Abstract: [Objective] To study the effects of different primers and annealing temperatures on real quantitative PCR amplification efficiency of reference gene β-actin with the halophyte Halostachys caspica as research material.[Method]Standard curves of β-actin and peroxidase gene,POD(representative gene responding to salt stress)from this species were built with well-designed two pairs of primers named β-actin1 and β-actin2 for β-actin as internal reference gene,and a pair of primers for POD gene and amplification curves were obtained under different conditions for these genes.[Result]Results showed that the amplification efficiency of the primers β-actin1 was higher than that of the primers β-actin2 in the Halostachys caspica branches under the 55℃ or 58℃ annealing temperature,and amplification efficiency at 55℃ annealing temperature was higher than that at 58℃.On the other hand,it was found there were some differences in the relative expression levels of Halostachys caspica POD gene using the internal reference gene β-actin with two pairs of primers β-actin1 and β-actin2 under the 55℃ annealing temperature.[Conclusion]The research indicates that primers and annealing temperatures can influence the fluorescence quantitative PCR amplification efficiency and then affect relative expression level of candidate genes at a certain extend.

摘要： [目的]以盐生植物盐穗木为材料,研究不同引物对和不同退火温度对盐穗木内参基因β-actin扩增效率的影响.[方法]设计扩增β-actin基因的两对引物,标记为β-actin1和β-actin2,分别建立这两对引物扩增盐穗木β-actin基因和特异性引物扩增该物种的过氧化物酶基因POD(盐响应的代表性基因),在不同退火温度下的标准曲线和扩增曲线.[结果]不论55还是58℃退火温度,引物对β-actin1比引物对β-actin2有更好的扩增效率;在55℃退火温度下,引物对β-actin1有更高的扩增效率.在这两个退火温度下,分别以β-actin1和β-actin2引物对扩增β-actin基因作为内参,盐穗木POD基因的相对表达水平具有一定的差异性.[结论]引物和退火温度会影响荧光定量PCR的扩增效率,进而会影响靶基因的相对表达水平.