藜科植物藜和灰绿藜实时荧光定量PCR内参基因的选择

Screening of qRT-PCR reference genes of chenopodiaceae species -Chenopodium album L. and Chenopodium glaucum L.

关键词:  关键词：藜，灰绿藜，实时荧光定量PCR，内参基因，胁迫

Key words:  Key words: Chenopodium album, Chenopodium glaucum, quantitative real-time PCR (qRT-PCR), reference gene, stress

摘要:

摘 要：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。本研究利用GeNorm、NormFinder、BestKeeper三个内参基因分析软件对已选择过的β-tubulin、β-actin、GAPDH三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。GeNorm、NormFinder内参软件分析结果显示：在NaCl和PEG胁迫下，GAPDH是在是藜和灰绿藜中科植株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比较结果显示，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分析结果显示，内参基因的选择需根据实际的实验材料和实验条件而定。基于三个分析软件对以上三个常用内参基因的分析结果，本实验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，这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。

Abstract:

Abstract: 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 we compared three commonly used candidate reference genes β-actin, β-tubulin, and GAPDH of the stability in Chenopodium album and Chenopodium glaucum under NaCl and PEG treatments. The results showed that, under NaCl and PEG stress, β-actin expressed stable in C. album;while β-tubulin expressed stable in C. glaucum; GAPDH showed stable both in C. album and C. glaucum. The results suggests that same reference gene expressed stable in same species. For the same stress in two species, results showed that selection of the most stable reference gene depended on the experimental materials and the conditions. Taken together, our results may provide reference for further study in qRT-PCR analysis of stress-relevant gene expression in Chenopodiaceae species.