Identification and functional analysis of the SET domain-containing gene family in Cordyceps militaris

The SET domain is the core domain catalyzing histone lysine methyltransferases. The SET domain-containing genes family plays an important role in various developmental and physiological processes of fungi, such as regulating fungal pathogenicity, affecting the synthesis of secondary metabolites, and responding to environmental stress. Although the related functions of SET genes have been studied in Aspergillus nidulans, Neurospora crassa, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and some other filamentous fungi, the identification and functional analysis of the SET gene family in Cordyceps militaris have not been reported. Because SET genes regulate fungal secondary metabolite synthesis through histone methylation modification, a systematic study of the SET gene family in C. militaris is crucial for deciphering the regulatory mechanisms of bioactive metabolites in this fungus. In this study, 22 SET domain-containing genes (excluding one tandem repeat gene) in C. militaris were identified through genome-wide analysis. The chromosome distribution, gene structure, protein domains, and physicochemical properties of the C. militaris SET domain-containing genes (CmSETs) were systematically analyzed and predicted. We constructed a multi-species phylogenetic tree of SET genes from C. militaris and four other model fungi, compared structural differences in SET domain-containing regions among these species, and explored homology relationships within the SET family. The phylogenetic tree showed that Suv4-20, SET1, SETD2 and SET_EZH subfamily members were highly homologous among the five fungi. Meanwhile we analyzed the expression levels of SET family members in C. militaris under salt stress and exogenous alanine treatment, suggesting that CmSETs are closely related to the cordycepin biosynthesis. To further confirm the function of CmSETs in cordycepin biosynthesis, CmSET14‌ and ‌CmSET16‌, showing up-regulation under both conditions, were overexpressed in C. militaris. The results showed that these two genes considerably enhanced the cordycepin production. This study provides the foundation for the role and molecular mechanism of SET domain genes in cordycepin biosynthesis.