Identification and Selection of Reference Genes for Quantitative Transcript Analysis in Corydalis yanhusuo

Zhenzhen Bao,Xiurong Zhao,Changjian Li,Jie Wu,Hanfeng Lin,Sihui Nian,Kaidi Zhang

doi:10.3390/genes11020130

Zhenzhen Bao, Xiurong Zhao + Show 5 more

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https://doi.org/10.3390/genes11020130

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Abstract

Corydalis yanhusuo is a medicinal plant frequently used in traditional Chinese medicine, which has effective medical effects in many aspects. Real-time polymerase chain reaction (RT-PCR) has been one of the most widely used methods in biosynthesis research due to its high sensitivity and quantitative properties in gene expression analysis. To obtain accurate normalization, reference genes are often selected in advance; however, no reference genes are available in C. yanhusuo. Herein, 12 reference gene candidates, named cyclophilin 2 (CYP2), elongation factor 1-α (EF1-α), protein phosphatase 2 (PP2A), SAND protein family (SAND), polypyrimidine tract-binding protein (PTBP), TIP41-like protein (TIP41), lyceraldehyde-3-phosphate hydrogenase (GAPDH), ubiquitin-conjugating enzyme 9 (UBC9), cyclophilin 1 (CYP1), tubulin beta (TUBA), thioredoxin (YLS8), and polyubiquitin 10 (UBQ10), were selected for stability analysis. After being treated with hormone, UV, salt, metal, oxidative, drought, cold (4 °C), and hot stresses (40 °C), the qRT-PCR data of the selected genes was analyzed with NormFinder, geNorm, and BestKeeper. The result indicated that GAPDH, SNAD, and PP2A were the top three most stable reference genes under most treatments. This study selected and validated reliable reference genes in C. yanhusuo under various environmental conditions, which can provide great help for future research on gene expression normalization in C. yanhusuo.

Highlights

Corydalis yanhusuo is a plant from the Papaveraceae family and is widely used to treat drug addiction and as analgesics in China [1]
Six candidate genes were obtained by comparing them with previous studies, and the remaining six candidate reference genes were selected according to the TAIR database
ubiquitin-conjugating enzyme 9 (UBC9), tubulin beta (TUBA), and elongation factor 1-α (EF1-α) can be considered as the least stable reference genes among the 12 candidates

Summary

Introduction

Corydalis yanhusuo is a plant from the Papaveraceae family and is widely used to treat drug addiction and as analgesics in China [1]. Abundant pharmacological effects on humans have been identified in the extracts of C. yanhusuo (tuber), such as pain relief, anti-tumor effects, and promotion of blood circulation [2,3]. Benzylisoquinoline alkaloids are the main biological components produced by C. yanhusuo, and previous studies have found that dehydrocorybulbine, which is found in the extract of C. yanhusuo, is a kind of alkaloid that can exhibit antagonistic activity by acting on dopamine receptors [4,5]. Few studies have focused on the gene expression of C. yanhusuo. Some pivotal genes involved in the biosynthetic pathways of important components are not yet clear. Various medical functions have been proven by previous research for D-glaucine, but the genes involved in

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