[根癌农杆菌介导的尿嘧啶营养缺陷型烟曲霉转化是一种有效的靶基因敲除方法]
[Agrobacterium tumefaciens-mediated transformation of uracil auxotroph Aspergillus fumigatus is an efficient method for target gene knockout].
作者信息
Qiao Jian Jun, Liu Wei, Ma Yan, Wan Zhe, Li Ruo Yu
机构信息
Department of Dermatology, Peking University First Hospital;Research Center for Medical Mycology, Peking University, Beijing, China.
出版信息
Beijing Da Xue Xue Bao Yi Xue Ban. 2008 Jun 18;40(3):330-3.
OBJECTIVE
To investigate the efficiency of Agrobacterium tumefaciens mediated transformation 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 uridine at 24 degrees C for 48 h. To inhibit growth of A. tumefaciens and to select transformants, the cultures were transferred to 37 degrees C and incubated for another 48 h.
RESULTS
In this study, A. tumefaciens-mediated 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.
目的
以pyrG作为隐性选择标记，研究根癌农杆菌介导的烟曲霉转化效率。
方法
将由选择标记pyrG以及FAP1和SHO1基因侧翼序列组成的FAP1和SHO1基因靶序列分别克隆到二元质粒pDHt/sk中。将构建好的质粒转化到根癌农杆菌中。将根癌农杆菌与尿嘧啶营养缺陷型烟曲霉在不含尿嘧啶和尿苷的诱导培养基中于24℃共培养48小时。为抑制根癌农杆菌生长并筛选转化子，将培养物转移至37℃再培养48小时。
结果
在本研究中，根癌农杆菌介导的烟曲霉转化产生了较高的同源重组率，FAP1为44%（16个中有7个），SHO1为35%（20个中有7个）。
结论
我们的研究表明，以pyrG作为隐性选择标记的根癌农杆菌介导转化是烟曲霉靶基因缺失的有效工具。
Zotero 插件