【摘要】 【目的】探索重叠延伸PCR法在昆虫化学感受蛋白基因点突变上的应用,研究半胱氨酸(Cys32)对小菜蛾(Plutella xylostella)化学感受蛋白1(PlxyCSP1)结合特性的影响。【方法】用计算机软件模拟PlxyCSP1上Cys32突变前后的蛋白三级结构以及与配体化合物的结合情况,确定点突变位点。根据扩增得到的PlxyCSP1序列设计并合成重叠引物,利用重叠延伸PCR法,将PlxyCSP1上的Cys32突变成色氨酸(Trp32),获得突变体PlxyCSP1-M。将突变后的基因与载体pET-32a(+)连接,转化大肠杆菌BL21(DE3)。【结果】分子对接图显示,配体可以顺利进入PlxyCSP1的结合口袋,而无法进入PlxyCSP1-M的结合口袋。结合能预测显示,配体化合物与PlxyCSP1的结合能均为正值,与PlxyCSP1-M的结合能均为0。突变后的重组质粒测序结果表明,Cys32(TGC)已突变为Trp32(TGG),经IPTG诱导,该菌表达了分子质量约为36 ku的融合蛋白。【结论】Cys32是影响PlxyCSP1结构和功能的重要氨基酸残基。Cys32突变为Trp32后,PlxyCSP1-M结合特性发生了变化,不能与配体化合物正常结合。更多还原

【Abstract】 【Objective】 This study explored overlap extension PCR based site-directed mutant of insects chemosensory and studied the effects of the cysteine(Cys32) in binding characteristics of PlxyCSP1.【Method】 The tertiary structure and binding characteristics of PlxyCSP1 and PlxyCSP1-M were simulated by computer software in order to confirm the mutation site.Overlapping primes were designed according to the sequence of PlxyCSP1 to obtain PlxyCSP1-M which the Cys32 was mutated to the Trp32.PlxyCSP1-M was linked with pET-32a(+) and transformed into E.coli BL21(DE3).【Result】 The result of molecular docking displayed that ligand compounds could easily enter binding pocket of PlxyCSP1 but could not enter the binding pocket of PlxyCSP1-M.The binding energies of ligand compounds and PlxyCSP1 were all positive while the binding energies of ligand compounds and PlxyCSP1-M were zero.Recombinant plasmid sequencing showed that the Cys32 was mutated to Trp32 in PlxyCSP1-M and the recombinant strain could express fusion protein which was 36 ku by IPTG induction.【Conclusion】 The Cys32 is important to the structure and function of PlxyCSP1.Binding characteristics of PlxyCSP1-M were changed and could not bind with ligand compounds.更多还原