Identification and validation of stable reference genes for gene expression analysis in sword-leaf dogbane using quantitative reverse transcriptase polymerase chain reaction

Abstract

Sword-leaf dogbane (Apocynum venetum) is a traditional Chinese herb with increasingly recognized potential to enhance health, but no study of stable reference genes in this herb has been reported. Based on a homologous cloning strategy, we have successfully cloned five candidate reference genes from sword-leaf dogbane: glyceraldehyde-3-phosphate dehydrogenase (AvGAPDH), beta tubulin (AvbTUB), polyubiquitin (AvUBQ), elongation factor 1-alpha (AvEF1a), and actin (AvACTIN). Three distinct algorithms, geNorm, NormFinder, and BestKeeper, were used to estimate the expression stability of candidate reference primer pairs. We found that AvACTIN-2 and AvACTIN-3 presented the highest stability of expression in different tissue samples, and AvGAPDH-2 was most stable under salinity stress. In addition, we illustrated the application of these new reference genes by assaying the expression levels of two hyperoside biosynthesis terminal enzyme genes, flavonoid 3#-hydroxylase (F3#H) and flavonol synthase (FLS), under salinity stress. Our study is the first to report stable expression of internal reference genes in sword-leaf dogbane in multiple experimental sample sets.