摘要
为了建立洋虫β-actin基因实时荧光定量RT-PCR体系,本实验采用MJ Research OpticonTM 2型实时荧光定量PCR仪,利用SYBR Green Ⅰ染料法,根据GenBank上其他昆虫β-actin基因的保守序列设计引物,对PCR退火温度、引物浓度、模板浓度等各反应因子进行优化,结合扩增曲线和熔解曲线进行分析。结果显示,在20μL体系下,当2×SYBRR Premix Ex TaqTM为10μL时,引物和cDNA模板的最佳浓度分别为1μmol/L和50ng/μL。最佳PCR反应程序为:94℃预变性30s,44个循环包括94℃变性10s,45℃退火30s,72℃延伸40s,最后加做熔解曲线82℃1s。结果表明,在洋虫不同发育时期β-actin基因表达水平基本稳定,因此β-actin基因可以作为洋虫实时荧光定量RT-PCR的内参基因。本研究成功建立了2-ΔΔCT相对定量法的洋虫β-actin基因实时荧光定量RT-PCR体系,并分析了优化PCR反应体系的重要性,建立的洋虫β-actin基因荧光定量RT-PCR方法简便、特异性强,该体系的建立可用于洋虫蜕皮相关基因表达差异的深入研究。
In this paper, we constructed a real-time fluorescence quantitative RT-PCR system for the β-actin gene of Palembus dermestoides with MJ Research Opticon^TM 2 instrument, using SYBR Green I method. The primers of β-aetin 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. The results showed that the optimum reaction conditions were 1μmol/L of each primer, 50 ng/μL of cDNA templet, and 10 μL of 2xSYBR^R Premix Ex Taq^TM 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 developmental stages. The 2-AAcv 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 fl-actin as a reference gene in quantitative analysis of ecdysis relevent gene expression of P. dermestoides.
出处
《中国农学通报》
CSCD
2013年第12期157-163,共7页
Chinese Agricultural Science Bulletin
基金
林业公益性行业科研专项"植物源杀虫剂开发与利用技术"(201004003-7)
