【摘要】 马尔尼菲青霉是温度依赖双相性条件性致病真菌,可感染免疫缺陷人群,区域流行于东南亚和我国南方。马尔尼菲青霉已成为研究真菌双相形态转换和病原-宿主相互作用分子机制的模式系统。功能基因组学研究为深入探讨马尔尼菲青霉双相转换和致病分子机制提供了线索,大量的候选基因功能需要高通量的基因修饰方法验证。在许多真菌中已建立基于阻断DNA非同源重组修复途径提高基因打靶效率的实验研究模型。本文报道在马尔尼菲青霉中通过缺失马尔尼菲青霉非同源重组修复途径的关键组分PKUB同源基因pkuB构建了一个高效基因打靶系统。结果表明,以pkuB基因缺失菌株(△pkuB)为出发菌株能降低外源DNA片段的非同源末端连接重组的概率而显著提高基因打靶效率,pkuB基因缺失不影响菌落形态和双相转换能力。高效基因打靶分子遗传实验模型的建立为大规模进行马尔尼菲青霉基因功能研究提供了有力的工具。更多还原

【Abstract】 Penicillium marne ffei is temperature dependent dimorphic opportunity pathogenic fungi, can cause infections in immunocompromised population, endemic to Southeast Asia and southern China. P. marneffei has been developed as a model system for investigating the molecular mechanisms controlling the dimorphic phase transition and host-pathogen interactions. Functional genomics studies are providing a wealth information and clues for probing the molecular mechanisms of dimorphic phase transition and pathogenicity in P. marneffei; the function of candidate genes need to be identified by high throughput and efficiency experimental models for gene modification. The excellent experimental models for high efficiency gene targeting based on blocking the nonhomologous end joining DNA repair pathway has been established in many species of fungi. Here, we report construction of a high efficiency gene targeting system by deletion KU80 ortholog, pku B, encodes a key component of the non-homologous end joining DNA repair pathway in P. marneffei. Results show the efficiency of gene targeting was significantly increased in △pku B strain by decreasing the non-homologous end joining recombination frequency, and the pku B deletion strain was indistinguishable from the parental strain SPM4 in colony morphology and ability to the dimorphic transition. The establishment of high efficiency gene targeting experimental model for molecular genetic manipulation provides a powerful tool for large-scale analysis of gene functions in P. marneffei.更多还原