[Agrobacterium tumefaciens-mediated transformation of uracil auxotroph Aspergillus fumigatus is an efficient method for target gene knockout].
[Agrobacterium tumefaciens-mediated transformation of uracil auxotroph Aspergillus fumigatus is an efficient method for target gene knockout].
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发表时间：
2008-06
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通讯作者：
Jian Qiao;Wei Liu;Yansen Ma;Z. Wan;Ruo-yu Li
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作者：
Jian Qiao;Wei Liu;Yansen Ma;Z. Wan;Ruo-yu Li
目的 以pyrG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FAP1和SHO1基因靶序列由选择标记pyrG和FAP1和SHO1基因的侧翼序列组成，分别克隆到二元质粒pDHt/sk中。将产生的质粒转化至根癌农杆菌中。将根癌农杆菌和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。为了抑制根癌农杆菌的生长并选择转化体，将培养物转移至37℃并再培养48小时。结果在这项研究中，根瘤农杆菌介导的烟曲霉转化产生了高同源重组率，FAP1 为 44%（16 中的 7 次），SHO1 为 35%（20 中的 7 次）。结论我们的研究表明，使用pyrG作为隐性选择标记的根癌农杆菌介导的转化是烟农杆菌靶基因删除的有效工具。
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.