primer concentrations was designed,which was set as 10μmol/L,5μmol/L,2.5μmol/L,1.25μmol/L and 0.625μmol/L,respectively.The experimental results showed that the amplification efficiency was the highest when the template concentration was 120 ng/μL and the primer concentration was 10μmol/L.Thus,the optimal real-time quantitative PCR system for the rhizome of Panax japonicus var.major withβ-actin gene as the internal reference gene was established.