四川农业大学学报2025，Vol.43Issue(3)：618-629,12.DOI:10.16036/j.issn.1000-2650.202502562
基于转录组测序对元宝枫叶片内参基因的筛选与验证
Selection and Verification of Reference Genes for Leaves of Acer truncatum Based on Transcriptome Sequencing
摘要
Abstract
[Objective]To identify optimal reference genes for real-time quantitative PCR(RT-qPCR)analysis in Acer truncatum Bunge leaves.[Method]Twenty-six candidate reference genes from eight gene families,including ACT,EF1α,EXA1,GAPDH,H3,PUB,UBRP and ZFP,were initially screened from the transcriptome of drought stress,and their stability was evaluated by geNorm,Norm-Finder,BestKeeper and weighted average methods.Subsequently,eight stable members from these gene families were selected for RT-qPCR validation across different tissues(stems,leaves,petioles,wing fruits)and leaves developmental stages(green leaves,red leaves),with their stability further evaluated by geNorm,NormFinder,BestKeeper and RefFinder softwares.Furthermore,twelve anthocyanin synthe-sis pathway genes were analyzed to validate the stability of reference genes.[Result]Transcriptome analysis identified EXA1-1 and EXA1-2 as the most stable reference genes under drought stress.RT-qPCR analysis revealed ZFP-1 and GAPDH-1 exhibited the highest expression stability in different leaf organs(green leaves,petioles,red leaves),while EF1α-1 showed the poorest stability.Comprehensive soft-ware evaluation ranked ACT7 as the most suitable reference for all sample groups.When normalizing an-thocyanin pathway gene expression in green versus red leaves using ZFP-1,GAPDH-1 and EF1α-1,genes with large expression fold-changes showed consistent patterns across all three references,whereas genes with small fold-changes displayed marked deviations when normalized with the least stable EF1α-1.[Conclusion]Among the 26 candidates,ZFP-1 demonstrated consistent stability in both drought-stressed leaves and across leaf organs at different developmental stages,establishing it as an optimal ref-erence gene for A.truncatum leaf studies.关键词
元宝枫叶片/内参基因/转录组测序/实时荧光定量PCR/稳定性评价Key words
Acer truncatum leaves/reference genes/transcriptome sequencing/RT-qPCR/stability evaluation分类
农业科技引用本文复制引用
蒋仕淳,李建波,吕英民,夏永秀..基于转录组测序对元宝枫叶片内参基因的筛选与验证[J].四川农业大学学报,2025,43(3):618-629,12.基金项目
中国林业科学研究院中央级公益性科研院所基本科研业务费专项(CAFYBB2018QA004、CAFYBB2024MA016) （CAFYBB2018QA004、CAFYBB2024MA016）
国家自然科学基金(32101553). （32101553）
