关键词: 耐盐性, 耐盐性

Abstract: In this paper, we constructed a real-time fluorescence quantitative RT-PCR system for the β-actin gene of Palembus dermestoides with MJ Research OpticonTM 2 instrument, using SYBR Green I method. The primers of β-actin were designed by the conserved sequence of other insects available in GenBank. PCR annealing temperature, primers concentration, template concentration and other reaction factors was optimized, and the amplification curve and melt curve was analyzed. Our results showed that the optimum reaction conditions were 1 μM of each primer, 50 ng/μL of cDNA templet, and 10 μL of 2×SYBRR Premix Ex TaqTM in a final volume of 20 μL. The optimum PCR procedure conditions were initial denaturation 94℃ for 30 s followed by 44 cycles of 94℃ for 10 s, 57℃ for 30 s and 72℃ for 40 s. A melt curve was acquired at 82℃ for 1 s finally. It found that β-actin could be used as reference gene of FQ RT-PCR of P. dermestoides because of its stable expression during different development stages. The 2-ΔΔCT relative quantitative RT-PCR system was established for the β-actin of P. dermestoides and the importance of the optimization of PCR reaction system was analyzed. The FQ RT-PCR method was simple and had a highly specificity and could be further applied to the usage of the β-actin as a reference gene in quantitative analysis of ecdysis relevent gene expression of P. dermestoides.