Cloning and functional analysis of a gene related to conidiospore formation in Botrytis cinerea

Abstract:

[Objective] To further investigate molecular mechanism of conidiospore formation, infection and pathogenicity of Botrytis cinerea, the gene related to conidiospore formation was cloned and characterized. [Methods] A mutant with no conidiospore, named BCt78, was found by screening T-DNA insertional mutant library of B. cinerea and it was testified by PCR and Southern Blotting techniques. The flanking sequence of T-DNA insertion site was acquired by using TAIL-PCR. The sequence of the T-DNA inserted gene was obtained by scanning the B. cinerea gene bank. The mutant was further identified by PCR and RT-PCR respectively. The function of the gene was studied by analysing the colony morphology, growth rate, cell wall degrading enzyme activity, biological activity of crude toxin and pathogenicity of the mutant strain on tomato leaves as well as the expression level of genes related to pathogenicity. [Results] T-DNA insertion site was defined in the initiation codon of BC1G_12707.1 gene, and the mutant gene was identified as BC1G_12707.1 by RT-PCR technology. The full-length DNA sequence of BC1G_12707.1 was 135 bp, and encoded a 44 amino acids hypothetical protein. Compared to the wild type strain, the mutant strain colony was white, growed slowly, did not produce conidium and sclerotia on PDA medium, but showed stronger pathogenicity on tomato leaves and cell wall degrading enzyme activity, higher expression level of genes related to pathogenicity, such as cell wall degrading enzyme gene cutA and Bcpg, genes (PKA1, PKA2, Bmp3 and Bac) involved in signal pathway, gene (BcBOT2) encoding sesquiterpene synthase, gene (Lac1) encoding melanin and transmembrane protein gene Btp1. [Conclusion] The BC1G_12707.1 gene was involved in conidiation, sclerotia formation and pathogenicity in B. cinerea.