藜科植物藜和灰绿藜实时荧光定量PCR内参基因的选择

选择合适的内参基因是实时荧光定量PCR(qRT-PCR)研究的关键,目前对藜科耐盐(盐生)植物胁迫相关基因的表达分析中所用内参基因的报道较为有限.该研究利用GeNorm、NormFinder和BestKeeper 3个内参基因分析软件,对已选择过的β-TUBULIN、β-ACTIN、GAPDH 3个常用候选内参基因进行了比较分析,筛选出在NaCl和PEG胁迫下藜和灰绿藜中表达相对稳定的内参基因.结果表明:GeNorm、NormFinder内参软件分析在NaCl和PEG胁迫下,GAPDH是藜和灰绿藜中均共同稳定表达的内参基因,同时在藜和灰绿藜中也有各自表达较稳定的内参基因,β-ACTIN在藜中稳定表达,β-TUBULIN则在灰绿藜中稳定表达.对相同科不同种的植物内参基因表达差异进行比较,内参基因在相同科中具有相同稳定表达的内参;对相同胁迫下两种不同植物内参基因表达稳定性进行分析,内参基因的选择需根据实际的实验材料和实验条件而定.基于3个分析软件对以上3个常用内参基因的分析结果,初步确定了在藜科植物藜和灰绿藜中相对稳定的内参基因,为藜和灰绿藜胁迫相关基因的定量表达分析提供了参考依据.

Screening of qRT-PCR reference genes for Chenopodium album and C. glaucum of Chenopodiaceae

Selection of suitable reference gene is a critical step in real-time quantitative PCR (qRT-PCR) analysis. So far, reports on reference gene screening on stress-tolerant gene expression of Chenopodiaceae species are limited. In the present study, by using three reference-gene-analysis softwares-GeNorm, NormFinder, BestKeeper, the stability of three commonly used candidate reference genesβ-ACTIN,β-TUBULIN and GAPDH of Chenopodium album and C. glaucum under NaCl and PEG treatments were compared. The results showed that under NaCl and PEG stresses,GAPDH showed stable both in C. al-bum and C. glaucum. The results also demonstated that the same reference genes expressed stable in the same family.β-AC-TIN expressed stable in C. album, whileβ-TUBULIN expressed stable in C. glaucum. We also studied the plants of different gera in the same family and the same stress of the stability of the expression of reference genes. The results suggested thatsame reference gene expressed stable in the same species. For the same stress in these two species, the results showed that selection of the most stable reference gene depended on the experimental materials and the conditions. The analysis results of the three candidate reference genes were based on the three analysis softwares. In our study, we determined the ralatively stable reference genes in C. album and C. glaucum. This study provides the reference for the further study on qRT-PCR analysis of stress-relevant gene expression in Chenopodiaceae species.