Spatio-temporal selection of reference genes in the two congeneric species of Glycyrrhiza

Abstract

Glycyrrhiza, a genus of perennial medicinal herbs, has been traditionally used to treat human diseases, including respiratory disorders. Functional analysis of genes involved in the synthesis, accumulation, and degradation of bioactive compounds in these medicinal plants requires accurate measurement of their expression profiles. Reverse transcription quantitative real-time PCR (RT-qPCR) is a primary tool, which requires stably expressed reference genes to serve as the internal references to normalize the target gene expression. In this study, the stability of 14 candidate reference genes from the two congeneric species G. uralensis and G. inflata, including ACT, CAC, CYP, DNAJ, DREB, EF1, RAN, TIF1, TUB, UBC2, ABCC2, COPS3, CS, R3HDM2, were evaluated across different tissues and throughout various developmental stages. More importantly, we investigated the impact of interactions between tissue and developmental stage on the performance of candidate reference genes. Four algorithms, including geNorm, NormFinder, BestKeeper, and Delta Ct, were used to analyze the expression stability and RefFinder, a comprehensive software, provided the final recommendation. Based on previous research and our preliminary data, we hypothesized that internal references for spatio-temporal gene expression are different from the reference genes suited for individual factors. In G. uralensis, the top three most stable reference genes across different tissues were R3HDM2, CAC and TUB, while CAC, CYP and ABCC2 were most suited for different developmental stages. CAC is the only candidate recommended for both biotic factors, which is reflected in the stability ranking for the spatio (tissue)-temporal (developmental stage) interactions (CAC, R3HDM2 and DNAJ). Similarly, in G. inflata, COPS3, R3HDM2 and DREB were selected for tissues, while RAN, COPS3 and CS were recommended for developmental stages. For the tissue-developmental stage interactions, COPS3, DREB and ABCC2 were the most suited reference genes. In both species, only one of the top three candidates was shared between the individual factors and their interactions, specifically, CAC in G. uralensis and COPS3 in G. inflata, which supports our overarching hypothesis. In summary, spatio-temporal selection of reference genes not only lays the foundation for functional genomics research in Glycyrrhiza, but also facilitates these traditional medicinal herbs to reach/maximize their pharmaceutical potential.

Identification of the histone demethylases gene family in Glycyrrhiza inflata reveals genes responding to abiotic stresses

“…RR820D, Takara) on LightCycler 480 (Roche) according to the manufacturer's instructions. The 2 −ΔΔCT method 21 was used to normalize and calculate the expression level of each tested gene relative to internal reference genes ( R3HDM2 and COPS3 ) 22,23 . All the primers of GiHDMT genes are listed in Supporting Information: Table S1.…”

“…The 2 −ΔΔCT method 21 was used to normalize and calculate the expression level of each tested gene relative to internal reference genes (R3HDM2 and COPS3). 22,23 All the primers of GiHDMT genes are listed in Supporting Information: Table S1. Three biological replicates and three technical replicates were performed for each treatment.…”

Glycyrrhizae Radix et Rhizoma: The popular occurrence of herbal medicine applied in classical prescriptions

“…Factors affecting quality stability also include developmental stages and tissues. Moreover, spatial (tissue)-temporal (developmental stage) interaction conditions have been comprehensively considered (Li, Liang, et al, 2021). Another study of the selection of reference genes in Glycyrrhiza Linn.…”

“…Accurate gene quantification for Glycyrrhiza species has been achieved using reverse transcription quantitative real‐time PCR (RT‐qPCR), which provides a new identification method (Li, Liang, et al, 2020; Li, Liang, et al, 2021). This method requires constitutively expressed genes as references to regulate or standardise the expression of the target genes.…”

Histone Deacetylase GiSRT2 Negatively Regulates Flavonoid Biosynthesis in Glycyrrhiza inflata

“…RNA extraction and qRT-PCR were carried out as previously described [20]. GiCOPS3 was used as internal reference gene [21]. All primers used in qRT-PCR are listed in Table S1.…”