大豆实时荧光定量PCR内参基因的筛选与验证  

Screening and validation of reference genes for real-time quantitative PCR in soybean

作　　者：张芷睿 张耀华[1] 王秋实 于慧[1] 杨素欣[1] ZHANG Zhirui;ZHANG Yaohua;WANG Qiushi;YU Hui;YANG Suxin(Key Laboratory of Soybean Molecular Design Breeding,Northeast Institute of Geography and Agroecology,Chinese Academy of Sciences,Changchun 130102,China;University of Chinese Academy of Sciences,Beijing 100049,China)

机构地区：[1]中国科学院东北地理与农业生态研究所,大豆分子设计育种重点实验室,长春130102 [2]中国科学院大学,北京100049

出　　处：《植物生理学报》2020年第9期1963-1973,共11页Plant Physiology Journal

基　　金：国家自然科学基金(31571692和31971901)。

摘　　要：选择合适的内参基因可以提高定量分析中目标基因表达的准确性。本研究选取大豆(Glycine max)不同发育时期的18个组织样本,利用实时荧光定量PCR技术,分析了大豆60S、ABC、ACT2/7、ACT11、CYP2、ELF1A、ELF1B、Fbox、G6PD、MTP、PEPKR1、TUB4、TUA5、UBC4等14个内参基因在不同组织中表达量的变化,并利用geNorm、NormFinder和BestKeeper三种软件对各基因在四个重要发育时期以及在叶片组织中的表达稳定性进行了评价。研究表明:大豆内参基因Fbox、ABC、G6PD和ACT11分别为出苗期(VE)、第一片三叶期(V1)、始花期(R1)和初荚期(R3)最稳定的内参基因;60S为叶片组织中最稳定的内参基因;在全部样本的整体评价中,Fbox为最稳定的内参基因。此外,通过分析大豆噻唑合成酶基因GmPGL1的表达量,进一步证明了内参基因筛选结果的可靠性和在大豆基因表达分析中的适用性。本研究为准确定量分析大豆关键发育时期不同组织中的基因表达提供参考基因。Appropriate reference genes can improve the accuracy of target gene expression in quantitative analysis.In this study,the expression of fourteen candidate reference genes(60 S,ABC,ACT2/7,ACT11,CYP2,ELF1 A,ELF1 B,Fbox,G6 PD,MTP,PEPKR1,TUB4,TUA5 and UBC4)were analyzed in eighteen tissues from different development stages of soybean(Glycine max)by real-time quantitative PCR(RT-qPCR),and their expression stabilities in four development stages and leaf tissues were evaluated by three algorithms(geNorm,NormFinder and BestKeeper).The results show that Fbox,ABC,G6 PD and ACT11 were the most stable reference genes expressed in VE,V1,R1 and R3 stages,respectively.60 S was the most consistent reference gene expressed in leaves.Fbox was the most stable reference gene expressed in all studied tissues.In addition,the result of thiazole synthase gene(GmPGL1)expression validates that the recommended reference genes were reliable and applicable for gene expression analysis in soybean.These findings would provide a set of reference genes for accurately quantifying gene expression in various tissues of soybean at the main development stages.

关 键 词：大豆 内参基因 发育时期 实时荧光定量PCR 

分 类 号：S565.1[农业科学—作物学]