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
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.
[1] 丁苏芹, 李玺, 唐东芹. 2020. 小苍兰实时荧光定量PCR中的内参基因筛选[J]. 南京林业大学学报(自然科学版), 44(03): 19-25. (Ding S Q, Li X, Tang D Q.2020. Screening on reference genes for real-time fluorescent quantitative PCR of Freesia hybrida[J]. Journal of Nanjing Forestry University (Natural Sciences Edition), 44(03): 19-25.) [2] 段国敏, 李田园, 田敏, 等. 2021. 扇脉杓兰实时荧光定量PCR内参基因的筛选[J]. 核农学报, 35(03): 576-585. (Duan G M, Li T Y, Tian M, et al.2021. Reference gene selection of real-time fluorescence quantitative PCR in Cypripedium japonicum[J]. Journal of Nuclear Agricultural Sciences, 35(03): 576-585.) [3] 葛鑫涛, 殷淯梅, 高丽丹, 等. 2021. 菰黑粉菌实时荧光定量PCR分析中内参基因的选择及应用[J]. 农业生物技术学报, 29(02): 402-412. (Ge X T, Yin Y M, Gao L D, et al.2021. Selection and application of reference genes for qRT-PCR in Ustilago esculenta[J]. Journal of Agricultural Biotechnology, 29(02): 402-412.) [4] 侯天泽, 易双双, 张志群, 等. 2022. 秋石斛RT-qPCR内参基因的筛选与验证[J]. 园艺学报, 49(11): 2489-2501. (Hou T Z, Yi S S, Zhang Z Q, et al.2022. Selection and validation of reference genes for RT-qPCR in phalaenopsis-type Dendrobium hybrid[J]. Acta Horticulturae Sinica, 49(11): 2489-2501.) [5] 姜婷, 苏乔, 安利佳. 2015. 多重胁迫下玉米实时定量PCR内参基因的筛选与验证[J]. 植物生理学报, 51(09): 1457-1464. (Jiang T, Su Q, An J L.2015. Screening and validation of reference genes of qPCR in maize under multiple stresses[J]. Plant Physiology Journal, 51(09): 1457-1464.) [6] 李永平, 叶新如, 王彬, 等. 2021. 黄秋葵实时荧光定量PCR内参基因的克隆与筛选评价[J]. 核农学报, 35(01): 60-71. (Li Y P, Ye R X, Wang B, et al.2021. Cloning and selection evaluation of reference gene for quantitative real-time PCR in Hibiscus esculentus L.[J]. Journal of Nuclear Agricultural Sciences, 35(01): 60-71.) [7] 刘洪峰, 高乐旋, 胡永红. 2015. 牡丹不同发育阶段种子和花瓣组织实时荧光定量PCR中内参基因的挖掘与筛选[J]. 农业生物技术学报, 23(12): 1639-1648. (Liu H F, Gao L X, Hu Y H.2015. Reference genes discovery and selection for quantitative real-time PCR in tree peony seed and petal tissue of different development stages[J]. Journal of Agricultural Biotechnology, 23(12): 1639-1648.) [8] 明如宏, 李良波, 姚绍嫦, 等. 2022. 绞股蓝实时荧光定量PCR内参基因筛选和验证[J]. 中药材, (05): 1082-1087. (Ming R H, Li L B, Yao S C, et al. 2022. Selection and validation of reference genes for quantitative real-time PCR analysis in Gynostemma pentaphyllum[J]. Journal of Chinese Medicinal Materials, (05): 1082-1087.) [9] 彭思静, 高燕燕, 张明生, 等. 2021. 杜鹃兰种子非共生萌发中的形态结构变化[J]. 种子, 40(12): 1-8. (Peng S J, Gao Y Y, Zhang M S, et al.2021. Morphological and structural changes during seeds asymbiotic germination of Cremastra appendiculata[J]. Seed, 40(12): 1-8.) [10] 任锐, 戴鹏辉, 李萌, 等. 2016. 珙桐实时定量PCR内参基因的筛选及稳定性评价[J]. 植物生理学报, 52(10): 1565-1575. (Ren R, Dai P H, Li M, et al.2016. Selection and stability evaluation of reference genes for real-time quantitative PCR in dove tree (Davidia involucrata)[J]. Plant Physiology Journal, 52(10): 1565-1575.) [11] 孙亚丽, 张德辉, 赵亮, 等. 2014. 铜胁迫下天蓝苜蓿根组织实时定量PCR内参基因的选择[J]. 农业生物技术学报, 22(10): 1223-1231. (Sun Y L, Zhang D H, Zhao L, et al.2014. Reference gene selection for real-time quantitative PCR in black medic (Medicago lupulina L.) root tissue under copper stress[J]. Journal of Agricultural Biotechnology, 22(10): 1223-1231.) [12] 王汪中, 张明生, 吕享, 等. 2017. 杜鹃兰种子萌发适宜培养基的筛选[J]. 北方园艺, (11): 157-161. (Wang W Z, Zhang M S, Lv X, et al. 2017. Media screening on seeds germination of Cremastra appendiculata[J]. Northern Horticulture, (11): 157-161.) [13] 吴建阳, 何冰, 杜玉洁, 等. 2017. 利用geNorm、NormFinder和BestKeeper软件进行内参基因稳定性分析的方法[J]. 现代农业科技, 3(05): 278-281. (Wu J Y, He B, Du Y J, et al.2017. Analysis method of systematically evaluating stability of reference genes using geNorm, NormFinder and BestKeeper[J]. Modern Agricultural Science and Technology, 3(05): 278-281.) [14] 许明, 伊恒杰, 赵帅, 等. 2017. 显齿蛇葡萄实时荧光定量PCR内参基因的筛选与验证[J]. 