Selection and Validation of Reference Genes for qRT-PCR in Bothriochloa ischaemum seedlings under Abiotic Stresses

doi:10.11733/j.issn.1007-0435.2025.11.008

Acta Agrestia Sinica ›› 2025, Vol. 33 ›› Issue (11): 3559-3570.DOI: 10.11733/j.issn.1007-0435.2025.11.008

Selection and Validation of Reference Genes for qRT-PCR in Bothriochloa ischaemum seedlings under Abiotic Stresses

GAO Shou-yu, XIANG Qing-yuan, MA Jing-ru, LI Yu-ying, XIA Fang-shan

1. College of Grassland Science, Shanxi Agricultural University, Taigu, Shanxi Province 030801, China

Received:2025-01-20 Revised:2025-03-04 Published:2025-11-13

非生物胁迫下苗期白羊草荧光定量PCR内参基因的筛选及验证

高守舆, 向清源, 马靖茹, 李钰莹, 夏方山

1. 山西农业大学草业学院, 山西太谷 030801

通讯作者: 李钰莹,E-mail:liyuy_ing@163.com
作者简介:高守舆（2000-），男，汉族，内蒙古巴彦淖尔人，硕士研究生，主要从事植物逆境研究，E-mail：gaoshouyu2022@163.com
基金资助:
山西农业大学科技创新基金项目：芒荻类植物修复山西重金属水污染的潜力分析（2017YJ06）；山西农业大学“十四五”生物育种工程项目（YZGC134）中央引导地方科技发展资金项目（YDZJSX2022C011）；山西省重点研发计划项目（201903D221091）资助

Abstract

Abstract: Due to its high sensitivity， strong specificity， and simple operation， real-time fluorescence quantitative PCR （qRT-PCR） is an important tool for gene expression analysis. In order to identify suitable reference genes for qRT-PCR in Bothriochloa ischaemum under abiotic stresses， this study selected seven candidate reference genes （18S， GAPDH， PAL， EF-1a， ACT， UBQ and TUB） based on previous transcriptome data. The expression levels （Ct values） of these seven candidates in leaves and roots were determined under salt， drought， and cold stresses using qRT-PCR. Their stability was then evaluated using the comparative ΔCt method and four software programs （geNorm， NormFinder， BestKeeper and RefFinder）. The results showed that the optimal reference genes varied with different stresses and tissues. Under drought stress， the best reference genes were ACT and PAL in leaves， and 18S and GAPDH in roots. Under salt stress， PAL and EF-1a were most stable in leaves， whereas TUB and UBQ were most stable in roots. Under cold stress， ACT and GAPDH were the top candidates in leaves， while PAL and ACT were the best in roots. Finally， the expression of the salt tolerance-related gene BiADH28 was validated using the selected reference genes， confirming the reliability of the screening results. This study provides a solid foundation for gene expression analysis of B. ischaemum under abiotic stresses.

Key words: Bothriochloa ischaemum, Reference genes, Quantitative real-time PCR （qRT-PCR）, Abiotic stress

摘要： 荧光定量PCR技术因其高灵敏度、强特异性、操作简洁等优点，是基因表达分析的重要工具。为筛选出在非生物胁迫下白羊草（Bothriochloa ischaemum）实时荧光定量PCR（qRT-PCR）适用的内参基因，本研究基于前期的转录组数据，选取了7个候选内参基因（18S，GAPDH，PAL，EF-1a，ACT，UBQ和TUB）。在盐胁迫、干旱胁迫和冷胁迫下，采用qRT-PCR技术检测了白羊草叶片和根系中这7个候选基因的Ct值，并使用比较ΔCt法和4个分析软件（geNorm，NormFinder，BestKeeper和RefFinder）对7个候选内参基因进行稳定性排序。结果表明，不同胁迫下、不同组织中的最佳内参基因有所不同。干旱胁迫下，叶片中最佳内参基因为ACT和PAL，根系中为18S和GAPDH；盐胁迫下，叶片中最佳内参基因为PAL和EF-1a，根系中为TUB和UBQ；冷胁迫下，叶片中最佳内参基因为ACT和GAPDH，根系中为PAL和ACT。最后，利用筛选出的内参基因对耐盐相关基因BiADH28的表达进行了验证，结果表明内参基因筛选结果可靠。本研究为白羊草在非生物胁迫条件下的基因表达分析提供了坚实的基础。

关键词: 白羊草, 内参基因, 荧光定量PCR, 非生物胁迫

CLC Number:

S543.9

GAO Shou-yu, XIANG Qing-yuan, MA Jing-ru, LI Yu-ying, XIA Fang-shan. Selection and Validation of Reference Genes for qRT-PCR in Bothriochloa ischaemum seedlings under Abiotic Stresses[J]. Acta Agrestia Sinica, 2025, 33(11): 3559-3570.

高守舆, 向清源, 马靖茹, 李钰莹, 夏方山. 非生物胁迫下苗期白羊草荧光定量PCR内参基因的筛选及验证[J]. 草地学报, 2025, 33(11): 3559-3570.

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Selection and Validation of Reference Genes for qRT-PCR in Bothriochloa ischaemum seedlings under Abiotic Stresses

非生物胁迫下苗期白羊草荧光定量PCR内参基因的筛选及验证

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