Effects of Different Primers and Annealing Tempearatures on the Amplification Eefficiency of Real-Time PCR Reference Gene β-actin in the Halophyte Halostachys Caspica

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.MethodStandard 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.ResultResults 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.ConclusionThe 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.