Genome-wide analysis of the ERF Family in Stephania japonica provides insights into the regulatory role in Cepharanthine biosynthesis

Cepharanthine (CEP), a bisbenzylisoquinoline alkaloid (bisBIA) extracted from , has received significant attention for its anti-coronavirus properties. While ethylene response factors (ERFs) have been reported to regulate the biosynthesis of various alkaloids, their role in regulating CEP biosynthesis remains unexplored. Genome-wide analysis of the ERF genes was performed with bioinformatics technology, and the expression patterns of different tissues, were analyzed by transcriptome sequencing analysis and real-time quantitative PCR verification. The nuclear-localized ERF gene cluster was shown to directly bind to the promoters of several CEP-associated genes, as demonstrated by yeast one-hybrid assays and subcellular localization assays. In this work, 59 SjERF genes were identified in the genome and further categorized into ten subfamilies. Notably, a SjERF gene cluster containing three genes was found on chromosome 2. Yeast one-hybrid assays confirmed that the SjERF gene cluster can directly bind to the promoters of several CEP-associated genes, suggesting their crucial role in CEP metabolism. The SjERFs cluster-YFP fusion proteins were observed exclusively in the nuclei of leaves. Tissue expression profiling revealed that 13 exhibit high expression levels in the root, and the qRT-PCR results of six were consistent with the RNA-Seq data. Furthermore, a co-expression network analysis demonstrated that 24 were highly positively correlated with the contents of various alkaloids and expression levels of CEP biosynthetic genes. This study provides the first systematic identification and analysis of ERF transcription factors in the genome, laying the foundation for the future functional research of SjERFs transcription factors.