Objective Chalcone synthase is a key enzyme in flavonoid biosynthesis. In this study, we identified the CHS gene family in hexaploid Camellia oleifera, performed expression analysis, and predicted its interacting proteins, aiming to provide a theoretical foundation for elucidating the molecular mechanisms underlying CHS regulation of flavonoid biosynthesis in C. oleifera.

Method Based on the genomic data of hexaploid C. oleifera ‘Changlin 40’, CoCHS genes were identified, and a comprehensive analysis of the gene family was performed, including physicochemical properties, phylogenetic evolution, gene structure, cis-acting regulatory elements in promoters, collinearity, tissue-specific expression patterns. And interacting proteins of CoCHS were predicted using AlphaFold3.

Result (1) A total of 45 CoCHS genes were identified from the hexaploid oil-Camellia genome. CoCHS proteins were clustered into two major subfamilies, the CHS subfamily and CHS-like subfamily. All members of the CHS subfamily contained the PLN03173 conserved domain, while those in the CHS-like subfamily contained the PLN03169 conserved domain. (2) Cis-acting element analysis showed that the promoters of the CoCHS genes contained a large number of elements related to plant growth and development, plant hormone response, as well as adversity stress response, which suggests that the CoCHS gene family is likely regulated by phytohormones and plays important roles in both stress responses and developmental processes in oil-tea plants. (3) Collinearity analysis revealed that segmental duplication was the primary driver of gene family expansion, and all hexaploid CoCHS genes exhibited collinearity strictly within the same subfamily. Interspecific collinearity analysis showed that tetraploid C. oleifera and hexaploid C. oleifera shared the highest number of collinear gene pairs. (4) Transcriptome analysis indicated that ubiquitous expression of CoCHS genes in multiple tissues and seed developmental phases, while CoCHS16,CoCHS17, CoCHS18, CoCHS19, CoCHS20, CoCHS21 exhibited significantly elevated expression specifically in mid-to-late embryogenesis, implying their involvement in modulating flavonoid accumulation in oil-tea camellia seeds. (5) CoCHS16-MYB transcription factor protein complex was modeled based on the dominantly expressed CoCHS16 protein during mid-to-late seed development and identified five potential MYB-interacting transcription factors.

Conclusion 45 CoCHS proteins are evolutionarily conserved, and their members are likely to play distinct roles in flavonoid synthesis and associated stress responses. This study provides key targets for elucidating the molecular mechanisms of flavonoid biosynthesis and lays a theoretical foundation for molecular breeding aimed at improving oil tea quality in C. oleifera.