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| Screening and Stability Evaluation on qRT-PCR Reference Genes of Cremastra appendiculata |
| ZHANG Yu-Jin1, JI Jun1, XIAO Xin1, ZHANG Jing-Yi1, WANG Li-Qin1, GAO Yan-Yan2, TIAN Yu-Hang1, ZHANG Ming-Sheng1,* |
1 School of Life Sciences/Key Laboratory of Plant Resource Conservation and Germplasm Innovation in Mountainous Region (Ministry of Education), Guizhou University, Guiyang 550025, China;
2 College of Pharmacy, Guizhou Medical University, Guiyang 550025, China |
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Abstract As a rare medicinal plant of orchid family, Cremastra appendiculata has been widely concerned for its high medicinal value, but its resources are depleted by severe reproductive barriers and predatory human extraction, using modern biomolecular technology to solve the reproduction difficulties of C. appendiculata species is one of the effective ways of resource conservation. In this study, different organs (root, stem and leaf) and seeds at different stages (the symbiotic and non-symbiotic germination) of C. appendiculata were used as research materials, qRT-PCR was used to detect the expression of 7 commonly used household genes of β-actin (ACT), translation elongation factor 1 beta (ef-1β), cyclophilin (CYP), ribosomal protein (RPL), ribosomal protein S8 (RPS), ubiquitin C (UBC), α-tubulin (TUB). The stability of candidate genes was comprehensively evaluated by geNorm, NormFinder and BestKeeper software, and finally the reference genes suitable for qRT-PCR of C. appendiculata were selected. The stability of the reference genes was verified by the target genes GA 3-beta dioxygenase (GA3ox) and lectin protein coding gene (Lectin10). The result showed that 2 reference genes could be introduced into different organs and treatments of C. appendiculata; The expression of RPL and ef-1β in different organs was relatively consistent, with good stability, and suitable as reference genes; The expression of UBC and TUB were the most stable in the symbiotic germinated seeds at different germination stages; RPL and ef-1β were the most stable reference genes in the non-symbiotic germination seeds at different germination stages. When the relatively stable UBC and TUB genes were used as internal reference genes, the target genes GA3ox and Lectin10 showed similar expression trends in the seed of C. appendiculata at different symbiotic germination stages, while the poorly stable ACT genes could not effectively correct the expression analysis of target genes, and the data showed extremely obvious deviations. This study provides correction and standardization genes for the expression analysis of related genes in C. appendiculata under different research conditions, which would effectively improve the accuracy and reliability of subsequent studies.
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Received: 27 March 2023
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Corresponding Authors:
*mshzhang@163.com
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