AUTHOR=Yang Hanting , Liu Baimei , Ding Haiyan , Liu Zhaoyu , Li Xiaodong , He Tianxing , Wu Ya , Zhang Yuxuan , Wang Can , Leng Liang , Chen Shilin , Song Chi 

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

JOURNAL=Frontiers in Plant Science

VOLUME=15

YEAR=2024

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2024.1433015

DOI=10.3389/fpls.2024.1433015

ISSN=1664-462X

ABSTRACT=<sec><title>Introduction</title><p>Cepharanthine (CEP), a bisbenzylisoquinoline alkaloid (bisBIA) extracted from <italic>Stephania japonica</italic>, 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.</p></sec><sec><title>Methods</title><p>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.</p></sec><sec><title>Results</title><p>In this work, 59 SjERF genes were identified in the <italic>S. japonica</italic> genome and further categorized into ten subfamilies. Notably, a SjERF gene cluster containing three <italic>SjERF</italic> 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 <italic>Nicotiana benthamiana</italic> leaves. Tissue expression profiling revealed that 13 <italic>SjERFs</italic> exhibit high expression levels in the root, and the qRT-PCR results of six <italic>SjERFs</italic> were consistent with the RNA-Seq data. Furthermore, a co-expression network analysis demonstrated that 24 <italic>SjERFs</italic> were highly positively correlated with the contents of various alkaloids and expression levels of CEP biosynthetic genes.</p></sec><sec><title>Conclusion</title><p>This study provides the first systematic identification and analysis of ERF transcription factors in the <italic>S.japonica</italic> genome, laying the foundation for the future functional research of SjERFs transcription factors.</p></sec>