氟胁迫条件下茶树叶部实时荧光定量PCR分析中内参基因的筛选与验证

李庆会; 李睿; 温晓菊; 倪德江; 王明乐; 陈玉琼

doi:10.13305/j.cnki.jts.2024.01.001

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茶叶科学 ›› 2024, Vol. 44 ›› Issue (1) : 27-36. DOI: 10.13305/j.cnki.jts.2024.01.001

研究报告

氟胁迫条件下茶树叶部实时荧光定量PCR分析中内参基因的筛选与验证

李庆会, 李睿, 温晓菊, 倪德江, 王明乐, 陈玉琼^*

作者信息 +

Selection and Validation of Internal Reference Genes for qRT-PCR Analysis under Fluoride Stress in Camellia sinensis Leaves

LI Qinghui, LI Rui, WEN Xiaoju, NI Dejiang, WANG Mingle, CHEN Yuqiong^*

Author information +

文章历史 +

摘要

为了筛选氟胁迫条件下茶树（Camellia sinensis）叶部用于实时荧光定量PCR（qRT-PCR）分析的内参基因,以课题组前期筛选的低氟茶树品种福鼎大白茶和高氟茶树品种金观音为试验材料,利用qRT-PCR技术结合geNorm、NormFinder和BestKeeper软件,分析氟胁迫条件下（0.42 mmol·L^-1 NaF）8个候选内参基因（CsACTIN、CsEF-1α、CseIF-4α、CsGAPDH、CsPP2A、CsTBP、CsTIP41和CsUBC）在茶树不同叶位（新梢和老叶）、不同胁迫时间（0、1、3、7 d）的表达稳定性。结果显示,在氟胁迫条件下,茶树新梢中最优内参基因组合是CsEF-1α、CsTIP41、CsTBP和CsACTIN,老叶中最优内参基因组合是CsPP2A和CsUBC。利用筛选得到的最优内参基因组合分析氟输出蛋白基因（CsFEX）的表达情况,发现CsFEX在两个茶树品种的新梢和老叶中的表达趋势一致,说明筛选的内参组合可用于氟胁迫条件下茶树新梢和老叶中目的基因的检测。

Abstract

In order to screen the internal reference genes for quantitative real-time PCR analysis in tea leaves under fluoride stress, the low-fluoride cultivar ‘Fuding Dabaicha’ and the high-fluoride cultivar ‘Jinguanyin’ were used as experimental materials according to the fluoride evaluation results in these tea cultivars previously. The qRT-PCR technology combined with three Excel-based algorithms (geNorm, NormFinder and BestKeeper) were used to analyze the expression stabilities of eight candidate reference genes (CsACTIN, CsEF-1α, CseIF-4α, CsGAPDH, CsPP2A, CsTBP, CsTIP41 and CsUBC) in tea leaves (shoots and old leaves) under fluoride treatment (0.42 mmol·L^-1 NaF) for different time periods (0, 1, 3, 7 d). The results indicate that under fluoride stress, the optimal combination of reference genes in tea shoots was CsEF-1α, CsTIP41, CsTBP and CsACTIN and the optimal combination of reference genes in old leaves was CsPP2A and CsUBC. Moreover, to further confirm the stability of the selected reference genes, the expression levels of CsFEX in tea shoots and old leaves were analyzed using their corresponding optimal internal reference gene combinations. The expression profiles of CsFEX in tea shoots or old leaves between the two cultivars were consistent, indicating that the combinations of four and two internal reference genes were sufficient for normalizing the target gene expression in tea shoots and old leaves under fluoride stress, respectively.

导出引用

李庆会, 李睿, 温晓菊, 倪德江, 王明乐, 陈玉琼. 氟胁迫条件下茶树叶部实时荧光定量PCR分析中内参基因的筛选与验证[J]. 茶叶科学. 2024, 44(1): 27-36 https://doi.org/10.13305/j.cnki.jts.2024.01.001

LI Qinghui, LI Rui, WEN Xiaoju, NI Dejiang, WANG Mingle, CHEN Yuqiong. Selection and Validation of Internal Reference Genes for qRT-PCR Analysis under Fluoride Stress in Camellia sinensis Leaves[J]. Journal of Tea Science. 2024, 44(1): 27-36 https://doi.org/10.13305/j.cnki.jts.2024.01.001

