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

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-1α)、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,同时使用2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.该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.

Screening of reference genes of pollinated Zingiber zerumet （ L. ） Smith female reproductive organ at development process is crucial for analyzing the expression of key regulatory genes for Zingiber zerumet （ L. ） Smith abortion. According to the transcriptome database of Z. zerumet Smith and researches on 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-1α）, Cyclophilin （CYP）, and Histone （H2A）, and 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 Z. zerumet Smith female reproductive organ. These two genes were suitable to be reference genes, and it was more accurate for qRT-PCR normalization analysis using these two reference genes at the same time. This work indicated that GAPDH and UBQ are effective reference genes for qRT-PCR normalization analysis in pollinated Z. zerumet Smith female reproductive organ at different development stages, which can be used for the study of molecular mechanism of abortive in Z. zerumet Smith, and for the screening of reference genes in other Zingiber species.