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

茶叶科学 >

2024 , Vol. 44 >Issue 1: 27 - 36

DOI: https://doi.org/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

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华中农业大学园艺林学学院,果蔬园艺作物种质创新与利用全国重点实验室,湖北武汉 430070

李庆会,女,博士研究生,主要从事茶叶安全生产与品质调控方面的研究。

收稿日期: 2023-12-16

修回日期: 2024-01-03

网络出版日期: 2024-03-13

基金资助

国家自然科学基金项目（31972463）

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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

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National Key Laboratory for Germplasm Innovation and Utilization of Horticultural Crops, College of Horticulture and Forestry Sciences, Huazhong Agricultural University, Wuhan 430070, China

Received date: 2023-12-16

Revised date: 2024-01-03

Online published: 2024-03-13

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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。

关键词： 茶树; 氟; 内参基因; 实时荧光定量PCR; 基因表达

本文引用格式

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 . DOI: 10.13305/j.cnki.jts.2024.01.001

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.

Key words： Camellia sinensis; fluoride; reference genes; quantitative real-time PCR; gene expression

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