西红花实时荧光定量PCR内参基因筛选

Screening of Reference Genes Based on Real-Time Quantitative PCR Analysis in Saffron(Crocus sativus)

为筛选西红花不同组织间稳定表达的最佳内参基因,以西红花的根、球茎、主芽、花丝、叶片、侧芽共6个组织为试验材料,运用实时荧光定量PCR方法分析了18S核糖体RNA (18S rRNA)、肌动蛋白基因(ACT)、转录延伸因子-1α(EF1α)、3-磷酸-甘油醛脱氢酶基因(GAPDH)、微管蛋白基因(TUB)和多聚泛素基因(UBQ)共6个内参基因在不同组织中的表达差异情况。基于Ct值,利用geNorm、NormFinder和BestKeeper分析6个内参基因表达稳定性。结果表明,3个软件筛选出的最佳内参基因略有不同。综合分析结果显示,GAPDH和UBQ表达较为稳定,其次为18S rRNA和ACT,而EF1α和TUB不适合作为西红花不同组织的内参基因。本研究确定西红花q RT-PCR分析的合适内参基因为GAPDH和UBQ,可用于种球发育、花发育、次生代谢产物形成和积累等机理研究。

In order to identify the suitable reference genes for the gene expression analysis in Crocus sativa, the expression of 6 housekeeping genes, including 18 S rRNA, ACT, EF1α, GAPDH, TUB and UBQ were assessed by using real time quantitative PCR in roots, corms, main buds, filaments, leaves and lateral buds of Crocus sativa.Based on the Ct value, the stability of 6 reference genes was analyzed by using geNorm, NormFinder and BestThe results showed that the suitable reference genes screened by the three softwares were slightly different. The results of comprehensive analysis showed that the expression of GAPDH and UBQ was stable, followed by 18 S rRNA and ACT, while EF1α and TUB were not suitable for different tissues of saffron. In this study, we determined that the suitable reference genes for saffron q RT-PCR analysis were GAPDH and UBQ, which can be used for the study of bulb development, flower development, secondary metabolite formation and accumulation.