Institute of Biotechnology, Provincial Key Laboratory of Agrobiology, Jiangsu Academy of Agricultural Sciences, Nanjing, 210014, China.
Plant Mol Biol. 2014 Nov;86(4-5):543-54. doi: 10.1007/s11103-014-0246-1. Epub 2014 Sep 11.
In the wake of recent progress of high throughput transcriptome profiling technologies, extensive housekeeping gene mining has been conducted in humans. However, very few studies have been reported in maize (Zea mays L.), an important crop plant, and none were conducted on a genome -wide level. In this study, we surveyed housekeeping genes throughout the maize transcriptome using RNA-seq and microarray techniques, and validated the housekeeping profile with quantitative polymerase chain reaction (qPCR) under a series of conditions including different genotypes and nitrogen supplies. Seven microarray datasets and two RNA-seq libraries representing 40 genotypes at more than 20 developmental stages were selected to screen for commonly expressed genes. A total of 1,661 genes showed constitutive expression in both microarray and RNA-seq datasets, serving as our starting housekeeping gene candidates. To determine for stably expressed housekeeping genes, NormFinder was used to select the top 20 % invariable genes to be the more likely candidates, which resulted in 48 and 489 entries from microarray and RNA-seq data, respectively. Among them, nine genes (2OG-Fe, CDK, DPP9, DUF, NAC, RPN, SGT1, UPF1 and a hypothetical protein coding gene) were expressed in all 40 maize diverse genotypes tested covering 16 tissues at more than 20 developmental stages under normal and stress conditions, implying these as being the most reliable reference genes. qPCR analysis confirmed the stable expression of selected reference gene candidates compared to two widely used housekeeping genes. All the reference gene candidates showed higher invariability than ACT and GAPDH. The hypothetical protein coding gene exhibited the most stable expression across 26 maize lines with different nitrogen treatments with qPCR, followed by CDK encoding the cyclin-dependent kinase. As the first study to systematically screen for housekeeping genes in maize, we identified candidates by examining the transcriptome atlas generated from RNA-seq and microarray technologies. The nine top-ranked qPCR-validated novel housekeeping genes provide a valuable resource of reference genes for maize gene expression analysis.
在高通量转录组分析技术取得最新进展的背景下，人们对人类的管家基因进行了广泛的挖掘。然而，在重要的作物玉米（Zea mays L.）中，仅有很少的研究报道，且均未在全基因组水平上进行。本研究采用 RNA-seq 和微阵列技术对玉米转录组中的管家基因进行了全面调查，并通过定量聚合酶链反应（qPCR）在不同基因型和氮供应条件下对管家基因表达谱进行了验证。我们选择了 7 个微阵列数据集和 2 个 RNA-seq 文库，代表 20 多个发育阶段的 40 个基因型，以筛选表达丰度高的基因。共有 1661 个基因在微阵列和 RNA-seq 数据集中均呈组成性表达，作为我们的起始管家基因候选基因。为了确定稳定表达的管家基因，我们使用 NormFinder 选择前 20%不变的基因作为更可能的候选基因，结果从微阵列和 RNA-seq 数据中分别得到 48 个和 489 个条目。其中，有 9 个基因（2OG-Fe、CDK、DPP9、DUF、NAC、RPN、SGT1、UPF1 和一个假定的蛋白质编码基因）在正常和胁迫条件下，在涵盖 16 种组织的 40 个不同玉米基因型中均有表达，表明这些基因是最可靠的参考基因。qPCR 分析证实，与两个广泛使用的管家基因相比，所选参考基因候选基因的表达更为稳定。与 ACT 和 GAPDH 相比，所有候选参考基因的稳定性更高。在具有不同氮处理的 26 个玉米品系中，用 qPCR 检测到假定的蛋白质编码基因表达最稳定，其次是编码细胞周期蛋白依赖性激酶的 CDK 基因。作为首次系统筛选玉米管家基因的研究，我们通过检查 RNA-seq 和微阵列技术生成的转录组图谱来鉴定候选基因。经过 qPCR 验证的 9 个排名靠前的新型管家基因是玉米基因表达分析的宝贵参考基因资源。
