授粉后红球姜雌性生殖器官qRT-PCR的内参基因筛选

Screening of Reference Genes for qRT-PCR Analysis in Zingiber zerumet (L.) Smith Female Reproductive Organ after Pollination

  • 摘要: 在研究分析红球姜(Zingiber zerumet (L.) Smith)败育关键调控基因的表达中,筛选授粉后雌性生殖器官发育过程中的内参基因至关重要。本研究根据授粉后不同时间点的红球姜转录组数据库以及相关文献报道的传统内参基因,筛选出10个表达相对稳定的基因Actin-2(ACT2)、Actin-7(ACT7)、Beta tubulin-1(TUB1)、Beta tubulin-5(TUB5)、Alpha tubulin-3(TUA3)、Ubiquitin(UBQ)、Glyceraldehyde-3-phosphate dehydrogenase(GAPDH)、Elongation factor 1-alpha(EF-1)、Cyclophilin(CYP)、Histone(H2A)作为候选内参基因,采用qRT-PCR技术,结合GeNorm、NormFinder和BestKeeper软件对候选内参基因的表达稳定性进行分析。结果表明,在红球姜雌性生殖器官授粉后的发育过程中,GAPDH和UBQ的表达稳定性最好,均适合作为内参基因,且同时使用两种作为内参基因能使实时荧光定量PCR标准化分析结果更精确。因此,最终选择GAPDH和UBQ作为实时荧光定量PCR标准化分析红球姜雌性生殖器官相关基因表达的内参基因。本研究将为探究红球姜败育的分子机理奠定基础,也为近源姜属植物内参基因的筛选提供线索。

     

    Abstract: Screening reference genes of pollinated Zingiber zerumet (L.) Smith female reproductive organ at development process is crucial, when we study and analyze the expression of key regulatory genes for Zingiber zerumet (L.) Smith abortion. According to the transcriptome database of Zingiber zerumet (L.) Smith and researches about traditional reference genes, we selected 10 relatively stable genes as candidate reference genes, including Actin-2 (ACT2), Actin-7 (ACT7), Beta tubulin-1 (TUB1), Beta tubulin-5 (TUB5), Alpha tubulin-3 (TUA3), Ubiquitin (UBQ), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Elongation factor 1-alpha (EF-1), Cyclophilin (CYP), Histone (H2A), and then analyzed their expression stability by qRT-PCR and three statistic programs: GeNorm, NormFinder and BestKeeper. The results showed that GAPDH and UBQ are the most stable genes during the development process of pollinated Zingiber zerumet (L.) Smith female reproductive organ. These two genes were suitable to be reference genes, and it was more accurate for qRT-PCR normalization analysis to utilize two reference genes at the same time. Consequently, GAPDH and UBQ were able to be reference genes for qRT-PCR normalization analysis in pollinated Zingiber zerumet (L.) Smith female reproductive organ at different development stages. This work is contributable to lay a foundation to study the molecular mechanism of abortive in Zingiber zerumet (L.) Smith, and also acts as a resource for other Zingiber to screen reference genes.

     

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