秋石斛RT-qPCR内参基因的筛选与验证

Selection and Validation of Reference Genes for RT-qPCR in Phalaenopsistype Dendrobium Hybrid

为了筛选出秋石斛中稳定表达的内参基因用于实时荧光定量PCR(RT-qPCR)分析,以不同品种和不同发育阶段花芽(包括花瓣、唇瓣和萼片)为材料,使用geNorm、NormFinder和BestKeeper软件计算了常用的7个候选内参基因表达的稳定性,使用RefFinder对其稳定性进行综合评价,并对筛选出的内参基因进行了验证。结果表明,各内参基因在秋石斛花芽中的表达稳定性差异较大,在不同品种同一发育阶段的花芽中β-actin、TUA和GADPH表达最稳定;在花芽发育过程中β-actin、TUA和CYP表达最稳定;而对于花器官不同组织,β-actin、TUA和GADPH在萼片中,β-actin、CYP和PGK在花瓣中,CYP、TUA和PGK在唇瓣中表达最稳定。

In order to screen reference genes stably expressing in Phalaenopsis-type Dendrobium hybrid for analysis of real-time fluorescent quantitative PCR,the whole floral buds,including the sepals,petals and labellums,from various cultivars and the floral bud at different development stages of dendrobium were used as materials.The expression stability of seven candidate gene was calculated by three commonly used software(geNorm,NormFinder and BestKeeper),comprehensive stability rankings were merged by RefFinder and the selected reference genes were verified.The results suggested that the expression stability of each reference gene in dendrobium differed greatly.In floral buds of different cultivars at the same developmental stage,β-actin,TUA,and GADPH were the most stable reference genes.The expression ofβ-actin,TUA,and CYP were the most stable during the development of the floral buds.For different tissues of floral organ,β-actin,TUA,and GADPH were the most stable reference genes in the sepals,β-actin,CYP,and PGK were in the petals and CYP,TUA,and PGK were in the labellum.