Title |
An efficient Agrobacterium-mediated transformation method for aflatoxin generation fungus Aspergillus flavus |
Author |
Guomin Han1,2, Qian Shao1, Cuiping Li1, Kai Zhao1, Li Jiang1, Jun Fan1,2, Haiyang Jiang1,2, and Fang Tao1* |
Address |
1School of Life Sciences, Anhui Agricultural University, Hefei 230036, P. R. China, 2The National Engineering Laboratory of Crop Stress Resistance Breeding, Anhui Agricultural University, Hefei 230036, P. R. China |
Bibliography |
Journal of Microbiology, 56(5),356–364, 2018,
|
DOI |
10.1007/s12275-018-7349-3
|
Key Words |
Agrobacterium tumefaciens-mediated transformation,
Aspergillus flavus, zeocin resistance, optimization,
mutant library |
Abstract |
Aspergillus flavus often invade many important corps and
produce harmful aflatoxins both in preharvest and during
storage stages. The regulation mechanism of aflatoxin biosynthesis
in this fungus has not been well explored mainly
due to the lack of an efficient transformation method for
constructing a genome-wide gene mutant library. This challenge
was resolved in this study, where a reliable and efficient
Agrobacterium tumefaciens-mediated transformation (ATMT)
protocol for A. flavus NRRL 3357 was established. The results
showed that removal of multinucleate conidia, to collect
a homogenous sample of uninucleate conidia for use as the
transformation material, is the key step in this procedure.
A. tumefaciens strain AGL-1 harboring the ble gene for zeocin
resistance under the control of the gpdA promoter from
A. nidulans is suitable for genetic transformation of this fungus.
We successfully generated A. flavus transformants with
an efficiency of ~ 60 positive transformants per 106 conidia
using our protocol. A small-scale insertional mutant library
(~ 1,000 mutants) was constructed using this method and
the resulting several mutants lacked both production of conidia
and aflatoxin biosynthesis capacity. Southern blotting
analysis demonstrated that the majority of the transformants
contained a single T-DNA insert on the genome. To the best
of our knowledge, this is the first report of genetic transformation
of A. flavus via ATMT and our protocol provides an
effective tool for construction of genome-wide gene mutant
libraries for functional analysis of important genes in A.
flavus. |