Abstract: Anthocyanins exhibit diverse health benefits in humans, including antioxidant and anticancer effects, making them a key target for functional crop breeding. Anthocyanin synthase (ANS), a pivotal enzyme in anthocyanin biosynthesis, has been demonstrated to regulate anthocyanin accumulation in various crops. However, functional studies on ANS genes in tea plants (Camellia sinensis) remain limited. In this study, we identified a specifically highly expressed CsANS1 gene in the purple leaves of tea cultivar ‘Zijuan' through comparative transcriptome analysis of four tea cultivars with distinct leaf colors. Gene expression analysis reveals that CsANS1 expression in ‘Zijuan' was significantly higher than in other cultivars and other ANS family members (CsANS2 and CsANS3) in tea plants. Tissue-specific expression analysis further demonstrats that CsANS1 exhibited the highest expression level in ‘Zijuan' leaves, showing an expression trend of first increasing and then decreasing with the development of buds and leaves. Phylogenetic analysis indicats that sequence similarity among ANS gene copies within species is higher than that among interspecies homologs, and all members of the tea ANS family possess the highly conserved Fe(Ⅱ)/2OG dioxygenase domain. Comparative gene sequencing and protein tertiary structure prediction analyses reveal two non-synonymous mutations in the coding region of CsANS1 in ‘Zijuan', with the amino acid variation at position 165 potentially affecting local protein conformation. Following VIGS-mediated silencing of CsANS1, the anthocyanin content in ‘Zijuan' shoots significantly decreased, accompanied by a lightening of leaf color. In conclusion, this study confirms that CsANS1 is a key gene regulating high-level anthocyanin accumulation in ‘Zijuan' buds and leaves, providing an important molecular target for breeding high-anthocyanin tea cultivars.

Key words: Camellia sinensis, anthocyanin synthase, anthocyanin, virus-induced gene silencing, gene function