Abstract:

【Objective】The objective of this study is to establish a highly efficient ATMT mutagenesis system of Fusarium verticillioides, and to construct ATMT mutant library in which mutant contains green fluorescent protein (GFP). And then this library was used for screening and analysis, which can lay a foundation for studying the infection pathway and molecular pathogenesis of F. verticillioides on maize ear. 【Method】 The inhibitory concentration of cefotaxime sodium (Cefo) and ampicillin sodium (Amp) against Agrobacterium tumefaciens AGL-1 and the sensitive concentration of hygromycin B against F. verticillioides were screened for ATMT mutagenesis system. A shuttle plasmid containing GFP and hygromycin phosphotransferase (HPH) genes was used as a vector to construct the ATMT mutants library of F. verticillioides. The T-DNA insertion and stability of transformant were detected and analyzed through hygromycin B resistance, PCR identification of GFP-specific primers, and fluorescence microscopy. Nine transformants were randomly selected and the sporulation number, conidial germination rate, and pathogenicity were measured. 【Result】When the concentration of Cefo/Amp was 150/150 μg·mL ^-1, the growth of AGL-1 was inhibited, and when the concentration of hygromycin B was 150 μg·mL ^-1, the growth of F. verticillioides was completely incapacitated. Using the optimized ATMT, a total of 2 465 GFP-labeled transformants were obtained, these transformants could still grow normally on PDA medium containing hygromycin B after cultured 5 generations on hygromycin-free PDA medium, which indicated that HPH had been integrated into wild-type (WT) genome of F. verticillioides and the transformants were stable in their characteristics of genetics. The PCR detection results amplified with GFP-specific primers showed that the homology of transformants with GFP (accession number: LC420351.1) in NCBI was 99.26%, and the hyphae and conidia of transformants showed green fluorescence under the fluorescence microscope, while no fluorescence was observed in WT, indicating that GFP had been integrated into the WT genome and successfully expressed. Compared with WT strain, the sporulation number of transformant 54 increased significantly, about 1.9 times of that of WT strain, and the conidial germination rate of transformant 24 decreased obviously in the same time. The pathogenicity of transformant 13 was enhanced, the disease grade reached 9, the pathogenicity of transformant 33 and 16 was reduced to grade 3, and the pathogenicity of transformant 4 was the weakest, the disease grade was 1. There was no significant change in biological characters of partial transformants.【Conclusion】ATMT mutant library with GFP of F. verticillioides was constructed, and the mutants with changed sporulation number, conidia germination rate and pathogenicity were obtained through primarily screening. It will lay a foundation for further study on the infection pathway and pathogenic molecular mechanisms of F. verticillioides on maize ear in the future.

Key words: maize, Fusarium verticillioides, ATMT, mutant library, green fluorescent protein, transformant