An efficient Agrobacterium-mediated transformation method for aflatoxin generation fungus Aspergillus flavus

Guomin Han; Qian Shao; Cuiping Li; Kai Zhao; Li Jiang; Jun Fan; Haiyang Jiang; Fang Tao

doi:10.1007/s12275-018-7349-3

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An efficient Agrobacterium-mediated transformation method for aflatoxin generation fungus Aspergillus flavus: Guomin Han ^1,2, Qian Shao ¹, Cuiping Li ¹, Kai Zhao ¹, Li Jiang ¹, Jun Fan ^1,2, Haiyang Jiang ^1,2, Fang Tao ¹; Journal of Microbiology 2018;56(5):356-364.
DOI: https://doi.org/10.1007/s12275-018-7349-3
Published online: May 2, 2018

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. China¹School 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

Corresponding author: Fang Tao , Tel: +86-551-6578-6907,

Received: 16 August 2017 • Revised: 13 February 2018 • Accepted: 19 February 2018

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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.

Keywords: Agrobacterium tumefaciens-mediated transformation;Aspergillus flavus;zeocin resistance;optimization;mutant library

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An efficient Agrobacterium-mediated transformation method for aflatoxin generation fungus Aspergillus flavus

J. Microbiol. 2018;56(5):356-364. Published online May 2, 2018

DOI: https://doi.org/10.1007/s12275-018-7349-3

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