香蕉枯萎病菌2个生理小种PL基因真核表达载体的构建与表达

【目的】构建香蕉枯萎病菌1号小种（FOC1）和4号小种（FOC4）果胶裂解酶（Pectate lyases, PL）基因的真核表达载体，并进行诱导表达，为进一步研究PL在病原菌致病过程中的作用奠定基础。【方法】将质粒pMD18-pl-foc1、pMD18-pl-foc4和真核表达穿梭载体pPICZαA进行EcoRⅠ和XbaⅠ双酶切反应，回收目的片段连接，转化至DH5α进行筛选扩繁，对菌落进行PCR鉴定后抽提质粒进行PCR和EcoRⅠ、XbaⅠ双酶切鉴定。将阳性载体线性化后电击转化毕赤酵母 SMD1168感受态细胞，用甲醇进行诱导表达，对表达产物进行SDS-PAGE分析。【结果】成功构建了FOC1和FOC4 PL基因的真核表达载体pPICZαA-pl-foc1和pPICZαA-pl-foc4，经甲醇诱导表达后分别获得了重组的PL蛋白，其分子质量约为23.4 ku。【结论】FOC1和FOC4 2个香蕉枯萎病菌小种的PL基因在酵母中得到了成功表达。

Construction of eukaryotic expression vector with pectate lyase gene from Fusarium oxysporum f.sp.cubense race 1 and race 4 and their eukaryotic expression

【Objective】 The study constructed Fusarium oxysporum f.sp.cubense race 1(FOC1) and Fusarium oxysporum f.sp.cubense race 4(FOC4) pectate lyases(PLs) genes’ eukaryotic expression vectors,and induced expression in order to provide further research of the pathgenicity in the fungi.【Method】 The plasmid pMD18-pl-foc1,pMD18-pl-foc4 and eukaryotic expression shuttle vector pPICZαA were linearized by double digestion of EcoRⅠ and XbaⅠ.Then the ligation fragment was subcloned into DH5α.The plasmids were identified by PCR and double restriction enzyme digestion with EcoRⅠ and XbaⅠ.The positive vector linearized Pichia pastoris SMD1168 electroporation competent cells.After induction by methanol,the reaction was analyzed by SDS-PAGE.【Result】 The eukaryotic expression vectors of pPICZαA-pl-foc1 from FOC1 and pPICZαA-pl-foc4 from FOC4 were constructed.Protein was expressed by methanol induction after clone selection by yeast genome PCR and the PL bands of the recombinant were obtained respectively.Protein of molecular mass was about 23.4 ku.【Conclusion】 The PLs of two races had been functionaly expressed in yeast cells.