Expression characteristics and functional analysis of RniSDH3 gene in raspberry

【Objective】Ellagic acid (EA) is a significant polyphenolic secondary metabolite with notable health-promoting properties, including antioxidant, anti-inflammatory, and anticancer activities, as well as preventive effects on cardiovascular and metabolic disorders. Raspberries (Rubus L.) are the richest natural sources of EA and serve as an ideal model for studying its biosynthetic pathways. However, efforts in raspberry breeding in China began relatively late, and the scarcity of elite cultivars has become a major constraint on industrial development. Rubus niveus Thunb., a wild raspberry species widely distributed in Southwest China, exhibits strong environmental adaptability, ease of cultivation, and high EA content, making it a valuable candidate for genetic improvement. The biosynthesis of EA in plants involves three main stages: the formation of gallic acid (GA) through the shikimate pathway, the conversion of GA to ellagitannins, and the hydrolysis of ellagitannins to produce EA. Shikimate dehydrogenase (SDH) catalyzes the conversion of 3- dehydroshikimate (3-DHS) to GA and represents a key enzyme in this pathway. Previous studies in grapevine and tea have confirmed the role of SDH fami-ly genes in GA biosynthesis; however, their function in raspberry remains unclear. Using a high-quality genome assembly of R. niveus, we identified four members of the SDH gene family. Expression analysis across fruit developmental stages revealed a significant positive correlation between RniSDH3 expression levels and EA accumulation. Further analyses, including bioinformatics, subcellular localization, and expression profiling of RniSDH3, were performed. The function of RniSDH3 was validated using an overexpression system in transgenic raspberry callus. This research aims to clarify the role of RniSDH3 in EA biosynthesis and to provide a theoretical foundation and genetic resources for the molecular breeding of raspberry cultivars with elevated EA content.【Methods】The RniSDH3 gene was identified and cloned from the genome of the red raspberry cultivar‘Rubus niveus Thunb.’. Sequence and domain analyses were performed, and homology to known SDH proteins was assessed by multiple sequence alignment and phylogenetic analysis. Subcellular localization of RniSDH3 was determined by transient expression of a RniSDH3-GFP fusion in tobacco leaf cells. EA content in fruits at 24, 30, 36, and 45 days after flowering was quantified by HPLC, and RniSDH3 expression was measured by RT-qPCR. Transgenic raspberry calli overexpressing RniSDH3 were generated via Agrobacterium- mediated transformation to assess their effect on EA accumulation.【Results】This study identified four genes homologous to shikimate dehydrogenase (SDH) in raspberry, which were named RniSDH1-1, RniSDH1-2, RniSDH3, and RniSDH4, respectively. The expression levels of these genes during different developmental stages of raspberry fruit were determined using RT-qPCR, and their correlation with EA content at each stage was analyzed. The results showed that among the candidate genes, the expression pattern of RniSDH3 was consistent with the trend of EA accumulation, suggesting that RniSDH3 may play a key role in EA biosynthesis in raspberry. Based on this, RniSDH3 was selected for further functional investigation. The full-length CDS of RniSDH3 was cloned and subjected to sequence analysis. The results revealed that the encoded protein contains typical conserved domains: Shikimate_dh_N and NAD_bind_Shikimate_DH, both of which are highly conserved among SDH proteins in various plants such as rose, strawberry, and blackberry. Physicochemical analysis showed that RniSDH3 encodes a stable, non-transmembrane protein composed of 530 amino acids, with neutral hydrophilicity. Sequence alignment indicated that RniSDH3 shares over 70% sequence identity with homologous SDH proteins from related Rosaceae species (e.g., strawberry and rose), suggesting its conserved role in the biosynthesis of aromatic amino acids and phenolic compounds. Subcellular localization analysis showed that RniSDH3 is mainly localized in the cytoplasm and nucleus. To further investigate the effect of RniSDH3 on EA accumulation, an RniSDH3 overexpression vector was constructed and used to generate transgenic raspberry calli via the CDB method. The results showed that the EA content in RniSDH3-overexpressing transgenic tissues was significantly higher than that in the empty vector control, indicating that RniSDH3 overexpression promotes EA accumulation in raspberry. Taken together with previous studies, the findings demonstrate that RniSDH3, a member of the SDH gene family, plays a pivotal role in the biosynthesis of EA in raspberry. This research provides a molecular basis for the development of raspberry cultivars enriched in EA through genetic improvement.【Conclusion】RniSDH3 is a key SDH gene involved in EA biosynthesis in raspberry. Its expression is closely associated with EA accumulation during fruit development, and its overexpression can significantly elevate EA levels. This study provides new insights into the molecular regulation of EA metabolism and lays a theoretical foundation for the genetic improvement of raspberry cultivars with enhanced nutritional and functional properties.