开花期水稻颖花实时定量RT-PCR分析中内参基因的选择

Reference Gene Selection for Quantitative Real-Time RT-PCR Normalization in Rice Florets during Anthesis

实时定量RT-PCR(qRT-PCR)技术现已发展成为基因表达分析的首选方法,然而为了得到精确和可靠的qRT-PCR分析结果,需要应用稳定表达的内参基因对样品进行校正。以粳稻‘武运粳7号’和籼稻‘明恢63’的颖花为试验材料,应用geNorm算法对6个常用的内参基因(ACT1、eEF1-α、β-TUB、UBQ5、GAPDH和UBC)在颖花开放过程中的表达稳定性进行分析。结果发现:eEF1-α和β-TUB在‘武运粳7号’颖花样品中的表达最为稳定,UBC和GAPDH在‘明恢63’颖花样品中的表达最为稳定,而ACT1在2个品种的颖花样品中表达均最不稳定。此外,同时选取2个最稳定表达的内参基因作内参照可获得更精确的校准。分析不同内参基因对JA生物合成关键基因OsAOC表达定量的影响。OsAOC表达的相对定量会随所选内参基因的不同而发生变异,进一步强调选择合适的内参基因进行校准是获得可靠qRT-PCR结果的重要前提。

Quantitative real-time reverse transcription-polymerase chain reaction（qRT-PCR） has become the most common method for gene expression analysis.In order to get accurate and reliable qRT-PCR results,stably expressed reference genes are required for normalization of tested samples.The expression stabilities of 6 commonly used reference genes（ACT1,eEF1-α,β-TUB,UBQ5,GAPDH and UBC） were carefully assessed by geNorm algorithm in florets during anthesis in japonica rice ‘Wuyunjing 7’ and indica rice ‘Minghui 63’.The results showed that the expression of eEF1-α and β-TUB was the most stable in the floret samples of ‘Wuyunjing 7’.UBC and GAPDH exhibited the most stable expression in ‘Minghui 63’.In contrast,ACT1 was found to be the least stable expression among the tested samples from both rice varieties.Moreover,a combination of two most stable genes was found to be better as internal control for normalization of the data.The effect of normalization with different internal controls on quantification of OsAOC,a key gene in JA biosynthesis,was also examined.The relative quantification of expression of OsAOC varied according to the internal controls used,thus highlighting that choice of stable reference genes for normalization was a critical precondition for reliable qRT-PCR data.