Construction and analysis of mutant library of cotton Fusarium wilt

Abstract:

[Background] Cotton Fusarium wilt has gradually become a major disease threatening the development of Gossypium barbadense cotton industry, but the related functional genes of cotton Fusarium wilt are not very clear. [Objective] In this study, a mutant library of F. oxysporum marked by green fluorescent protein (GFP) was constructed to screen mutants with random insertion of T-DNA, which could provide materials for the screening and research of functional genes of F. oxysporum. library of F. oxysporum marked by GFP was constructed.[Methods] The mutant library of GFP-labeled F. oxysporum was constructed by Agrobacterium tumefaciens mediated transformation (ATMT), and the T-DNA insertion and transformation stability were detected and analyzed. The colony morphology, growth rate, sporulation, germination rate, T-DNA insertion copy number and pathogenicity of randomly selected mutants were analyzed, so as to screen mutants with obvious variation and stability. [Results] 1 600 GFP-labeled F. oxysporum transformants were obtained by using the optimized ATMT mediated system. The transformants were transferred to PDA medium without hygromycin B for 7 generations and then transferred to the medium with hygromycin B. The transformants could still grow normally, indicating that hygromycin gene Hyg was successfully inserted into the wild-type genome and stably inherited. Finally, 17 mutants with different colony phenotypes were screened, including slow growth type, dark purple mycelium type, light purple mycelium type, and light yellow mycelium type. Compared with the wild type, the pathogenicity of mutant A-1 increased by 21.98%, the sporulation of mutant A-1 increased by 103.54%, and the sporulation of mutant C-6 and C-7 decreased by 61.90%. [Conclusion] The mutant library of cotton Fusarium wilt marked by GFP mediated by Agrobacteriumwas constructed, and the mutants with changes in colony morphology, growth rate, sporulation and pathogenicity were screened and analyzed, which laid a foundation for further study of functional genes related to cotton fusarium wilt.

Key words:cotton;Fusarium oxysporum;ATMT;mutant library;green fluorescent protein