Genome-wide Identification and Characterization of the 3- Dehydroquinate Dehydratase/Shikimate Dehydrogenase Gene Family in Rubus chingii Hu

Objective 3-Dehydroquinic acid dehydrogenase/shikimate dehydrogenase (DHD/SDH) is the sole bifunctional enzyme in the plant shikimate pathway, catalysing both shikimic acid formation and its subsequent conversion to gallic acid. As a key regulatory node in gallic acid biosynthesis, DHD/SDH is implicated in the production of downstream derivatives, including ellagic acid. In this study, we systematically analysed the RcDHD/SDH gene family in Rubus chingii Hu ( R. chingii ), with the aim of elucidating their regulatory features and expression patterns across metabolic pathways, characterising tissue- and fruit developmental stage-specific expression profiles, and evaluating their potential association with gallic acid and ellagic acid accumulation. Methods In this study, bioinformatics approaches were employed to identify members of the DHD/SDH gene family in R. chingii . The physicochemical properties, predicted subcellular localisations, chromosomal distributions, phylogenetic relationships and promoter cis-acting element compositions of the encoded proteins were systematically analysed. Quantitative real-time PCR (qRT-PCR) was used to evaluate the expression levels of individual DHD/SDH genes across different tissues and organs, during fruit developmental stages, and in response to exogenous hormone treatments. In parallel, high-performance liquid chromatography (HPLC) was conducted to quantify gallic acid and ellagic acid contents in the corresponding tissues and fruit developmental stages. These integrated analyses enabled assessment of potential associations between DHD/SDH gene expression patterns and metabolite accumulation in R. chingii . Results A total of four DHD/SDH family members ( RcDHD/SDH1 – RcDHD/SDH4 ) were identified in Rubus chingii Hu. These genes were evenly distributed across two chromosomes and were predicted to encode proteins ranging from 440 to 1,325 amino acids in length. Phylogenetic analysis indicated that all RcDHD/SDH proteins clustered within subfamily IV. Subcellular localisation predictions suggested that RcDHD/SDH proteins are localised to both the nucleus and the cytoplasm, and that all members contain the conserved DHD/SDH functional domain. Promoter analysis revealed that RcDHD/SDH genes harbour diverse cis-acting elements, including hormone-responsive, light-responsive, and low-temperature-responsive elements. qRT-PCR analysis demonstrated that the RcDHD/SDH gene family exhibits tissue-specific expression patterns in R. chingii . RcDHD/SDH1 - RcDHD/SDH4 exhibited relatively stable expression in roots, whereas RcDHD/SDH4 displayed comparatively high expression levels in stems and leaves. During fruit development, the overall expression of RcDHD/SDH genes was highest at the early stages and subsequently declined. This expression trend closely corresponded with the rapid accumulation of gallic acid and ellagic acid from the green fruit stage to the colour transition stage, followed by stabilisation at fruit maturity. These results suggest that RcDHD/SDH genes play an important regulatory role during the early stages of fruit growth and development. Conclusion This study systematically characterised the composition, structural features, and expression patterns of the RcDHD/SDH gene family in R. chingii, providing a theoretical foundation for further investigation of their biological functions and roles in secondary metabolite regulation.