十字花科黑斑病菌生合成tenuazonic acid之PKS-NRPS基因鑑定

Abstract

Brassica black spot disease fungus, Alternaria brassicicola is an important plant pathogenic fungus that produces plenty of secondary metabolites under both in vitro and in vivo conditions, including 3 most well-known mycotoxins: alternariol (AOH), alternariol-9-methyl ether (AME) and tenuazonic acid (TA). Thus far, very limited information is available concerning their biosynthesis and roles in pathogenesis. In this study, we aim to identify the genes involved in the biosynthesis of TA in A. brassicicola. With the knowledge of structural discovery and isotope feeding researches on TA, the activities of both polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) are speculated to be essential in the biosynthesis of TA while isoleucine and acetoacetate were identified to be its precursors. We first examined the TA production in A. brassicicola wild type strain by agar diffusion assay on honey foulbrood disease pathogen, Paenibacillus larvae subsp. larvae. Candidate genes were identified from the genome database of A. brassicicola and applied to bioinformatical analysis on evolutionary relationship and functional domain prediction with known PKS/NRPS genes. Further, AB04556.1 gene was chosen from candidate genes for constructing a gene-disrupted mutant on basis of gene expression level examined by qPCR. The transformants which constructed via biolistic transformation were purified and compared gene expression as well as TA production with wild type. Additionally, we construct a heterologous expression system on Aspergillus nidulans LO2026 strain in order to find out the role that AB04556.1 gene played in the biosynthesis of TA. In this study, we found a potentially candidate gene from the genomic database of A. brassicicola and generated a gene-disrupted mutant for analyzing of the biosynthesis of TA. Ultimately, inoculation was held to understand how TA affects to the pathogenesis during infection.