Wang Hao, Cai Qi-Zhong, Liu Lu, Yang Quan, Zhou Liang-Yun
Guangdong Engineering Research Center of Good Agricultural Practice, Comprehensive Development for Cantonese Medicinal Materials,Guangzhou Comprehensive Experimental Station of National Industrial Technology System for Chinese Materia Medica,Key Laboratory of State Administration of Traditional Chinese Medicine for Production, Development of Cantonese Medicinal Materials, School of Traditional Chinese Medicine, Guangdong Pharmaceutical University Guangzhou 510006, China.
Zhongguo Zhong Yao Za Zhi. 2021 Jan;46(1):80-85. doi: 10.19540/j.cnki.cjcmm.20201024.103.
To select suitable references gene of Polygonum multiflorum for gene expression analysis in different tissues, five candidate reference genes like Actin,GAPDH,SAND,PP2A,TIP41 were selected from the transcriptome data of P. multiflorum, then the specific primers were designed. The expression stability of the five reference genes in different tissues of P. multiflorum was analyzed by Real-time quantitative PCR through avilable analysis methods such as geNorm, NormFinder, BestKeeper, Delta CT and RefFinder, to ensure the reliability of the analysis results. The results showed that there were significant differences in the expression levels and stability of candidate genes in different tissues of P. multiflorum. Ct distribution analysis of the expression levels of candidate genes showed that the expression levels of Actin and GAPDH genes were relatively high in different tissues, while the expression levels of SAND, PP2A and TIP41 were lower. The stability of each candidate gene was analyzed by different methods. The results of geNorm analysis showed that the expression of PP2A and GAPDH was the most stable, the expression stability of SAND was the worst, the stability of PP2A was the highest in both NormFinder and Delta CT, the stability of SAND was the lowest, and the stability of Actin was the most stable in BestKeeper analysis. Through the comprehensive evaluation and analysis of the stability of candidate genes by RefFinder, it is concluded that the stability of PP2A gene is the highest, followed by GAPDH, Actin, TIP41, SAND, and SAND gene is the worst. Therefore, the PP2A gene is an ideal reference gene for the analysis of gene expression in different tissues of P. multiflorum.
为筛选出适合何首乌不同组织基因表达分析的内参基因，从何首乌转录组数据中选取肌动蛋白（Actin）、甘油醛-3-磷酸脱氢酶（GAPDH）、SAND、蛋白磷酸酶2A（PP2A）、TIP41-like蛋白（TIP41）5个候选内参基因，并设计特异性引物。采用实时荧光定量PCR技术，通过geNorm、NormFinder、BestKeeper、Delta CT和RefFinder等分析方法，分析5个内参基因在何首乌不同组织中的表达稳定性，以确保分析结果的可靠性。结果表明，候选基因在何首乌不同组织中的表达水平和稳定性存在显著差异。候选基因表达水平的Ct分布分析显示，Actin和GAPDH基因在不同组织中的表达水平相对较高，而SAND、PP2A和TIP41的表达水平较低。采用不同方法分析各候选基因的稳定性。geNorm分析结果显示，PP2A和GAPDH的表达最稳定，SAND的表达稳定性最差；NormFinder和Delta CT分析均显示PP2A的稳定性最高，SAND的稳定性最低；BestKeeper分析显示Actin的稳定性最稳定。通过RefFinder对候选基因稳定性进行综合评价分析，得出PP2A基因稳定性最高，其次是GAPDH、Actin、TIP41、SAND，SAND基因稳定性最差。因此，PP2A基因是何首乌不同组织基因表达分析的理想内参基因。
