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

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.

Selection of Reference Genes for qRT-PCR in Bletilla striata under Heat Stress

“…No single reference gene has been determined that is always expressed stably under any condition (Argyropoulos et al 2006). Accordingly, it is crucial to discover suitable internal control genes with stable expression for the study of specific transcriptional profiles of genes of interest under a certain experimental condition for a certain species (Yang et al 2019). There are many reports on screening for proper reference genes in plants, however, there has been no report regarding reference genes for the study of B. striata.…”

Reference gene selection for real‐time quantitative PCR assays in different tissues of Huperzia serrata based on full‐length transcriptome sequencing

“…Many studies have shown that GAPDH was often applied as stable RG in different tissues or under various experimental conditions (Chen et al, 2017 ; Zhuang et al, 2015 ). Especially for H. serrata , GAPDH also was proved as stable RG by Yang et al ( 2019 ). TBP was also identified as stable RG for H. serrata by Yang et al ( 2019 ).…”

“…Especially for H. serrata , GAPDH also was proved as stable RG by Yang et al ( 2019 ). TBP was also identified as stable RG for H. serrata by Yang et al ( 2019 ). Unfortunately, TBP gene could not be amplified in our materials.…”

“…Especially for the selection of optimum reference genes, little research has been reported. TATA binding protein (TBP) and GAPDH were identified as reference genes for H. serrata in Yang et al's ( 2019 ) report. Unfortunately, TBP gene could not be amplified when we study the expression of HupA‐biosynthetic genes in our materials.…”

“…So, researchers reach a consensus that specific RG for a given species and treatment needs to be identified first (Bansal et al, 2015 ). Unfortunately, the previously reported RGs are not suitable for the research of HupA‐biosynthetic genes in our materials (Yang et al, 2019 ).…”