中图分类号： S571.1 Q52

参考文献

[1] Fan Z P, Gao Y H, Wang W, et al.Prevalence of brick tea-type fluorosis in the Tibet Autonomous region[J]. Journal of Epidemiology, 2016, 26(2): 57-63.
[2] 沙济琴, 郑达贤. 茶树黄棪对氟的生物积累特征[J]. 福建茶叶, 1993(3): 25-28.
Sha J Q, Zheng D X.The bioaccumulation characteristics of fluoride in Camellia sinensis ‘Huangdan’[J]. Tea in Fujian, 1993(3): 25-28.
[3] 马立锋. 茶树对氟吸收累积特性及降氟措施研究[D]. 杭州: 浙江大学, 2004.
Ma L F.Study on accumulation and distribution of fluoride in tea plants (Camellia sinensis) and its measure of reducing fluoride uptake [D]. Hangzhou: Zhejiang University, 2004.
[4] Li C L, Ni D J.Effect of fluoride on the amino acid composition of tea leaves[J]. Fluoride, 2016, 49(3): 266-270.
[5] Yang X, Yu Z, Zhang B, et al.Effect of fluoride on the biosynthesis of catechins in tea [Camellia sinensis (L.) O. Kuntze] leaves[J]. Scientia Horticulturae, 2015, 184: 78-84.
[6] Ruan J Y, Ma L F, Shi Y Z, et al.Uptake of fluoride by tea plant (Camellia sinensis L) and the impact of aluminium[J]. Journal of the Science of Food and Agriculture, 2003, 83(13): 1342-1348.
[7] Bustin S A.Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems[J]. Journal of Molecular Endocrinology, 2002, 29(1): 23-39.
[8] 乔永刚, 王勇飞, 曹亚萍, 等. 药用蒲公英低温和高温胁迫下内参基因筛选与相关基因表达分析[J]. 园艺学报, 2020, 47(6): 1153-1164.
Qiao Y G, Wang Y F, Cao Y P, et al.Reference genes selection and related genes expression analysis under low and high temperature stress in Taraxacum officinale[J]. Acta Horticulturae Sinica, 2020, 47(6): 1153-1164.
[9] 马璐琳, 段青, 崔光芬, 等. 钝裂银莲花花色素合成相关基因qRT-PCR内参基因的筛选[J]. 园艺学报, 2021, 48(2): 377-388.
Ma L L, Duan Q, Cui G F, et al.Selection and validation of reference genes for qRT-PCR analysis of the correlated genes in flower pigments biosynthesis pathway of Anemone obtusiloba[J]. Acta Horticulturae Sinica, 2021, 48(2): 377-388.
[10] Nolan T, Hands R E, Bustin S A.Quantification of mRNA using real-time RT-PCR[J]. Nature Protocols, 2006, 1(3): 1559-1582.
[11] Bustin S A, Benes V, Garson J A, et al.The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments[J]. Clinical Chemistry, 2009, 55(4): 611-622.
[12] Wu Z J, Tian C, Jiang Q, et al.Selection of suitable reference genes for qRT-PCR normalization during leaf development and hormonal stimuli in tea plant (Camellia sinensis)[J]. Scientific Reports, 2016, 6: 19748. doi: 10.1038/srep19748.
[13] Nikalje G C, Srivastava A K, Sablok G, et al.Identification and validation of reference genes for quantitative real-time PCR under salt stress in a halophyte, Sesuvium portulacastrum[J]. Plant Gene, 2018, 13: 18-24.
[14] 杨婷, 薛珍珍, 李娜, 等. 铁十字秋海棠斑叶发育过程内参基因筛选及验证[J]. 园艺学报, 2021, 48(11): 2251-2261.
Yang T, Xue Z Z, Li N, et al.Reference genes selection and validation in Begonia masoniana leaves of different developmental stages[J]. Acta Horticulturae Sinica, 2021, 48(11): 2251-2261.
[15] 张秋悦, 刘昌来, 于晓晶, 等. 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, 2022, 49(7): 1557-1570.
Zhang Q Y, Liu C L, Yu X J, et al.Screening of reference genes for differentially expressed genes in Pyrus betulaefolia plant under salt stress by qRT-PCR[J]. Acta Horticulturae Sinica, 2022, 49(7): 1557-1570.
[16] Hao X, Horvath D P, Chao W S, et al.Identification and evaluation of reliable reference genes for quantitative real-time PCR analysis in tea plant (Camellia sinensis (L.) O. Kuntze)[J]. International Journal of Molecular Sciences, 2014, 15(12): 22155-22172.
[17] Wang M L, Li Q H, Xin H H, et al.Reliable reference genes for normalization of gene expression data in tea plants (Camellia sinensis) exposed to metal stresses[J]. PLoS One, 2017, 12(4): e0175863. doi: 10.1371/journal.pone.0175863.
[18] Zhou Z W, Deng H L, Wu Q Y, et al.Validation of reference genes for gene expression studies in post-harvest leaves of tea plant (Camellia sinensis)[J]. Peer Journal, 2019, 7: e6385. doi: 10.7717/peerj.6385.
[19] Xu W, Dong Y, Yu Y, et al.Identification and evaluation of reliable reference genes for quantitative real-time PCR analysis in tea plants under differential biotic stresses[J]. Scientific Reports, 2020, 10(1): 2429. doi: 10.1038/s41598-020-59168-z.
