Selection of Reference Genes for qRT-PCR Analysis in Medicinal Plant Glycyrrhiza under Abiotic Stresses and Hormonal Treatments.

Yuping Li,Xiaoju Liang,Xuguo Zhou,Yongqing Li,Ying Wang,Ling Yuan,Zhigeng Wu

doi:10.3390/plants9111441

Abstract

Best known as licorice, Glycyrrhiza Linn., a genus of herbaceous perennial legume, has been used as a traditional herbal medicine in Asia and a flavoring agent for tobacco and food industry in Europe and America. Abiotic stresses and hormonal treatments can significantly impact the development and metabolism of secondary metabolites in Glycyrrhiza. To better understand the biosynthesis of the trace-amount bioactive compounds, we first screened for the suitable reference genes for quantitative real-time reverse transcription PCR (qRT-PCR) analysis in Glycyrrhiza. The expression profiles of 14 candidate reference genes, including Actin1 (ACT), Clathrin complex AP1 (CAC), Cyclophilin (CYP), Heat-shock protein 40 (DNAJ), Dehydration responsive element binding gene (DREB), Translation elongation factor1 (EF1), Ras related protein (RAN), Translation initiation factor (TIF1), β-Tubulin (TUB), Ubiquitin-conjugating enzyme E2 (UBC2), ATP binding-box transpoter 2 (ABCC2), COP9 signal compex subunit 3 (COPS3), Citrate synthase (CS), and R3H domain protein 2 (R3HDM2) from two congeneric species, Glycyrrhiza uralensis F. and Glycyrrhiza inflata B., were examined under abiotic stresses (osmotic and salinity) and hormonal treatments (Abscisic acid (ABA) and methyl jasmonic acid (MeJA)) using a panel of software, including geNorm, NormFinder, BestKeeper, and Delta CT. The overall stability, however, was provided by RefFinder, a comprehensive ranking system integrating inputs from all four algorithms. In G. uralensis, the most stable reference genes under osmotic stress, salt stress, ABA treatment, and MeJA treatment were TIF1, DNAJ, CS, and ABCC2 for leaves and DNAJ, DREB, CAC, and CAC for roots, respectively. In comparison, the top ranked genes were TUB, CAC, UBC2, and RAN for leaves and TIF1, ABCC2, CAC, and UBC2 for roots, respectively, under stress and hormonal treatments in G. inflata. ACT and TIF1, on the other hand, were the least stable genes under the most experimental conditions in the two congeneric species. Finally, our survey of the reference genes in legume shows that EF, ACT, UBC2, and TUB were the top choices for the abiotic stresses while EF, UBC2, CAC, and ABCC2 were recommended for the hormonal treatments in Leguminosae. Our combined results provide reliable normalizers for accurate gene quantifications in Glycyrrhiza species, which will allow us to exploit its medicinal potential in general and antiviral activities in particular.

Highlights

Medicinal plants have been documented as an important source for discovering new pharmaceutical molecules [1]
Abscisic acid (ABA) and methyl jasmonic acid (MeJA) are two important abiotic elicitors involved in plant responses to drought, salt or osmotic stresses [24], and secondary metabolism [25]
Our results showed the suitable reference genes were not universal among the three Glycyrrhiza species; Ubiquitin-conjugating enzyme E2 (UBC2) was the only optimal reference gene shared among G. glabra, G. uralensis, and G. inflata under the osmotic stress (Figure 5, Table 1)

Summary

Introduction

Medicinal plants have been documented as an important source for discovering new pharmaceutical molecules [1]. Included in the Pharmacopoeia of the People’s Republic of China, three congeneric species, Glycyrrhiza uralensis F., G. inflata B., and Glycyrrhiza glabra L. Glycyrrhizin, a glycoside and the major bioactive component, has showed promise against a broad spectrum of respiratory, hepatic, and systemic viral diseases. The eminent threats to public health and national security by global pandemics of viral diseases, including influenza; HIV; and recently, the two highly contagious respiratory diseases caused by coronaviruses, severe acute respiratory syndrome (SARS) and COVID-19, and the increasing issues with drug resistances (e.g., influenza and HIV) demand the development of effective antiviral alternatives with different mode of actions [3].

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Conclusion