Genome-wide identification of internal reference genes for normalization of gene expression values during endosperm development in wheat
Genome-wide identification of internal reference genes for normalization of gene expression values during endosperm development in wheat
复制标题
全基因组鉴定内参基因，用于小麦胚乳发育过程中基因表达值的标准化
DOI：
10.1007/s13353-019-00503-0
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发表时间：
2019-11-01
影响因子：
2.4
通讯作者：
Li, Xiaohui
中科院分区：
文献类型：
--
作者：
Mu, Junyi;Chen, Lin;Li, Xiaohui
稳定表达的内参基因对于比较表达分析中的标准化是必不可少的。然而，基因表达在物种、生物体、组织、发育阶段、应激和处理之间变化显著。因此，在小麦胚乳发育过程中寻找稳定表达的参考基因对胚乳基因的表达分析具有重要意义。作为首次系统地筛选小麦胚乳不同发育阶段的参考基因的研究，基于高通量RNA测序数据从76个相对稳定的基因中筛选出9个基因。这些候选基因和5个传统的参考基因的表达稳定性进行了评估，通过实时定量PCR结合三个独立的算法：geNorm，Normalization，和BestKeeper。结果表明，在小麦胚乳发育过程中，ATG 8d最稳定，其次是Ta 54227，而常用作内参的管家基因GAPDH最不稳定，ATG 8d和Ta 54227共同构成了内参基因的最佳组合。对发育胚乳中麦谷蛋白基因的比较表达分析表明，对发育胚乳中的ATG 8进行归一化处理可以实现可靠的定量。本研究所鉴定的稳定表达基因可作为小麦胚乳基因表达分析的内参，特别是与养分和养分合成相关的基因。
Internal reference genes that are stably expressed are essential for normalization in comparative expression analyses. However, gene expression varies significantly among species, organisms, tissues, developmental stages, stresses, and treatments. Therefore, identification of stably expressed reference genes in developmental endosperm of bread wheat is important for expression analysis of endosperm genes. As the first study to systematically screen for reference genes across different developmental stages of wheat endosperm, nine genes were selected from among 76 relatively stable genes based on high-throughput RNA sequencing data. The expression stability of these candidate genes and five traditional reference genes was assessed by real-time quantitative PCR combined with three independent algorithms: geNorm, NormFinder, and BestKeeper. The results showed thatATG8dwas the most stable gene during wheat endosperm development, followed byTa54227, while the housekeeping geneGAPDH, commonly used as an internal reference, was the least stable.ATG8dandTa54227together formed the optimal combination of reference genes. Comparative expression analysis of glutenin genes indicated that credible quantification could be achieved by normalization againstATG8din developmental endosperm. The stably expressed gene characterized here can act as a proper internal reference for expression analysis of wheat endosperm genes, especially nutrient- and nutrient synthesis–related genes.