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 (AvEF1α), 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.