稻粒黑粉病菌实时荧光定量PCR内参基因筛选

Selection of reference genes for quantitative real-time PCR in Tilletia horrida

实时荧光定量PCR是一种被广泛应用于基因表达研究的分子技术,内参基因的选择对试验结果的可靠性起重要作用。稻粒黑粉病是一种全球性水稻真菌病害,目前没有关于稻粒黑粉病菌内参基因的报道。本文选择6个常用内参基因,对其在稻粒黑粉病菌不同菌株、不同生活史阶段和不同浓度Basta处理3组生物学样本中的稳定性进行比较分析。经geNorm3.5、NormFinder0.953及BestKeeper1.0软件综合评价,结果显示在不同菌株、不同生活史阶段、不同浓度Basta处理生物学样本中,最稳定的内参基因分别为UBQ、GAPDH和EF1α,最优组合分别为UBQ/GAPDH、UBQ/GAPDH和EF1α/GAPDH。本研究结果为稻粒黑粉病菌基因表达研究奠定了基础,同时对其他真菌内参基因的选择提供了参考。

Quantitative real-time PCR （q RT-PCR） is a molecular technique that has been widely used in gene expression analysis. The reliable result of qRT-PCR requires appropriate and stable internal reference gene（s）. The rice kernel smut, caused by Tilletia horrida, is a global rice fungal disease and currently, the internal reference gene for analysis of gene express of the pathogen is unavailable. In this paper, six frequently used internal reference genes were selected in T. horrida and their expression stabilities in different strains, life stages and treatments with various concentrations of Basta were compared via geNorm3.5, Normfinder1.0 and BestKeeper 0.953. Our results show that UBQ is the most stable internal reference gene followed by GAPDH and EF1α, and that UBQ/GAPDH, UBQ/GAPDH, GAPDH/EF1α are the optimal combinations in the above mentioned conditions. The results lay a foundation for analysis of gone expression in T. horrida and provide a reference for selection of internal reference genes in other fungi.