《植物生理学报》 2014, 50(12): 1903-1911

苹果着色期实时定量PCR内参基因的筛选和验证

樊连梅^1,2,*, 王超^1,*, 刘更森², 原永兵^2,**

青岛农业大学¹生命科学学院/山东省高校植物生物技术重点实验室, ²园艺学院/青岛市现代农业质量与安全工程重点实验室, 山东青岛266109

通信作者：樊连梅；E-mail: yyb@qau.edu.cn；Tel: 0532-88030513

摘　要：

苹果果实着色期基因表达水平的变化对果实品质形成具有重要影响, 选择适合的内参基因是提高实时荧光定量PCR分析准确性的首要条件。本试验以‘富士’苹果着色过程中不同取样时间的果皮为材料, 通过qRT-PCR分析了常用候选持家基因β-actin、EF-1α、GAPDH和18S rRNA的表达变化, 借助geNorm和NormFinder程序筛选出在果实着色期qRT-PCR分析的理想内参基因。结果表明, EF-1α的表达水平高且最稳定, 其次是18S rRNA, 而β-actin和GAPDH的相对表达水平较低; 同时, 用筛选的内参基因EF-1α和18S rRNA分析苹果花青苷合成途径的二氢黄酮醇-4-还原酶基因的表达水平, 其表达规律比较一致, 均呈现正态分布。因此, EF-1α和18S rRNA是研究苹果着色期表达的理想的内参基因。

关键词：‘富士’苹果; 内参基因; 实时荧光定量PCR; geNorm程序; NormFinder程序

收稿：2014-08-25   修定：2014-11-08

资助：青岛农业大学高层次人才科研基金资助(630929)、青岛农业大学大学生科技创新基金项目、公益性行业(农业)科研专项项目(201203075-04)和山东省现代农业产业体系水果创新团队建设基金(SDAIT-03-022-10)。 * 共同第一作者。

Screening and Validation of Reference Genes for Real-Time Fluorescence Quantitative PCR during Coloring Period in Apple (Malus domestica)

FAN Lian-Mei^1,2,*, WANG Chao^1,*, LIU Geng-Sen², YUAN Yong-Bing^2,**

¹College of Life Sciences/Key Lab of Plant Biotechnology in Universities of Shandong, ²College of Horticulture/Qingdao Key Lab of Modern Agriculture Quality and Safety Engineering, Qingdao Agricultural University, Qingdao, Shandong 266109, China

Corresponding author: FAN Lian-Mei; E-mail: yyb@qau.edu.cn; Tel: 0532-88030513

Abstract:

The change of gene expression level during coloring period is crucial for the formation of apple fruit quality. The selection of a suitable reference gene is an important factor for accurate gene expression analysis by real-time fluorescence quantitative PCR (qRT-PCR). In this study, peels of apple (Malus domestica cv. ‘Fuji’) were taken as materials sampled at different time during coloring stage. The expression level of four commonly used housekeeping genes β-actin, EF-1α, GAPDH and 18S rRNA were studied by qRT-PCR, and reliable reference gene were screened to use for gene expression during coloring stage of fruit by geNorm and NormFinder software. The results showed that the expression level of EF-1α was highest and most stable, followed by 18S rRNA. However, the expression level of β-actin and GAPDH were relatively low. Meanwhile, the expression of flavonoid-4-reductase gene (MdDFR) in the pathway of apple anthocyanin biosynthesis was analyzed, and the results indicated that the variation tendency of MdDFR was exactly consistent and presented normal distribution using EF-1α and 18S rRNA as reference gene. Therefore, EF-1α and 18S rRNA are suitable reference genes and could be used to normalize mRNA levels in peel tissues of apple during coloring period.

Key words: Malus domestica cv. ‘Fuji’; coloring stage; reference gene; qRT-PCR; geNorm software; Norm-Finder software