根癌农杆菌介导的尿嘧啶缺陷烟曲霉转化是基因敲除的有效方法  

Agrobacterium tumefaciens-mediated transformation of uracil auxotroph Aspergillus fumigatus is an efficient method for target gene knockout

作　　者：乔建军[1] 刘伟[1] 马彦[1] 万喆[1] 李若瑜[1] 

机构地区：[1]北京大学第一医院皮肤科,北京大学真菌和真菌病研究中心,北京100034

出　　处：《北京大学学报（医学版）》2008年第3期330-333,共4页Journal of Peking University:Health Sciences

基　　金：国家自然科学基金(30500027);教育部科学技术研究重点项目资助(107002)~~

摘　　要：目的:探讨用嘧啶营养缺陷作为筛选标记、根癌农杆菌介导的转化方法敲除烟曲霉基因。方法:以pyrG作为筛选标记,在双元载体pDHt/sk上分别构建烟曲霉FAP1和SHO1基因的打靶序列。构建好的质粒分别转化入根癌农杆菌。24℃条件下,含打靶质粒的根癌农杆菌与烟曲霉在不含尿嘧啶和尿苷的诱导培养基上共培养48h;然后将培养物转移到37℃,筛选转化子。结果:根癌农杆菌介导的烟曲霉转化同源重组率较高,FAP1基因为44%(7/16)、SHO1基因为35%(7/20)。结论:以pyrG作为筛选标记、根癌农杆菌介导的转化方法能高效敲除烟曲霉基因,为烟曲霉基因功能研究提供了有效方法。Objective： To investigate the efficiency of Agrobacterium tumefaciensmediated transfor-mation of Aspergillus fumigatus by using pyrG as a recessive selectable marker. Methods： FAP1 and SHO1 genes target sequences, composed of a selectable marker pyrG and the flanking sequences of the FAP1 and the SHO1 genes, were cloned into a binary plasmid pDHt/sk, respectively. The produced plasmids were transformed into A. tumefaciens. The A. tumefaciens and uracil auxotroph A. fumigatus were cocultured in induction medium without uricil and urldine at 24℃ for 48 h. To inhibit growth of A. tumefaciens and to select transformants, the cultures were transferred to 37 ℃ and incubated for another 48 h. Results： In this study, A. tumefaciens-medidated transformation of A. fumigatus produced high homologous recombination rates, which was 44% （7 of 16） for FAP1 and 35% （7 of 20） for SHO1. Conclusion： Our study showed that A. tumefaciens-medidated transformation by using pyrG as a recessive selectable marker is an efficient tool for target gene deletion of A. fumigatus.

关 键 词：根瘤菌 放射型 曲霉菌 烟 基因 

分 类 号：R756[医药卫生—皮肤病学与性病学]