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虫害胁迫下豇豆不同组织中实时荧光定量PCR内参基因的筛选

引用本文：高扬,何云川,何微,周瀛,祝增荣.虫害胁迫下豇豆不同组织中实时荧光定量PCR内参基因的筛选.植物保护学报,2025,52(2):446-455

DOI：10.13802/j.cnki.zwbhxb.2025.2024082

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作者	单位	E-mail
高扬	浙江大学海南研究院, 三亚 572025 浙江大学农业与生物技术学院昆虫科学研究所, 杭州 310058
何云川	浙江大学海南研究院, 三亚 572025 浙江大学农业与生物技术学院昆虫科学研究所, 杭州 310058 海军特色医学中心, 上海 200433
何微	浙江大学海南研究院, 三亚 572025 浙江大学农业与生物技术学院昆虫科学研究所, 杭州 310058
周瀛	浙江大学海南研究院, 三亚 572025	yzhyzb@zju.edu.cn
祝增荣	浙江大学海南研究院, 三亚 572025 浙江大学农业与生物技术学院昆虫科学研究所, 杭州 310058

中文摘要:为筛选适用于分析虫害处理下豇豆Vigna unguiculata根、茎、叶中和在不同组织间的内参基因，取三叶草斑潜蝇Liriomyza trifolii为害后的豇豆根、茎和叶样品，提取其总RNA并合成cDNA，根据虫害胁迫下大豆内参基因稳定性研究选择ACT11、CYP2、ELF1A、ELF1B、G6PD、TUA3、TUB4和UBC4作为候选基因，对这8个候选内参基因进行引物设计，检测引物的特异性、单一性和扩增效率，利用引物进行实时荧光定量PCR分析，利用GeNorm、NormFinder和BestKeeper三种常用软件筛选在豇豆根、茎和叶中表达稳定的内参基因，并分析同一内参基因在豇豆不同组织间的表达稳定性。结果表明：设计的8对候选内参基因引物均具有较高的特异性，扩增效率在104.84%~110.38%之间，扩增效果良好； 3种软件因算法逻辑差异导致筛选出的豇豆最优内参基因存在明显差异，经ReFinder软件综合评价确定CYP2为虫害处理下豇豆根、茎和叶中最稳定的内参基因，TUA3为不同组织间最稳定的内参基因；此外，CYP2的表达丰度在根、茎和叶中均最高。因此，建议采用CYP2作为内参基因分析虫害处理下豇豆根、茎、叶组织中目标基因的表达量，采用TUA3作为内参基因分析豇豆不同组织间目标基因的表达量。

中文关键词:豇豆虫害内参基因实时荧光定量PCR 引物特异性单一性扩增效率

Screening of reference genes for real-time quantitative PCR in different tissues of cowpea under pest stress

Author Name	Affiliation	E-mail
Gao Yang	Hainan Institute, Zhejiang University, Sanya 572025, Hainan Province, China Institute of Insect Sciences, College of Agriculture & Biotechnology, Zhejiang University, Hangzhou 310058, Zhejiang Province, China
He Yunchuan	Hainan Institute, Zhejiang University, Sanya 572025, Hainan Province, China Institute of Insect Sciences, College of Agriculture & Biotechnology, Zhejiang University, Hangzhou 310058, Zhejiang Province, China Naval Medical Centre of Chinese People' s Liberation Army, Shanghai 200433, China
He Wei	Hainan Institute, Zhejiang University, Sanya 572025, Hainan Province, China Institute of Insect Sciences, College of Agriculture & Biotechnology, Zhejiang University, Hangzhou 310058, Zhejiang Province, China
Zhou Ying	Hainan Institute, Zhejiang University, Sanya 572025, Hainan Province, China	yzhyzb@zju.edu.cn
Zhu Zengrong	Hainan Institute, Zhejiang University, Sanya 572025, Hainan Province, China Institute of Insect Sciences, College of Agriculture & Biotechnology, Zhejiang University, Hangzhou 310058, Zhejiang Province, China

Abstract:To screen reference genes for analyzing cowpea roots, stems, and leaves under insect herbivory and across tissues, total RNA was extracted from cowpea root, stem, and leaf samples infested by American serpentine leafminer Liriomyza trifolii, followed by cDNA synthesis. Based on previous studies of reference gene stability in soybean under insect herbivory, eight candidate genes (ACT11, CYP2, ELF1A, ELF1B, G6PD, TUA3, TUB4, and UBC4) were selected for primer design. The specificity, uniqueness, and amplification efficiency of the primers were validated, and qPCR analysis was conducted using these primers. The expression stability of the reference genes in cowpea roots, stems, and leaves was evaluated using three software packages (GeNorm, NormFinder, and BestKeeper), and their cross-tissue stability was also analyzed. Results showed that the eight candidate reference gene primers exhibited high specificity with amplification efficiencies ranging from 104.84% to 110.38%, indicating efficient amplification. Significant discrepancies in optimal reference gene selection were observed among the three software programs due to their distinct algorithms. Comprehensive evaluation using ReFinder software identified CYP2 as the most stable reference gene in cowpea roots, stems, and leaves under insect herbivory, while TUA3 showed optimal stability across different tissues. Additionally, CYP2 displayed the highest expression abundance in roots, stems, and leaves. Therefore, CYP2 is recommended as the reference gene for analyzing target gene expression in cowpea roots, stems, and leaves under insect herbivory, whereas TUA3 is suitable for cross-tissue gene expression analysis in cowpea.

keywords:cowpea insect herbivory reference gene real-time quantitative PCR primer specificity singularity amplification efficiency

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