禾谷镰孢碳源代谢调控因子FgCreA的功能

doi:10.3864/j.issn.0578-1752.2018.02.006

Abstract

Abstract: 【Objective】The objective of this study is to knock out the FgCreA of Fusarium graminearum, which is the carbon source metabolic regulation factor, to research the vegetative growth, sexual reproduction and pathogenicity of the knock out mutant, and to provide a basis for carbon source metabolic mechanism of F. graminearum. 【Method】 According to the budding yeast carbon source regulation factorMig1gene sequence on SGD and NCBI databases, the carbon source regulation factor FgCreA in F. graminearum was determined. the sequences of FgCreA orthologs of other species were retrieved from NCBI database. multiple sequence alignment of CreA orthologs was performed with ClustalW2 software, and phylogenetic tree was constructed by MEGA5 software to determine their evolutionary relationships. The protein properties of FgCreA were predicted via InterProScan. The promotor regions of the carbon absorption structure genes and DON biosynthesis genes were obtained from the NCBI database, then the potential FgCreA binding sites in these promotor regions were analyzed. Primer5 software was used to design primers, the FgCREA gene knockout construct was generated by Split-PCR and then introduced into the wild type strain PH-1 by PEG-mediated protoplast transformation. PCR and Southern blot were used to confirm the FgCREA gene deletion mutants (Fgcrea). The function of FgCREA was analyzed according to the vegetative growth, sexual reproduction and pathogenicity of the Fgcrea mutants. 【Result】There is only one carbon metabolism regulator gene FgCREA (FGSG_09715) in F. graminearum. The 416-amino acid protein encoded by FgCREA has two conserved C2H2 zinc finger regions. FgCreA homologue proteins have a certain degree of homology and are highly conserved in fungi. The promoter regions of the carbon absorption structure genes (XYL2, ARA1, ICL1, PG1, SUC2) and DON biosynthesis genes (TRI1, TRI3, TRI4, TRI5, TRI6, TRI7, TRI8, TRI10, TRI12, TRI101) contained FgCreA DNA binding sites. Two FgCREA gene knockout mutants were obtained and screened by PCR and Southern blot. The growth rate of Fgcrea mutants was 90% lower than that of wild type. The conidia morphology of mutants was normal, but the conidiation decreased by 88% compared with the wild type. In the sexual reproduction stage, the Fgcrea mutants could produce normal perithecia, ascus and ascospore, but it delayed 20-28 d longer than the wild type. The Fgcrea mutants were sensitive to salt stress, and were almost non-pathogenic on wheat. 【Conclusion】The carbon metabolic regulator FgCreA plays important roles in vegetative growth and sexual reproduction, and is essential for plant infection in F. graminearum. However, whether FgCreA affects transcription of the carbon absorption structure genes and DON biosynthesis genes remains to be verified.

Key words: Fusarium graminearum, FgCREA, gene knockout, phenotype, carbon metabolism

HOU Rui, WANG ChenFang. The Function of the Carbon Metabolism Regulator FgCreA in Fusarium graminearum[J].Scientia Agricultura Sinica, 2018, 51(2): 257-267.

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