Identification of Chalcone Synthase Genes and Their Responses to Salt and Cold Stress in Poncirus trifoliata

Chalcone Synthase (CHS) plays a vital role in flavonoid synthesis, influencing plant growth, development, and responses to both biotic and abiotic stress. In this study, 11 <i>CHS</i> genes were identified in <i>Poncirus trifoliata</i> using bioinformatics methods, with their distribution across five chromosomes and unassigned contigs. Each gene contains 2-3 exons and 3-8 conserved motifs. In silico prediction suggested that the PtrCHS proteins are localized in the cytoplasm. PtrCHS9 and PtrCHS11 share identical protein tertiary structures. Phylogenetic analysis classified the <i>CHS</i> family members into four subgroups. Synteny analysis revealed one set of collinear gene pairs within <i>Poncirus trifoliata</i>. Between <i>Poncirus trifoliata</i> and <i>Arabidopsis thaliana</i>, two sets of collinear gene pairs were identified, while one such set was found between <i>Poncirus trifoliata</i> and <i>Oryza sativa</i>. Promoter element analysis showed the presence of various hormone response and stress response elements within <i>PtrCHS</i> promoters. RNA-Seq data demonstrated tissue-specific expression patterns of <i>PtrCHSs</i>. RT-qPCR results indicated that all <i>CHS</i> genes, except <i>PtrCHS11</i>, respond to salt stress with dynamic, member-specific patterns. Additionally, four <i>PtrCHSs</i> (<i>PtrCHS3</i>, <i>PtrCHS5</i>, <i>PtrCHS7</i>, and <i>PtrCHS10</i>) were significantly upregulated in response to cold treatment. Notably, <i>PtrCHS7</i> and <i>PtrCHS10</i> maintained high expression levels at both 6 and 12 h, implying they may be key players in cold stress response in <i>Poncirus trifoliata</i>. Clones of <i>PtrCHS7</i> and <i>PtrCHS10</i> were obtained, and overexpression vectors were constructed in preparation for gene transformation. Overall, this study provides a solid foundation for future research into the functions of the <i>PtrCHSs</i>.