文献类型：期刊文献

中文题名：基于转录组测序对元宝枫叶片内参基因的筛选与验证

英文题名：Selection and Verification of Reference Genes for Leaves of Acer truncatum Based on Transcriptome Sequencing

作者：蒋仕淳[1] 李建波[2] 吕英民[1] 夏永秀[2]

第一作者：蒋仕淳

机构：[1]北京林业大学园林学院,北京100083;[2]中国林业科学研究院华北林业实验中心/北京九龙山暖温带森林国家长期科研基地,北京102300

年份：2025

卷号：43

期号：3

起止页码：618-629

中文期刊名：四川农业大学学报

外文期刊名：Journal of Sichuan Agricultural University

收录：;北大核心:【北大核心2023】；

基金：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);国家自然科学基金(32101553)。

语种：中文

中文关键词：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

外文关键词：Acer truncatum leaves;reference genes;transcriptome sequencing;RT-qPCR;stability evaluation

分类号：S687.1

摘要：【目的】筛选出元宝枫(Acer truncatum Bunge)叶片中理想的实时荧光定量PCR(RT-qPCR)内参基因。【方法】从干旱胁迫转录组中筛选出ACT、EF1α、EXA1、GAPDH、H3、PUB、UBRP和ZFP共8个基因家族26个候选内参基因,利用geNorm、NormFinder、BestKeeper及加权平均法对其稳定性进行评价,在此基础上,分别从8个基因家族中挖掘较稳定的8个成员,通过RT-qPCR检测其在不同组织(茎、叶片、叶柄、翅果)及不同发育时期叶片(绿叶、红叶)中的表达,并由geNorm、NormFinder、BestKeeper和RefFinder等软件评估其稳定性。进一步通过12个花青素合成途径基因,验证内参基因的稳定性。【结果】在转录组中,筛选出EXA1-1与EXA1-2作为干旱胁迫叶片中稳定表达的内参基因。在RT-qPCR检测中,ZFP-1与GAPDH-1在不同发育时期叶器官中(绿叶、叶柄、红叶)稳定性最高,EF1α-1稳定性最差。在软件评估结果中,ACT7是所有样本组中最适内参。用ZFP-1、GAPDH-1及EF1α-1校正花青素合成途径基因在绿叶与红叶中的表达,发现表达量差异倍数较大的基因用3个内参校正后的表达趋势一致,而表达量差异倍数较小的基因用最不稳定的EF1α-1校正后存在明显偏差。【结论】在26个候选内参基因中,综合软件分析排名并结合RT-qPCR验证,ZFP-1是在干旱胁迫叶片以及不同发育时期叶器官中均稳定表达的基因,可作为元宝枫叶片的理想内参基因。
【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,NormFinder,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 synthesis 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 software evaluation ranked ACT7 as the most suitable reference for all sample groups.When normalizing anthocyanin 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 reference gene for A.truncatum leaf studies.