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目的建立荧光染料(SYBR Green I)实时荧光定量PCR(real-timePCR)方法检测的家蝇抗菌肽基因diptericin时间表达模式方法。方法基于已克隆出的diptericin基因,利用软件Prmier5.0设计上下游引物,对未诱导,诱导后2h、4h、8h、12h、24h、36h、48h及不同发育阶段的家蝇三龄幼虫进行real-timePCR设计引物,然后利用10倍系列稀释含有目的基因的质粒,进行实时荧光定量PCR反应,制作标准曲线。结果未刺激的三龄幼虫diptericin基因表达量非常低,与诱导后2h的相比,差1个数量级,诱导后2h开始缓慢上升,12h后达到最高峰,24h后缓慢下降,36h后回到诱后2h的水平,在家蝇三龄幼虫的卵、1龄及2龄期的表达量非常低,3龄期达到最高峰。结论实时荧光定量PCR检测家蝇抗菌肽基因diptericin的方法具有特异性好、灵敏度高,为进一步探讨家蝇抗菌肽生物活性,研究新的抗菌药物打下初步基础。
Objective To establish a time-dependent expression pattern of diptericin gene in housefly Musca domestica detected by real-time quantitative PCR (SYBR Green I). Methods Based on the cloned diptericin gene, the upstream and downstream primers were designed by software Prmier5.0. The third instar larvae of housefly at 2h, 4h, 8h, 12h, 24h, 36h, 48h and different developmental stages -timePCR primers designed, and then use 10-fold serial dilutions containing the gene of interest plasmid, real-time fluorescence quantitative PCR reaction, the production of standard curve. Results The expression level of diptericin gene in third instar larvae was very low, which was about one order of magnitude worse than that at 2h after induction. The diptericin gene began to rise slowly at 2h after induction and peaked at 12h, then decreased slowly at 24h and returned to the induced at 36h After 2h, the expression level of eggs in 3rd instar larvae of housefly was very low at 1st and 2nd instar, and peaked at 3rd instar. Conclusion The method of real-time fluorescence quantitative PCR for detection of Diptericin in Musca domestica has good specificity and high sensitivity. This study laid the preliminary foundation for the further study on the bioactivity of Musca domestica antimicrobial peptides and new antibacterial drugs.