珠子参实时荧光定量PCR体系的建立与优化
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摘要
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 gene β-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 10 μmol/L,5 μmol/L,2. 5 μmol/L,1. 25 μmol/L and 0. 625 μmol/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 10 μmol/L. Thus,the optimal real-time quantitative PCR system for the rhizome of Panax japonicus var. major with β-actin gene as the internal reference gene was established.
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 gene β-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 10 μmol/L,5 μmol/L,2. 5 μmol/L,1. 25 μmol/L and 0. 625 μmol/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 10 μmol/L. Thus,the optimal real-time quantitative PCR system for the rhizome of Panax japonicus var. major with β-actin gene as the internal reference gene was established.
引文
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[15]苏晓娟,樊保国,袁丽钗,等.实时荧光定量PCR分析中毛果杨内参基因的筛选和验证[J].植物学报,2013,48(05),5075518.
[2]张志清,曹蕾,宋亮,珠子参类药物历史沿革与应用展望[J].云南中医中药杂志,2012(09):68-71.
[3]赵仁,赵毅,李东明,等.珠子参研究进展[J].中国现代中药,2008(07):3-6.
[4]赵新礼.珠子参生殖生物学特性的研究[J].中国现代中药,2015(12):1298-1301.
[5]郭乔仪,鲁菊芬,普荣,等.中海拔地区珠子参引种栽培生物学特性[J].江苏农业科学,2016(11):209-211.
[6]赵东东,宋小妹,汤海峰,等.珠子参叶的皂苷成分研究[J].中南药学,201302:85-88.
[7]何瑞,刘琦,刘银环,等.珠子参叶化学成分研究[J].中国中药杂志,2014(09).
[8]包旭,詹常森,肖倬殷.近十年来珠子参的皂甙成分及药理研究进展[J].华西药学杂志,1992(01):49-52.
[9]陈涛,陈茂华,胡月琴,等.珠子参多糖提取及抗癌活性研究[J].中国中药杂志,2010(07):9125914.
[10] LegoffJ,Bouhlal H,Gresenguet G,et al. realtime PCR quantification of genital shedding of HSV and HIV in women coinfected with HSV and HIV[J]. Journal of Clinical Microbiology,2006,44(02):423-432.
[11] Hong-Keun Choi,Jun Wen. A phylogenetic analysis of Panax(Araliaceae):Integrating cpDNA restriction site and nuclear r DNA ITS sequence data[J]. Plant Systematics and Evolution. 2000,224(1-2):109-120.
[12]吴亚运,赵小龙,陈平,等.珠子参β-香树素合成酶基因的克隆和生物信息学分析[J].生物技术通报,2017(02):109-117.
[13]陈涛,陈茂华,胡月琴,等.珠子参多糖提取及抗癌活性研究[J].中国中药杂志,2010(07):912-914.
[14]董恩妮,梁青,李利,等.实时荧光定量PCR内参基因的选择[J].中国畜牧杂志,2013(11):92-96.
[15]苏晓娟,樊保国,袁丽钗,等.实时荧光定量PCR分析中毛果杨内参基因的筛选和验证[J].植物学报,2013,48(05),5075518.