Screening and Stability Evaluation on qPCR Reference Genes of Camellia spp.
NIE Rui-Min, ZHAO Wan-Yue, CHEN Shao-Yuan, HU Yun-Chong, CHEN Sheng-Tong, CHEN Long-Qing, GENG Fang*
College of Landscape Architecture and Horticulture Sciences/Southwest Landscape Architecture Engineering Technology Research Center of National Forestry and Grassland Administration/Yunnan Functional Flower Resources and Industrialization Technology Engineering Research Center, Southwest Forestry University, Kunming 650224, China
Abstract：With the increasing research on the molecular biology of Camellia spp., it is crucial to screen the reference genes suitable for qPCR analysis of Camellia spp.. 3 Camellia varieties with different ornamental values were selected as experimental materials for this study. 7 common reference genes, including elongation factor-α (EF-1α), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Actin 7 (ACT), α- tubulin (TUA), β-tubulin (TUB), protein phosphatase 2C (PP2C), and phosphatase activator TIP41 (TIP41), were detected using reverse transcription polymerase chain reaction (RT-PCR) and qPCR in various Camellia spp. flower tissues and flower developmental periods. The stability of the candidate genes was analyzed using ΔCt, geNorm, NormFinder and BestKeeper programs, and RefFinder was used for comprehensive evaluation to screen out the candidate reference genes suitable for qPCR in Camellia spp., and to verify the stability of the reference genes by the key genes of flower color and floral aroma biosynthesis. The results showed that the average expression of EF-1α was found to be higher than that of other candidate reference genes. The analysis results from ΔCt, NormFinder and BestKeeper all showed that EF-1α was the most stable in different tissues and stages of Camellia spp. flower development, while the results from geNorm showed that GAPDH and TIP41 were more stable. Additionally, the results from all programs showed that ACT was the least stable reference gene. Based on the comprehensive evaluation of RefFinder, EF-1α and ACT were validated by the expression pattern analysis of the floral color genes, chalcone synthase (CrCHS) and chalcone isomerase (CrCHI), and the floral scent genes, 1-deoxy-D-xylulose 5-phosphate synthase (ChDXS1) and 1-deoxy-D-xylulose 5-phosphate reductoisomerase (ChDXR). The findings demonstrated that EF-1α with superior stability could be chosen as an internal reference gene in the study of Camellia spp. floral color and floral scent. The results of the study provide a theoretical basis and technical support for further research on the functional genes related to the formation process of Camellia spp. floral color and floral scent.
[1] 陈艺荃, 林榕燕, 孔兰, 等. 2024. 山茶花花香成分鉴定及相关基因的转录组分析[J]. 核农学报, 38(12): 2281-2293. (Chen Y Q, Lin R Y, Kong L, et al.2024. Identification of floral aroma components and transcriptome analysis of related genes in Camellia japonica L.[J]. Journal of Nuclear Agricultural Sciences, 38(12): 2281-2293.) [2] 陈艺荃, 潘宏, 魏云华. 2019. 山茶花品种观赏性状的主成分分析与观赏价值综合评价[J]. 福建农业学报, 34(05): 551-559. (Chen Y Q, Pan H, Wei Y H.2019. Principal component analysis and comprehensive evaluation on ornamental value of varieties of Camellia[J]. Fujian Journal of Agricultural Sciences, 34(05): 551-559.) [3] 邝雪琨, 郑倩, 孔庆博, 等. 2024. 山茶属植物系统发育及分类研究进展[J]. 园艺学报, 51(01): 53-66. (Kuang X K, Zheng Q, Kong Q B, et al.2024. Research progress on phylogeny and classification of Camellia[J]. Acta Horticulturae Sinica, 51(01): 53-66.) [4] 李凌燕, 陈红, 马玥, 等. 2022. 转基因玉米MON87419实时荧光定量PCR检测方法的建立[J]. 山东农业科学, 54(11): 121-126. (Li L Y, Chen H, Ma Y, et al.2022. Establishment of a real-time fluorescence quantitative PCR method for detecting genetically modified maize MON87419[J]. Shandong Agricultural Sciences, 54(11): 121-126.) [5] 梁芳, 许申平, 张燕, 等. 2022. 蝴蝶兰PhPP2Aa基因作为低温胁迫内参基因的研究[J]. 热带作物学报, 43(07): 1338-1346. (Liang F, Xu S P, Zhang Y, et al.2022. PhPP2Aa as reference gene in Phalaenopsis under low-temperature stress[J]. Chinese Journal of Tropical Crops, 43(07): 1338-1346.) [6] 梁蕤, 徐雷锋, 毕蒙蒙, 等. 2023. 柠檬色百合实时荧光定量PCR内参基因筛选[J]. 中国农业大学学报, 28(02): 59-73. (Liang R, Xu L F, Bi M M, et al.2023. Validation of reference genes for quantitative real-time PCR in Lilium leichtlinii[J]. Journal of China Agricultural University, 28(02): 59-73.) [7] 梁子英, 刘芳. 2020. 实时荧光定量PCR技术及其应用研究进展[J]. 现代农业科技,(06): 1-3+8. (Liang Z Y, Liu F.2020. Research progress on real-time quantitative PCR technology and its application[J]. Modern Agricultural Science and Technology,(06): 1-3+8.) [8] 廖初琴, 缪绅裕. 2023. 