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


CONCLUSIONS
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.
Qiao
Jian Jun
JJ
Department of Dermatology, Peking University First Hospital;Research Center for Medical Mycology, Peking University, Beijing, China.
Liu
Wei
W
Ma
Yan
Y
Wan
Zhe
Z
Li
Ruo Yu
RY
D016960:Agrobacterium tumefaciens / Q000235:genetics*; D001232:Aspergillus fumigatus / Q000235:genetics*; D055786:Gene Knockout Techniques / Q000379:methods*; D005800:Genes, Fungal; D014170:Transformation, Genetic; D014498:Uracil / Q000378:metabolism*
D014498:Uracil