中草药, 48(06): 1192-1198. (Xu M, Yi H J, Zhao S, et al.2017. Screening and validation of reference genes for quantitative RT-PCR analysis in Ampelopsis grossedentata[J]. Chinese Traditional and Herbal Drugs, 48(06): 1192-1198.) [15] 于晓松, 王晓红, 张明生, 等. 2021. 钩藤实时荧光定量PCR分析中内参基因的筛选及稳定性评价[J]. 农业生物技术学报, 29(03): 599-609. (Yu X S, Wang X H, Zhang M S,et al.2021. Screening and stability evaluation of reference genes in Uncaria rhynchophylla qRT-PCR analysis[J]. Journal of Agricultural Biotechnology, 29(03): 599-609.) [16] 张明生, 彭斯文, 杨小蕊, 等. 2009. 杜鹃兰人工种子技术研究[J]. 中国中药杂志, 34(15): 1894-1897. (Zhang M S, Peng S W, Yang X R, et al.2009. Preparation technique of Cremastra appendiculata synthetic seed[J]. China Journal of Chinese Materia Medica, 34(15): 1894-1897.) [17] 张娜. 2022. 杜鹃兰种子共生与非共生萌发研究[D]. 硕士学位论文, 陕西理工大学, 导师: 曹小勇, pp. 1-64. (Zhang N.2022. Symbiotic and asymbiotic germination of Cremastra appendiculata seeds[D]. Thesis for M.S., Shaanxi University of Technology, Suppervisor: Cao X Y, pp. 1-64.) [18] 张玉芳, 赵丽娟, 曾幼玲. 2014. 基因表达研究中内参基因的选择与应用[J]. 植物生理学报, 50(08): 1119-1125. (Zhang Y F, Zhao L J, Zeng Y L.2014. Selection and application of reference genes for gene expression studies[J]. Plant Physiology Journal, 50(08): 1119-1125.) [19] Chang E, Shi S Q, Liu J F, et al.2012. Selection of reference genes for quantitative gene expression studies in Platycladus orientalis (Cupressaceae) using real-time PCR[J]. PLOS ONE, 7(3): e33278. [20] Chen K T, Fessehaie A, Arora R.2012. Selection of reference genes for normalizing gene expression during seed priming and germination using qPCR in Zea mays and Spinacia oleracea[J]. Plant Molecular Biology Reporter, 30(2): 478-487. [21] Chowrasia S, Kaur H, Mujib A, et al.2019. Evaluation of Oryza coarctata candidate reference genes under different abiotic stresses[J]. Biologia Plantarum, 63(1): 496-503. [22] Cui B, Smooker P M, Rouch D A, et al.2016. Selection of suitable reference genes for gene expression studies in Staphylococcus capitis during growth under erythromycin stress[J]. Molecular Genetics and Genomics, 291(4): 1795-1811. [23] Fedick A, Su J, Jalas C, et al.2012. High-throughput real-time PCR-based genotyping without DNA purification[J]. BMC Research Notes, 5(1): 573-590. [24] Feng K, Liu J X, Xing G M, et al.2019. Selection of appropriate reference genes for RT-qPCR analysis under abiotic stress and hormone treatment in celery[J]. PeerJ, 7(7): 7925-7937. [25] Gao Y Y, Ji J, Zhang M S, et al.2022a. Biochemical and transcriptomic analyses of the symbiotic interaction between Cremastra appendiculata and the mycorrhizal fungus Coprinellus disseminatus[J]. BMC Plant Biology, 22(1): 15-28. [26] Gao Y Y, Peng S J, Zhang M S, et al.2022b. Mycorrhizal fungus Coprinellus disseminatus influences seed germination of the terrestrial orchid Cremastra appendiculata (D. Don) Makino[J]. Scientia Horticulturae, 293(5): 110724-110734. [27] Gutierrez L, Mauriat M, Guenin S, et al.2008. The lack of a systematic validation of reference genes: A serious pitfall undervalued in reverse transcription-polymerase chain reaction (RT-PCR) analysis in plants[J]. Plant Bio-technology Journal, 6(6): 609-618. [28] Hu A S, Yang X Y, Zhu J F, et al.2022. Selection and validation of appropriate reference genes for RT-qPCR analysis of Nitraria sibirica under various abiotic stresses[J]. BMC Plant Biology, 22(1): 592-606. [29] Joong S S, Jin H K, Jiyong L, et al.2004. Anti-angiogenic activity of a homoisoflavanone from Cremastra appendiculata[J]. Planta Medica, 70(2): 171-173. [30] Li H G, Zhu Y Q, Gu K X, et al.1996. A report on four cases of liver carcinoma treated by topical adhesive method[J]. Journal of Traditional Chinese Medicine, 16(4): 243-246. [31] Li W N, Zhao Q, Guo M H, et al.2022. Predicting the potential distribution of the endangered plant Cremastra