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W544是Cry1Ac蛋白上独特于其它Cry类蛋白的一个氨基酸,它与F578和F604一起组成一个“螺旋桨状”的疏水簇,通过疏水相互作用维持蛋白的三维结构稳定.本研究通过定点突变将W544保守地替换为苯丙氨酸,SDS-PAGE分析结果表明其纯化的原毒素对紫外照射、胰蛋白酶处理和室温存贮的稳定性相对于野生Cry1Ac都有一定程度的提高;经原子力显微镜观察,发现W544F产生的晶体两个顶点间的垂直距离比野生型Cry1Ac约长0.6μm,且晶体表面不及野生型光滑;此外,W544F与野生Cry1Ac的杀虫活性相似,但经过紫外光照射9 h后,其保留的杀虫活性比野生型高4倍以上.W544F突变较好地解决了Cry1Ac毒素蛋白田间应用不持久的问题,具有重要的应用价值.
W544 is an amino acid of Cry1Ac protein that is unique to other Cry proteins and forms a “propeller ” hydrophobic cluster together with F578 and F604 to maintain the three-dimensional structural stability of the protein through hydrophobic interaction.In this study, W544 conservatively replaced by phenylalanine, SDS-PAGE analysis showed that the purified protoxin to UV irradiation, the stability of trypsin treatment and storage at room temperature relative to wild Cry1Ac have a certain degree of increase; observed by atomic force microscopy The results showed that the vertical distance between the two vertices of W544F was about 0.6μm longer than that of wild type Cry1Ac, and the crystal surface was not as smooth as the wild type. In addition, the insecticidal activity of W544F was similar to that of wild Cry1Ac, but after UV irradiation for 9 h , Which retained more than 4 times more insecticidal activity than the wild type.The W544F mutation solved the problem of non-durable application of Cry1Ac toxin protein in fields, and has important application value.