山茶属植物传统及现代分类的研究进展[J]. 热带农业科学, 43(07): 37-45. (Liao C Q, Miao S Y.2023. Advances in the traditional and modern classification of Camellia[J]. Chinese Journal of Tropical Agriculture, 43(07): 37-45.) [9] 刘小飞, 于波, 黄丽丽, 等. 2020. 杜鹃红山茶实时定量PCR内参基因筛选及验证[J]. 广东农业科学, 47(12): 203-211. (Liu X F, Yu B, Huang L L, et al.2020. Screening and validation of reference genes of Camellia azalea by quantitative real-time PCR[J]. Guangdong Agricultural Sciences, 47(12): 203-211.) [10] 刘晓婷, 王顺利, 薛璟祺, 等. 2018. 朱顶红实时荧光定量PCR中不同组织器官内参基因的筛选[J]. 园艺学报, 45(05): 919-930. (Liu X T, Wang S L, Xue J Q, et al.2018. Selection of reference genes for quantitative real-time PCR in different tissue and organ of barbadoslily[J]. Acta Horticulturae Sinica, 45(05): 919-930.) [11] 刘圆, 王丽鸳, 韦康, 等. 2016. 不同氮处理茶树实时定量PCR内参基因筛选和验证[J]. 茶叶科学, 36(01): 92-101. (Liu Y, Wang L Y, Wei K, et al.2016. Screening and validation of reference genes for quantitative real-time PCR analysis in tea plant (Camellia sinensis) under different nitrogen nutrition[J]. Journal of Tea Science, 36(01): 92-101.) [12] 宋志波, 刘敏, 贾宝光, 等. 2014. 油茶总RNA的提取与内参基因的初选[J]. 经济林研究, 32(02): 93-98. (Song Z B, Liu M, Jia B G, et al.2014. RNA extraction and primary screening of reference genes in Camellia oleifera[J]. Non-wood Forest Research, 32(02): 93-98.) [13] 唐孝富, 孟银银, 杜莹, 等. 2024. '羌脆李'果实发育期qRT-PCR内参基因的筛选与验证[J]. 四川农业大学学报, 42(06): 1220-1225+1231. (Tang X F, Meng Y Y, Du Y, et al.2024. Screening and validation of reference genes for real-time PCR during 'Qiangcuili' fruit development[J]. Journal of Sichuan Agricultural University, 42(06): 1220-1225+1231.) [14] 王彦杰, 陈叶清, 薛泽云, 等. 2017. 荷花花瓣着色过程实时荧光定量PCR内参基因的筛选及验证[J]. 南京农业大学学报, 40(03): 408-415. (Wang Y J, Chen Y Q, Xue Z Y, et al.2017. Selection and validation of reference genes for RT-qPCR normalization in lotus (Nelumbo nucifera) during petal coloration[J]. Journal of Nanjing Agricultural University, 40(03): 408-415.) [15] 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, (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, (05): 278-281.) [16] 吴学琴, 申开成, 肖红星, 等. 2024. 50种茶花叶表型性状的多样性分析[J]. 中南林业科技大学学报, 44(10): 165-180. (Wu X Q, Shen K C, Xiao H X, et al.2024. Diversity analysis of leaf phenotypic traits in 50 camellias species[J]. Journal of Central South University of Forestry & Technology, 44(10): 165-180.) [17] 叶勇, Henry Lusekelo Ingwe, 郑子飞, 等. 2023. 梅花花色合成基因的qPCR内参基因筛选及验证[J]. 农业生物技术学报, 31(12): 2674-2684. (Ye Y, Ingwe H L, Zheng Z F, et al.2023. Screening and validation of reference genes for qPCR analysis of flower color synthesis genes in Prunus mume[J]. Journal of Agricultural Biotechnology, 31(12): 2674-2684.) [18] 张宝华. 2020. PCR技术在植物生物学中的应用研究[J]. 生物化工, 6(01): 130-132. (Zhang B H.2020. Application of PCR in plant biology[J]. Biological Chemical Engineering, 6(01): 130-132.) [19] 张秋悦, 刘昌来, 于晓晶, 等. 2022. 盐胁迫条件下杜梨叶片差异表达基因qRT-PCR内参基因筛选[J]. 园艺学报, 49(07): 1557-1570. (Zhang Q Y, Liu C L, Yu X J, et al.2022. Screening of reference genes for differentially expressed genes in Pyrus betulaefolia plant under salt stress by qRT-PCR[J]. Acta Horticulturae Sinica, 49(07): 1557-1570.) [20] 张玉琎, 吉军, 肖鑫, 等. 2024. 杜鹃兰qRT-PCR内参基因的筛选及稳定性评价[J]. 农业生物技术学报, 32(04): 926-938. (Zhang Y J, Ji J, Xiao X, et al.2024. Screening and stability evaluation on qRT-PCR reference genes of Cremastra appendiculata[J]. Journal of Agricultural Biotechnology, 32(04): 926-938.) [21] Du G G, Wang L Y, Li H W, et al.2019. Selection and validation of reference genes for quantitative gene expression analyses in persimmon (Diospyros kaki Thunb.) using real-time quantitative PCR[J]. Biologia Futura, 70(4): 261-267. [22] Shao C, Lao W, Liang Y.2022. Reference genes selection of Gymnosporangium yamadae during the interaction with apple leaves[J]. Journal of Fungi, 8(8): 830. [23] 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. [24] Yu W J, Tao Y, Luo L P, et al.2021. Evaluation of housekeeping gene expression stability in carnation (Dianthus caryophyllus)[J]. New Zealand Journal of Crop and Horticultural Science, 49(4): 347-360. [25] Zhang P L, Chen S Y, Chen S Y, et al.2024. Selection and validation of qRT-PCR internal reference genes to study flower color formation in Camellia impressinervis[J]. International Journal of Molecular Sciences, 25(5): 3029. [26] Zhu X L, Wang B Q, Wang X, et al.2021. Screening of stable internal reference gene of quinoa under hormone treatment and abiotic stress[J]. Physiology and Molecular Biology of Plants, 27: 2459-2470.