appendiculata (Orchidaceae) in China under multiple climate change scenarios[J]. Forests, 13(9): 1504-1514. [32] Lin Y A, Zhang C L, Lan H, et al.2014. Validation of potential reference genes for qPCR in maize across abiotic stresses, hormone treatments, and tissue types[J]. PLOS ONE, 9(5): e95445. [33] Liu L, Li J, Tu P F, et al.2016. Five new biphenanthrenes from Cremastra appendiculata[J]. Molecules, 21(8): 1089-1103. [34] Nicot N, Hausman J F, Hoffmann L, et al.2005. Housekeeping gene selection for Real-time RT-PCR normalization in potato during biotic and abiotic stress[J]. Journal of Experimental Botany, 56(421): 2907-2914. [35] Park S C, Kim Y M, Ji C Y, et al.2017. Stable internal reference genes for the normalization of real-time PCR in different sweetpotato cultivars subjected to abiotic stress conditions[J]. PLOS ONE, 7(12): 1795-1811. [36] Shukla P, Reddy R A, Ponnuvel K M, et al.2019. Selection of suitable reference genes for quantitative real-time PCR gene expression analysis in mulberry (Morus alba L.) under different abiotic stresses[J]. Molecular Biology Reports, 46(2): e51502. [37] Song H, Zhang X, Shi C, et al.2016. Selection and verification of candidate reference genes for mature microRNA expression by quantitative RT-PCR in the tea plant (Camellia sinensis)[J]. Genes, 7(6): 25-37. [38] Štajner N, Cregeen S, Javornik B.et al.2017. Evaluation of reference genes for RT-qPCR expression studies in hop (Humulus lupulus L.) during infection with vascular pathogen Verticillium albo-atrum[J]. PLOS ONE, 8(7): e68288. [39] Takahiro Y, Eriko F, Eiji N, et al.2013. Identification and symbiotic ability of Psathyrellaceae fungi isolated from a photosynthetic orchid, Cremastra appendiculata (Orchidaceae)[J]. American Journal of Botany, 100(9): 1823-1830. [40] Vandesompele J, De Preter K, Pattyn F, et al.2002. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes[J]. Genome Biology, 3(7): 0034.1. [41] Wang B, Duan H, Chong P, et al.2020. Systematic selection and validation of suitable reference genes for quantitative real-time PCR normalization studies of gene expression in Nitraria tangutorum[J]. Scientific Reports, 10(1): 15891-15903. [42] Wang G L, Tian C, Jiang Q, et al.2016. Comparison of nine reference genes for real-time quantitative PCR in roots and leaves during five developmental stages in carrot (Daucus carota L.)[J]. The Journal of Horticultural Science and Biotechnology, 91(3): 264-270. [43] Wang J, Xie J J, Chen H X, et al.2022. A draft genome of the medicinal plant Cremastra appendiculata (D. Don) provides insights into the colchicine biosynthetic pathway[J]. Communications Biology, 5(1): 1294-1306. [44] Wang M, Wang Z W, Wei S S, et al.2022. Identification of RT-qPCR reference genes suitable for gene function studies in the pitaya canker disease pathogen Neoscytalidium dimidiatum[J]. Scientific Reports, 12(1): 22357-22357. [45] Xiao X L, Ma J B, Wang J R, et al.2015. Validation of suitable reference genes for gene expression analysis in the halophyte Salicornia europaea by real-time quantitative PCR[J]. Frontiers in Plant Science, 5(5): 788-799. [46] Zhang L C, Liu L, Cheng P, et al.2017a. Identification and validation of reference genes for RT‐qPCR analysis in banana (Musa spp.) under Fusarium wilt resistance induction conditions[J]. Journal of Phytopathology, 165(11): 746-761. [47] Zhang M S, Peng S W, Wang W.2010. Macro research on growth and development of Cremastra appendiculata (D. Don.) Makino (Orchidaceae)[J]. Journal of Medicinal Plants Research, 4(18): 1837-1842. [48] Zhang Y X, Han X J, Chen S S, et al.2017b. Selection of suitable reference genes for quantitative real-time PCR gene expression analysis in Salix matsudana under different abiotic stresses[J]. Scientific Reports, 7(1): 40290-40305.