[20] Wang J X, Liu L L, Tang Q H, et al.Evaluation and selection of suitable qRT-PCR reference genes for light responses in tea plant (Camellia sinensis)[J]. Scientia Horticulturae, 2021, 289: 110488. doi: 10.21203/rs.3.rs-127369/v1.
[21] Zhang L Y, Zhang R M, Hu X L, et al.Reference gene selection for qRT-PCR analysis in the shoots and roots of Camellia sinensis var. sinensis under nutritional stresses[J]. Scientia Horticulturae, 2023, 320: 112237. doi: 10.1016/j.scienta.2023.112237.
[22] 孙美莲, 王云生, 杨冬青, 等. 茶树实时荧光定量PCR分析中内参基因的选择[J]. 植物学报, 2010, 45(5): 579-587.
Sun M L, Wang Y S, Yang D Q, et al.Reference genes for real-time fluorescence quantitative PCR in Camellia sinensis[J]. Chinese Bulletin of Botany, 2010, 45(5): 579-587.
[23] 郝姗. 茶树不同逆境条件下QRT-PCR适宜内参基因的筛选[D]. 南京: 南京农业大学, 2012.
Hao S.Selection of appropriate reference genes for expression studies in Camellia sinensis by real-time polymerase chain reaction [D]. Nanjing: Nanjing Agricultural University, 2012.
[24] 刘圆, 王丽鸳, 韦康, 等. 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, 2016, 36(1): 92-101.
Liu Y, Wang L Y, Wei K, et al.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, 2016, 36(1): 92-101.
[25] Wan Q, Xu R K, Li X H.Proton release by tea plant (Camellia sinensis L.) roots as affected by nutrient solution concentration and pH[J]. Plant Soil and Environment, 2012, 58(9): 429-434.
[26] Zhu J J, Xing A Q, Wu Z C, et al.CsFEX, a fluoride export protein gene from Camellia sinensis, alleviates fluoride toxicity in transgenic Escherichia coli and Arabidopsis thaliana[J]. Journal of Agricultural and Food Chemistry, 2019, 67(21): 5997-6006.
[27] 李永平, 叶新如, 王彬, 等. 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, 2021, 35(1): 60-71.
Li Y P, Ye X R, Wang B, et al.Cloning and selection evaluation of reference gene for quantitative real-time PCR in Hibiscus esculentus L.[J]. Journal of Nuclear Agricultural Sciences, 2021, 35(1): 60-71.
[28] 侯天泽, 易双双, 张志群, 等. 秋石斛RT-qPCR内参基因的筛选与验证[J]. 园艺学报, 2022, 49(11): 2489-2501.
Hou T Z, Yi S S, Zhang Z Q, et al.Selection and validation of reference genes for RT-qPCR in Phalaenopsis-type Dendrobium hybrid[J]. Acta Horticulturae Sinica, 2022, 49(11): 2489-2501.
[29] Vandesompele J, De Preter K, Pattyn F, et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes [J]. Genome Biology, 2002, 3(7): research0034.0031. doi: 10.1186/gb-2002-3-7-research0034.
[30] Andersen C L, Jensen J L, Ørntoft T F.Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets[J]. Cancer Research, 2004, 64(15): 5245-5250.
[31] Pfaffl M W, Tichopad A, Prgomet C, et al.Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper--Excel-based tool using pair-wise correlations[J]. Biotechnology Letters, 2004, 26(6): 509-515.
[32] Livak K J, Schmittgen T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2^-ΔΔCT method[J]. Methods, 2001, 25(4): 402-408.
[33] Gutierrez L, Mauriat M, Guenin S, et al.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 Biotechnology Journal, 2008, 6(6): 609-618.
[34] Bustin S A, Nolan T.Pitfalls of quantitative real-time reverse-transcription polymerase chain reaction[J]. Journal of Biomolecular Techniques, 2004, 15(3): 155-166.
[35] Derveaux S, Vandesompele J, Hellemans J.How to do successful gene expression analysis using real-time PCR[J]. Methods, 2010, 50(4): 227-230.
[36] 刘小飞, 于波, 黄丽丽, 等. 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, 2020, 47(12): 203-211.
Liu X F, Yu B, Huang L L, et al.Screening and validation of reference genes of Camellia azalea by quantitative real-time PCR[J]. Guangdong Agricultural Sciences, 2020, 47(12): 203-211.
[37] 刘瑞姣, Abdulrahman A.A.Amer, 高兴华, 等. 肌动蛋白结构与生物学功能的研究进展[J]. 中国细胞生物学学报, 2020, 42(10): 1870-1875.
Liu R J, Abdulrahman A, Gao X H, et al.Advances in research on the structure and biological function of actin[J]. Chinese Journal of Cell Biology, 2020, 42(10): 1870-1875.
[38] 王爽, 李海英. 植物E3泛素连接酶与非生物胁迫相关研究进展[J]. 中国农学通报, 2020, 36(29): 47-53.
Wang S, Li H Y.Plant E3 ubiquitin ligase and abiotic stress: research progress[J]. Chinese Agricultural Science Bulletin, 2020, 36(29): 47-53.