草地早熟禾荧光定量PCR分析中内参基因的筛选  

Screening of reference genes for real-time fluorescence quantitative PCR in Kentucky bluegrass

作　　者：张兰[1] 檀鹏辉 滕珂[1] 闫蒙举 何春燕[1] 甘露[1] 尹淑霞[1] 

机构地区：[1]北京林业大学林学院草坪研究所,北京100083

出　　处：《草业学报》2017年第3期75-81,共7页Acta Prataculturae Sinica

基　　金：国家自然基金项目(31302016);深圳市科技计划项目(JSGG20160229155434792)资助

摘　　要：为筛选出草地早熟禾实时荧光定量中的最适内参基因,利用实时荧光定量PCR技术,分析检测6个传统内参基因ACT、GAPDH、UBQ、EF-1α、18s rRNA和β-tublin在草地早熟禾不同组织器官、叶片不同发育期、非生物胁迫和不同激素诱导下mRNA的表达差异情况。利用geNorm、NormFinder和BestKeeper软件综合评价6个候选内参基因的表达稳定性。结果表明,在不同组织、叶片不同发育期、激素诱导和非生物胁迫下,候选内参基因的表达稳定性存在差异。GAPDH在草地早熟禾不同组织器官中表达最为稳定;EF-1α在非生物胁迫下表达最为稳定;不同激素下首选β-tublin;在叶片不同发育期ACT表达最为稳定。综上所述,通过筛选出不同条件下适宜的内参基因不仅有助于提高草地早熟禾基因表达分析的准确性,也为早熟禾属植物其他内参基因的开发提供了理论参考。To select the most stable reference genes of Kentucky bluegrass （Poa pratensis ） for real- time quanti -tative PCR, we assessed the mRNA expression in different tissues of six traditional candidate reference genes at various leaf development stages, under abiotic stress, or following various hormone applications. Genes as-sessed included ACT, GAPDH, UBQ , EF_la,18s r RNA and β - tubulin . The expression stabilities of these six reference genes were evaluated by geNorm, NormFinder and BestKeeper. It was found that mRNA expression of the candidate reference genes differed significantly between the tissue types,developmental stages,hormone applications or under abiotic stress. In Kentucky bluegrass leaves, GAPDH is recommended as the reference gene for analyzing the mRNA expression in different tissues; EF -1a was the most stable gene under abiotic stress; p-tubul in was most consistent with different hormones; ACT was stably expressed in the different leaf development stages. In conclusion, use of the most appropriate reference genes under different conditions would enhance the accuracy analysis of gene expression in the Kentucky bluegrass. These findings may be ap-plicable when selecting reference genes for analysis of mRNA expression in other members of the Poa genus.

关 键 词：草地早熟禾 实时荧光定量PCR 内参基因 稳定性评价 

分 类 号：S688.4[农业科学—观赏园艺]