滇重楼和长柱重楼实时荧光定量PCR内参基因的筛选和验证

Screening and validation of reference genes for real-time fluorescence quantitative PCR in Paris polyphylla var.yunnanensis and P.forrestii

重楼是传统中药,具有重要的药用和经济价值。筛选稳定可靠的内参基因是应用实时荧光定量PCR(qRT-PCR)技术分析重楼基因表达模式的前提。本研究基于滇重楼和长柱重楼的转录组数据,筛选出GAPDH、ACT、α-TUB、UBC、UBQ、PP2A、β-TUB、EF-1α共8个常用的管家基因作为候选内参基因,利用qRT-PCR分别检测上述基因在滇重楼和长柱重楼不同组织部位(根茎、茎和叶)中的表达水平。用geNorm、Normfinder、BestKeeper三个程序以及RefFinder网站综合分析各个候选内参基因的稳定性,并对筛选出的内参基因稳定性进行验证。结果显示,三种软件基于不同指标进行稳定性评估,结果并不相同,综合分析得出滇重楼中综合稳定性最好的是ACT,其次为UBQ;长柱重楼中稳定性最好的是GAPDH,PP2A次之。以ACT、UBQ、GAPDH和PP2A作为内参基因分别定量SMO1基因在滇重楼和长柱重楼不同组织部位的表达量,滇重楼中ACT和UBQ、长柱重楼中GAPDH和PP2A标准化SMO1基因的表达量时均显示出一致的相对表达水平,稳定性较差的内参基因不能有效地标准化目的基因的表达。综上,本研究发现ACT和UBQ、GAPDH和PP2A分别是滇重楼和长柱重楼基因表达分析适宜的内参基因,为后期探索这两种植物不同部位有效成分积累过程中基因的功能调控和差异表达奠定了基础。

Paris polyphylla is a traditional Chinese medicine with significant medicinal and economic value.Screening stable and reliable reference genes is the premise of applying quantitative real-time PCR(qRTPCR)technology to analyze gene expression pattern of P.polyphylla.In this study,based on the transcriptome data of P.polyphylla var.yunnanensis and P.forrestii,eight commonly used housekeeping genes including GAPDH,ACT,α-TUB,UBC,UBQ,PP2A,β-TUB and EF-1αwere selected as candidate reference genes.The expression levels of each candidate reference gene in different tissues(rhizome,stem and leaf)of P.polyphylla var.yunnanensis and P.forrestii were detected by qRT-PCR.The stability of each candidate reference gene was analyzed by geNorm,Normfinder,BestKeeper and RefFinder,and the stability of the selected reference genes was verified.The results showed that the stability of the three softwares are evaluated based on different indicators,and the results were not the same.The comprehensive analysis showed that the best comprehensive stability was ACT,followed by UBQ in P.polyphylla var.yunnanensis.GAPDH had the best stability,followed by PP2A in P.forrestii.ACT,UBQ,GAPDH and PP2A were used as internal reference genes to quantify the expression of SMO1 gene in different tissues of P.polyphylla var.yunnanensis and P.forrestii.The expression levels of ACT and UBQ in P.polyphylla var.yunnanensis and GAPDH and PP2A in P.forrestii showed consistent relative expression levels,and the internal reference genes with poor stability could not effectively standardize the expression of the target gene.In summary,this study found that ACT or UBQ,GAPDH or PP2A were suitable reference genes for the study of gene expression in P.polyphylla var.yunnanensis and P.forrestii,respectively.This study laid a foundation for the study of functional regulation and differential expression of genes in the accumulation of active components in different parts of these two plants.