Screening and Stability Evaluation of Reference Genes in Uncaria rhynchophylla qRT-PCR Analysis
YU Xiao-Song, WANG Xiao-Hong, LI Xue, JIA Na, SHANGGUAN Li-Yang, QIANG Wei, ZHANG Ming-Sheng*
Key Laboratory of Plant Resource Conservation and Germplasm Innovation in Mountainous Region (Ministry of Education)/College of Life Sciences, Guizhou University, Guiyang 550025, China
Abstract：The medicinal components of Uncaria rhynchophylla, rhynchophylline and isorhynchophylline, are regulated by various enzymatic reaction genes. Screening the best internal reference genes in the qRT-PCR system of U. rhynchophylla is of great significance for the study of gene expression of medicinal components in U. rhynchophylla. The present study aimed to select reference genes stably expressed in different tissues, different shading treatments and different ethylene treatments in U. rhynchophylla. U. rhynchophylla was selected as the material, and the expression of 8 reference genes of β-actin (β-act), α-tubulin (α-tub), 18S ribosomal RNA (18S rRNA), translation elongation factor (ef-1), glyceraldehyde-3-phosphate dehydrogenase (gapdh), β-tubulin (β-tub), ribosomal protein (rpl) and ubiquitin-ligase enzyme (ubc) in different tissues and different treatments were analyzed by qRT-PCR. The GeNorm program was used to determine the number of reference genes, and then NormFinder and BestKeeper programs were combined to evaluate the stability of the reference genes. Finally, the geometric mean method was used to integrate the results obtained from different software and obtained comprehensive ranking. The results showed that single reference gene could be used in different tissues and treatments. In different tissues, ef-1 should be selected as the reference gene; under shading treatments, gapdh should be selected as the reference gene; under ethylene treatments, both ubc and β-act could be used as the reference gene. If a relatively stable reference gene is used in multiple expression studies, ubc should be selected. This study provides correction and standardization genes for the expression analysis of related genes in different experimental systems of U. rhynchophylla, and is helpful to improve the accuracy and reliability of the experiments.
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