Cordyceps militaris (Hypocreales: Cordycipitaceae): transcriptional analysis and molecular characterization of cox1 and group I intron with putative LAGLIDADG endonuclease

Abstract

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.

Cordycepin from Cordyceps militaris prevents hyperglycemia in alloxan-induced diabetic mice

“…Cordycepin also showed significant antitumor and immunomodulation effects [15][16][17]. Cordyceps militaris, a well-known traditional Chinese herb mushroom, is one of the most representative and widely used species of Cordyceps [18]. For a long time, C militaris has gained considerable significance in several clinical applications.…”

Rhf1 gene is involved in the fruiting body production of Cordyceps militaris fungus

“…ATMT has been described as an efficient protocol for many filamentous fungi, not only applicable to C. militaris [50,63], but also to several other species, including M. anisopliae [43], A. awamori [34], Glomus intraradices [20], and Beauveria bassiana [14]. T-DNA insertion occurred efficiently as a single copy without AS in all transformational processes of C. militaris [62].…”

“…The culture conditions for mycelia in liquid or solid PPDA and for fruiting body in an artificial rice medium were described previously [63]. JM4 and the transformants were cultured on solid PPDA at 23 °C for 7 days.…”

“…The total fungal genomic DNA (gDNA) and the RNA of all C. militaris strains were, respectively, prepared by the E.Z.N.A.™ HP Fungal DNA Kit (Omega, USA) and the Trizol reagent (Invitrogen, USA) according to the manufacturer's instructions [63]. The mycelia from liquid PPDA at 22 °C in a shaking platform (120 rpm) for 7 days were harvested, washed twice with sterile distilled water and used for gDNA or total RNA isolation [50].…”

“…From the genome analysis, it was estimated that the Zn2Cys6-type transcription factors and MAPK pathway were induced during fruiting, but not the PKA pathway, and more than 63 % of the total 9684 genes were expressed during both mycelial growth and fruiting body formation [49,60]. Some C. militaris genes have been cloned in recent years, including the glyceraldehyde-3-phosphate dehydrogenase (GPD) gene [16], the Cu/ Zn superoxide dismutase gene (CmSOD) [64], coxI with putative LAGLIDADG endonuclease [63], the matingtype genes (MAT1-1-1 and MAT1-1-2) [7], geranylgeranyl diphosphate synthases [28], SSU rDNA group I introns [29], and the blue-light receptor gene Cmwc-1 [52]. The transcriptome and proteome for fruiting body production have been reported [53,61].…”