Liang Wenxian, Zou Xiaoxing, Carballar-Lejarazú Rebeca, Wu Lingjiao, Sun Weihong, Yuan Xueyuan, Wu Songqing, Li Pengfei, Ding Hui, Ni Lin, Huang Wei, Zou Shuangquan
1College of Forestry, Fujian Agriculture and Forestry University, Fuzhou, China.
2Fujian Colleges and Universities Engineering Research Institute of Conservation and Utilization of Natural Bioresources, Fujian Agriculture and Forestry University, Fuzhou, China.
Plant Methods. 2018 Jun 4;14:42. doi: 10.1186/s13007-018-0311-x. eCollection 2018.
Quantitative real-time reverse transcription-polymerase chain reaction has been widely used in gene expression analysis, however, to have reliable and accurate results, reference genes are necessary to normalize gene expression under different experimental conditions. Several reliable reference genes have been reported in plants of Traditional Chinese Medicine, but none have been identified for Hayata.
In this study, 12 candidate reference genes, including 3 common housekeeping genes and 9 novel genes based on Hayata transcriptome data were selected and analyzed in different tissues (root, branch, leaf, capsule and seed), capsule and seed development stages. Expression stability was calculated using geNorm and NormFinder, the minimal number of reference genes required for accurate normalization was calculated by Vn/Vn + 1 using geNorm. -- and were the two most stable reference genes for all samples, while and were the most stable reference genes for tissue samples. For seed development stages, and -- were the most stable genes, whereas and were identified as the two most stable genes in the capsule development stages. Two reference genes were sufficient to normalize gene expression across all sample sets.
Results of this study revealed that suitable reference genes should be selected for different experimental samples, and not all the common reference genes are suitable for different tissue samples and/or experimental conditions. In this study, we present the first data of reference genes selection for Hayata based on transcriptome data, our data will facilitate further studies in molecular biology and gene function on Hayata and other closely related species.
定量实时逆转录-聚合酶链反应已广泛应用于基因表达分析,然而,为了获得可靠和准确的结果,需要使用内参基因对不同实验条件下的基因表达进行标准化。在中药植物中已经报道了几种可靠的内参基因,但对于玉山柯(Hayata)尚未有相关报道。
在本研究中,基于玉山柯转录组数据选择了12个候选内参基因,包括3个常见管家基因和9个新基因,并在不同组织(根、枝、叶、果实和种子)、果实和种子发育阶段进行了分析。使用geNorm和NormFinder计算表达稳定性,使用geNorm通过Vn/Vn + 1计算准确标准化所需的最少内参基因数量。对于所有样本,[具体基因1]和[具体基因2]是最稳定的两个内参基因,而对于组织样本,[具体基因3]和[具体基因4]是最稳定的内参基因。在种子发育阶段,[具体基因5]和[具体基因6]是最稳定的基因,而在果实发育阶段,[具体基因7]和[具体基因8]被确定为两个最稳定的基因。两个内参基因足以对所有样本集的基因表达进行标准化。
本研究结果表明,应针对不同的实验样本选择合适的内参基因,并非所有常见的内参基因都适用于不同的组织样本和/或实验条件。在本研究中,我们基于转录组数据首次提供了玉山柯内参基因选择的数据,我们的数据将有助于对玉山柯及其他近缘物种进行进一步的分子生物学和基因功能研究。