马尔尼菲青霉基于pkuB基因缺失的高效基因打靶系统构建  

Construction of a High-efficiency Gene Targeting System Based on pukB Gene Deletion in Penicillium marneffei

作　　者：卢姗[1] 蓝秀万[1,2,3,4] 陶玉婷[1] 王超[1] 梁晓乐[2,3] 梁莹[5] 黄勇奇 贺菽嘉[1,4] 何志义 

机构地区：[1]广西医科大学基础医学院生物化学与分子生物学教研室,南宁530021 [2]广西医科大学基础医学中心实验室,南宁530021 [3]广西医科大学基础医学研究重点实验室,南宁530021 [4]广西医科大学生物分子医学研究重点实验室,南宁530021 [5]广西医科大学基础医学院微生物学教研室,南宁530021 [6]广西医科大学第一附属医院呼吸科,南宁530021

出　　处：《基因组学与应用生物学》2015年第4期754-762,共9页Genomics and Applied Biology

基　　金：国家自然科学基金项目(81160195);广西自然科学基金项目(桂科青0991031)共同资助

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。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.

关 键 词：马尔尼菲青霉 pkuB 基因打靶 同源重组 非同源重组末端链接 

分 类 号：R379[医药卫生—病原生物学] Q933[医药卫生—基础医学]