《植物生理学报》 2015, 51(9): 1457-1464

多重胁迫下玉米实时定量PCR内参基因的筛选与验证

姜婷, 苏乔^*, 安利佳

大连理工大学生命科学与技术学院, 辽宁大连116024

通信作者：苏；E-mail: dsuqiao@dlut.edu.cn；Tel: 13940903690

摘　要：

实时定量PCR (qPCR)技术广泛应用于基因表达量的检测, 为了保证其结果的准确性, 选择合适的内参基因是必不可少的环节。本研究分析了11个玉米候选内参基因的稳定性, 其中7个是常用内参基因(GAPDH、ACT、EF-1α、γ-TUB、18S rRNA、5S rRNA和U6 snRNA), 4个是microRNA (zma-miR171a、zma-miR171b、zma-miR172和zma-miR159a/b)。通过计算11个基因在正常生长条件与干旱、盐和碱共胁迫条件下的循环阈值(C_t), 输入软件geNorm、NormFinder和BestKeeper中进行分析。结果表明, microRNA比其他候选内参基因稳定, 其中zma-miR172和zma-miR171a最稳定, 18S rRNA最不稳定。另外, 选择zma-miR397a及其靶基因LAC4对候选内参基因进行验证, 发现不合适的内参基因可能会导致错误的结果。

关键词：玉米; 内参基因; 实时定量PCR; 干旱胁迫; 盐胁迫; 碱胁迫

收稿：2015-03-12   修定：2015-09-08

资助：辽宁省科学技术计划项目(2011208001)。

Screening and Validation of Reference Genes of qPCR in Maize under Multiple Stresses

JIANG Ting, SU Qiao^*, AN Li-Jia

School of Life Science and Biotechnology, Dalian University of Technology, Dalian, Liaoning 116024, China

Corresponding author: SU Qiao; E-mail: dsuqiao@dlut.edu.cn; Tel: 13940903690

Abstract:

qPCR is a powerful and widely used technique for the measurement of different gene expression levels. However, it is crucial to choose a suitable gene as an internal control for the proper analysis. In the present study, we analyzed the expression patterns of 11 genes which include seven common reference genes (GAPDH, ACT, EF-1α, γ-TUB, 18S rRNA, 5S rRNA and U6 snRNA) and four microRNAs (zma-miR171a, zma-miR171b, zma-miR172 and zma-miR159a/b) in maize (Zea mays) samples. Expression level of these genes was observed from two experimental conditions i.e. normal growth condition and co-stress of drought, salt and alkali. The threshold cycle (C_t) values of each sample were analyzed using geNorm, NormFinder and BestKeeper. In this study, the expression of microRNAs was found to be more stable than other reference genes. The results revealed that zma-miR172 and zma-miR171a were highly stable compared to other reference genes while 18S rRNA was the most unstable one in both experimental conditions. The expression levels of zma-miR397a and its target gene LAC4 (laccase 4) were used to confirm the effect of candidate reference genes, showing that the use of an inappropriate reference gene could induce erroneous results.

Key words: Zea mays; reference genes; qPCR; drought stress; salt stress; alkali stress