Selection of Reference Genes for Expression Analysis in Chinese Medicinal Herb Huperzia serrata.

Mengquan Yang,Wenjing You,Youli Xiao,Shiwen Wu,Amit Jaisi

doi:10.3389/fphar.2019.00044

Abstract

Huperzine A (HupA) is a powerful and selective inhibitor of acetylcholinesterase. It has attracted widespread attention endangering the ultimate plant sources of Lycopodiaceae family. In this study, we used Huperzia serrata, extensively used in Traditional Chinese medicine (TCM), a slow growing vascular plant as the model plant of the Lycopodiaceae family to develop and validate the reference genes. We aim to use gene expression platform to understand the gene expression of different tissues and developmental stages of this medicinal herb. Eight candidate reference genes were selected based on RNA-seq data and evaluated with qRT-PCR. The expression of L/ODC and cytochrome P450s genes known for their involvement in lycopodium alkaloid biosynthesis, were also studied to validate the selected reference genes. The most stable genes were TBP, GAPDH, and their combination (TBP + GAPDH). We report for the first time the reference gene of H. serrata’s different tissues which would provide important insights into understanding their biological functions comparing other Lycopodiaceae plants and facilitate a good biopharming approach.

Highlights

The Lycopodiaceae family comprises three main genera, namely, Huperzia, Phlegmariurus, and monotypic Phylloglossum
The presence of a single peak in Quantitative real-time PCR (qRT-PCR) melting curve analysis for each of the eight sets of primers indicated high specificity (Figure 4). qRT-PCR was performed to determine the expression levels of each candidate reference genes, and the cycle quantification (Cq) values showed differential transcript levels in the samples examined with low Cq values, which suggested transcript abundance
We proposed H. serrata as a model plant for functional genomics study in the Lycopodiaceae family

Summary

Introduction

The Lycopodiaceae family comprises three main genera, namely, Huperzia, Phlegmariurus, and monotypic Phylloglossum. The morphological variability between Phlegmariurus and Huperzia has presented a taxonomic challenge. They possess similar chemical diversity, especially lycopodium alkaloids, such as huperzine A (HupA), a highly potent, selective, and reversible inhibitor of AchE (Zhao and Tang, 2002), a lead candidate for Alzheimer’s disease. HupA was initially isolated from the traditional Chinese medicine Qian Ceng Ta (Huperzia serrata). H. serrata is an economically important traditional Chinese herb that is used extensively for treatment of contusions, strains, swellings, schizophrenia, myasthenia gavis, and organophosphate poisoning since the Tang Dynasty (Ma et al, 2007; Xu et al, 2017). Extensive harvest for HupA has endangered H. serrata and other species in the Lycopodiaceae family. Synthetic biology approach offers an Reference Genes for Huperzia serrata alternative potential source of HupA, but the inadequate understanding of its biosynthetic pathway restricts its production by metabolic engineering

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