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首页> 美国卫生研究院文献>Frontiers in Genetics >Genome-Wide Identification and Evaluation of New Reference Genes for Gene Expression Analysis Under Temperature and Salinity Stresses in Ciona savignyi
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Genome-Wide Identification and Evaluation of New Reference Genes for Gene Expression Analysis Under Temperature and Salinity Stresses in Ciona savignyi

机译：盐和盐胁迫下全基因组鉴定和温度和盐分胁迫下基因表达分析新参考基因的评估

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Rapid adaptation/accommodation to changing environments largely contributes to maximal survival of invaders during biological invasions, usually leading to success in crossing multiple barriers and finally in varied environments in recipient habitats. Gene expression is one of the most important and rapid ways during responses to environmental stresses. Selection of proper reference genes is the crucial prerequisite for gene expression analysis using the common approach, real-time quantitative PCR (RT-qPCR). Here we identified eight candidate novel reference genes from the RNA-Seq data in an invasive model ascidian Ciona savignyi under temperature and salinity stresses. Subsequently, the expression stability of these eight novel reference genes, as well as other six traditionally used reference genes, was evaluated using RT-qPCR and comprehensive tool RefFinder. Under the temperature stress, two traditional reference genes, ribosomal proteins S15 and L17 (RPS15, RPL17), and one novel gene Ras homolog A (RhoA), were recommended as the top three stable genes, which can be used to normalize target genes with a high and moderate expression level, respectively. Under the salinity stress, transmembrane 9 superfamily member (TMN), MOB kinase activator 1A-like gene (MOB) and ubiquitin-conjugating enzyme (UBQ2) were suggested as the top three stable genes. On the other hand, several commonly used reference genes such as α-tubulin (TubA), β-tubulin (TubB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) showed unstable expressions, thus these genes should not be used as internal controls for gene expression analysis. We also tested the expression level of an important stress response gene, large proline-rich protein bag6-like gene (BAG) using different reference genes. As expected, we observed different results and conclusions when using different normalization methods, thus suggesting the importance of selection of proper reference genes and associated normalization methods. Our results provide a valuable reference gene resource for the normalization of gene expression in the study of environmental adaptation/accommodation during biological invasions using C. savignyi as a model.
机译：快速适应/适应不断变化的环境在很大程度上有助于生物入侵期间入侵者的最大生存，通常会成功克服多种障碍，并最终在受体生境的各种环境中取得成功。基因表达是对环境压力作出反应时最重要和最快速的方法之一。选择合适的参考基因是使用通用方法实时定量PCR（RT-qPCR）进行基因表达分析的关键前提。在这里，我们在温度和盐度胁迫下，从侵袭性模型海鞘Ciona savignyi中的RNA-Seq数据中鉴定了八个候选新参考基因。随后，使用RT-qPCR和综合工具RefFinder评估了这8个新参考基因以及其他6个传统使用的参考基因的表达稳定性。在温度胁迫下，推荐使用两个传统的参考基因核糖体蛋白S15和L17（RPS15，RPL17），以及一个新的基因Ras同源物A（RhoA）作为最稳定的三个基因，可用于对靶基因进行标准化。高和中等表达水平。在盐分胁迫下，跨膜9超家族成员（TMN），MOB激酶激活因子1A样基因（MOB）和泛素结合酶（UBQ2）被认为是最稳定的三个基因。另一方面，几种常用的参考基因，例如α-微管蛋白（TubA），β-微管蛋白（TubB）和3-磷酸甘油醛脱氢酶（GAPDH），表现出不稳定的表达，因此这些基因不应该用作内部对照。基因表达分析。我们还使用不同的参考基因测试了重要的应激反应基因，即富含脯氨酸的大型bag6-like基因（BAG）的表达水平。正如预期的那样，当使用不同的标准化方法时，我们观察到了不同的结果和结论，从而表明选择合适的参考基因和相关标准化方法的重要性。我们的研究结果为利用萨维氏梭菌作为模型的生物入侵过程中的环境适应/适应研究提供了有价值的参考基因资源，可用于基因表达的标准化。

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