Stable Internal Reference Genes for Normalizing Real-Time Quantitative PCR in Baphicacanthus cusia under Hormonal Stimuli and UV Irradiation, and in Different Plant Organs.

Yuxiang Huang,Hexin Tan,Yong Diao,Qinglei Zhang,Lei Zhang,Junfeng Chen,Yue Chen,Zhiying Guo,Jian Yu,Guoquan Wang

doi:10.3389/fpls.2017.00668

Abstract

Baphicacanthus cusia (Nees) Bremek, the plant source for many kinds of drugs in traditional Chinese medicine, is widely distributed in South China, especially in Fujian. Recent studies about B. cusia mainly focus on its chemical composition and pharmacological effects, but further analysis of the plant's gene functions and expression is required to better understand the synthesis of its effective compounds. Real-time quantitative polymerase chain reaction (RT-qPCR) is a powerful method for gene expression analysis. It is necessary to select a suitable reference gene for expression normalization to ensure the accuracy of RT-qPCR results. Ten candidate reference genes were selected from the transcriptome datasets of B. cusia in this study, and the expression stability was assessed across 60 samples representing different tissues and organs under various conditions, including ultraviolet (UV) irradiation, hormonal stimuli (jasmonic acid methyl ester and abscisic acid), and in different plant organs. By employing different algorithms, such as geNorm, NormFinder, and BestKeeper, which are complementary approaches based on different statistical procedures, 18S rRNA was found to be the most stable gene under UV irradiation and hormonal stimuli, whereas ubiquitin-conjugating enzyme E2 was the best suitable gene for different plant organs. This novel study aimed to screen for suitable reference genes and corresponding primer pairs specifically designed for gene expression studies in B. cusia, in particular for RT-qPCR analyses.

Highlights

Baphicacanthus cusia (Nees) Bremek (Figure S1), widely distributed in Southern China, is the only plant belonging to the family Acanthaceae
They were glyceraldehyde3-phosphate dehydrogenase (GAPDH), malate dehydrogenase (MDH), ubiquitin 10 (UBQ), ubiquitin-conjugating enzyme E2 (UBC), actin (ACT), 18S rRNA (18S), elongation factor 1-alpha (EFa), cyclophilin (CYP), alpha-tubulin (TUBa), and beta-tubulin (TUBb) housekeeping genes, and these genes have all been reported as stable genes in other species
The full-length unigene sequences from transcriptome database, which were used to design the specific primers for Real-time quantitative polymerase chain reaction (RT-qPCR), could be obtained in National Center for Biotechnology Information (NCBI)

Summary

Introduction

Baphicacanthus cusia (Nees) Bremek (Figure S1), widely distributed in Southern China, is the only plant belonging to the family Acanthaceae. Numerous active compounds have been identified from B. cusia to date, such as indole alkaloids, quinazolinone alkaloids, monoterpenes, triterpenes, flavonoids, sterols, anthraquinones, benzoxazinones, and lignans. Of these ingredients, the pharmaceutical activities of indole alkaloids are most frequently reported for their leukocyteinhibitory, anti-inflammatory, and antiviral activities (Li et al, 1993; Wu et al, 2005; Huang et al, 2014). The genetic information of B. cusia for molecular biology research is limited in public databases, which makes further in-depth studies more difficult. The mRNA has recently been sequenced, and the partial contigs have already been deposited into the National Center for Biotechnology Information (NCBI) database (SRR4428209) to accelerate genetic studies in B. cusia

Methods

Results

Conclusion