Validation of reference genes for gene expression analysis in Valsa mali var. mali using real-time quantitative PCR
Validation of reference genes for gene expression analysis in Valsa mali var. mali using real-time quantitative PCR
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Valsa mali var. 基因表达分析参考基因的验证
DOI：
10.1007/s11274-013-1320-6
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发表时间：
2013-09-01
影响因子：
4.1
通讯作者：
Huang, Lili
中科院分区：
文献类型：
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作者：
Yin, Zhiyuan;Ke, Xiwang;Huang, Lili
苹果腐烂病菌mali（Vmm）是我国苹果腐烂病的优势种。参与毒力或致病性的基因的现代分析通常涉及最常使用实时定量聚合酶链反应（RT-qPCR）进行的基因表达分析。然而，对于相对基因表达分析，相关的参考基因必须在使用它们作为内部参考之前进行验证。到目前为止，还没有关于VMM的报告。因此，克隆了8个常用的管家基因（ACT、EFL、EF 1-α、G6 PDH、GAPDH、L13、TUB和UBQ），并通过geNorm和Normandem评估了其表达稳定性。总体而言，发现所有候选参考基因都适合于基因表达分析。通过对10个不同菌株和非生物胁迫处理样品的分析，G6 PDH似乎是最合适的参考基因，而GAPDH是最不合适的。以G6 PDH与L13或L14联合作为参考基因，可显著提高RT-qPCR的可靠性。通过分析编码内切多聚半乳糖醛酸酶基因的Vmmpg-1，证明了参考系统对表达数据的影响。果胶酶被认为是该真菌的关键致病因子。为了更好地了解果胶酶在Vmm致病性中的作用，使用RT-qPCR进行表达分析。本研究结果为进一步研究苹果枯萎病菌基因表达提供了参考。用RT-qPCR方法检测了苹果的DNA序列。
Valsa mali var. mali (Vmm), is the predominant species of apple valsa canker in China. Modern analysis of genes involved in virulence or pathogenicity usually implicate gene expression analysis most often performed using real-time quantitative polymerase chain reaction (RT-qPCR). However, for relative gene expression analysis pertinent reference genes have to be validated before using them as internal reference. This has not been reported for Vmm, so far. Therefore, eight commonly used housekeeping genes (ACT, CYP, EF1-alpha, G6PDH, GAPDH, L13, TUB, and UBQ) were cloned and evaluated for their expression stability by geNorm and NormFinder. Overall, all of the candidate reference genes were found to be suitable for gene expression analysis. After analysis of 10 samples from different strains and abiotic stress treatments, G6PDH appeared to be the most suitable reference gene, whereas GAPDH was the least suitable. Moreover, taking G6PDH combined with L13 or CYP as reference genes, improved the reliability of RT-qPCR significantly. The influence of the reference system on expression data was demonstrated by analyzing Vmmpg-1 encoding an endo-polygalacturonase gene. Pectinases are considered key pathogenicity factors for this fungus. In order to better understand the role of pectinases in pathogenicity of Vmm, RT-qPCR was used for expression analysis. Our results may provide a guideline for future studies on gene expression of V. mali var. mali by using RT-qPCR.