No abstract

A mutant library of Cordyceps militaris was constructed by improved Agrobacterium tumefaciens-mediated transformation and screened for degradation features. Six mutants with altered characters in in vitro and in vivo fruiting body production, and cordycepin formation were found to contain a single copy T-DNA. T-DNA flanking sequences of these mutants were identified by thermal asymmetric interlaced-PCR approach. ATP-dependent helicase, cytochrome oxidase subunit I and ubiquitin-like activating enzyme were involved in in vitro fruiting body production, serine/threonine phosphatase involved in in vivo fruiting body production, while glucose-methanol-choline oxidoreductase and telomerase reverse transcriptase involved in cordycepin formation. These genes were analyzed by bioinformatics methods, and their molecular function and biology process were speculated by Gene Ontology (GO) analysis. The results provided useful information for the control of culture degeneration in commercial production of C. militaris.

The full-length cytochrome c oxidase subunit I gene (cox1) containing a group I intron was isolated from an important medical fungus Cordyceps militaris (Cordycipitaceae). The open reading frame (ORF) of 1,593 nucleotides encoded a predicted protein COX1 of 530 amino acids. The group I intron encoded a putative homing endonuclease (HE) with two LAGLIDADG motifs. RT-PCR and Northern analysis showed a mature transcript of spliced cox1. Both 5'exon-intron and intron-3'exon junctions were also found by RT-PCR, suggesting the possible presence of unspliced cox1 RNA in total RNA. Sequence comparison by BLASTn showed that the coding region of cox1 (CRcox1) of C. militaris had significant similarities to those of related species (such as Cordyceps bassiana and C. brongniartii), while the intron had no significant homologous sequences of Cordycipitaceae fungi in NCBI database. The phylogenetic tree based on the CRcox1 confirmed the present taxonomic status of related species, but the cox1 introns were phylogenetically distinct. Compared to C. bassiana and C. brongniartii, the cox1 intron of C. militaris exhibited specific splicing site and different intronic ORF. The analysis of the folding RNA structures of the known cox1 introns from Cordyceps species showed different base pairs and conserved regions (P1-P10) in their structures. The present results provide useful information on the studies of cox1 intron splicing and Cordyceps evolution.