水稻虫害诱导相关基因实时定量PCR中内参基因的选择

摘要

实时定量PCR技术广泛应用于植物功能基因转录水平变化的研究,选择合适的内参基因进行相对定量分析是实验结果准确的关键因素.通过分析5个常用的内参基因(eEF-1α、18S rRNA、25S rRNA、Actin和UBQ5)在水稻(Oryza sativa)经过各种处理后表达的稳定性,结果表明,水稻经过机械损伤处理后eEF-1α基因的表达最稳定；二化螟处理后25S rRNA基因的表达最为稳定；稻纵卷叶螟处理后Actin基因的表达最稳定；两种刺吸式口器昆虫褐飞虱和白背飞虱危害后,UBQ5基因的表达最稳定.同时,利用OsHI-LOX基因在不同处理后的表达来评价这些内参基因.研究结果为水稻虫害诱导实时定量PCR分析中内参基因的选择提供了理论依据.%Quantitative real-time PCR is popularly used to study gene transcription levels in plants. Validation of candidate reference genes is the key step to ensure relative quantification. Here, we selected 5 reference genes (eEF-1α, 18S rRNA, 25S rRNA, Actin, and UBQ5) and tested their expression stability in rice under various treatments. eEF-1a showed the most stable expression with mechanical wounding, 25S rRNA with stripped stem borer treatment, and Actin with leaf folder treatment. UBQ5 showed the most stable expression on treatment with 2 phloem-feeding herbivores, brown plant hopper and white-backed plant hopper. We validated the expression profiles of these candidate reference genes by studying that of OsHI-LOX. We offer an available reference for studying gene transcription levels with herbivore infestation in rice.