Research Institute of Horticulture, Konstytucji 3 Maja 1/3, 96-100, Skierniewice, Poland.
Sci Rep. 2017 May 17;7(1):2034. doi: 10.1038/s41598-017-02078-4.
To study the expression of pathogenicity-related genes in Erwinia amylovora, seven candidate reference genes (ffh, glyA, gyrA, proC, pykA, recA, rpoB) were selected and validated with the following five different mathematic algorithms: geNorm, NormFinder, BestKeeper, the delta CT method and the RefFinder web-based tool. An overall comprehensive ranking output from each of the selected software programs revealed that proC and recA, followed by ffh and pykA, were the most stably expressed genes and can be recommended for the normalization of RT-qPCR data. A combination of the three reference genes, proC, recA and ffh, allowed for the accurate expression analysis of amsB and hrpN genes and the calculation of their fold change in E. amylovora after its infection of susceptible and resistant apple cultivars. To the best of our knowledge, this is the first study presenting a list of the most suitable reference genes for use in the relative quantification of target gene expression in E. amylovora in planta, selected on the basis of a multi-algorithm analysis.
为了研究果胶黏性菌（Erwinia amylovora）中致病性相关基因的表达情况，选择了 7 个候选参考基因（ffh、glyA、gyrA、proC、pykA、recA 和 rpoB），并使用以下 5 种不同的数学算法进行验证：GeNorm、NormFinder、BestKeeper、ΔCT 法和 RefFinder 网络工具。从每个选定软件程序的综合排名结果来看，proC 和 recA 紧随其后的是 ffh 和 pykA，是最稳定表达的基因，可以推荐用于 RT-qPCR 数据的归一化。这三个参考基因 proC、recA 和 ffh 的组合，可以准确分析 amsB 和 hrpN 基因的表达，并计算它们在果胶黏性菌感染易感和抗性苹果品种后的倍数变化。据我们所知，这是首次根据多算法分析，为在植物体内相对定量果胶黏性菌靶基因表达中选择最适合的参考基因列表而进行的研究。
