| Summary: | <i>Abelmoschus manihot</i> L. (HSK) is a rare and endangered species in the wild that grows on the cliffs of deep mountains. As a natural plant, the chemical composition of HSK is relatively complex, which mainly includes flavonoids, organic acids, polysaccharides, and various trace elements with good effects of clearing away heat, anti-inflammatory, analgesic, and calming nerves, and inhibiting tumor cells. In this experiment, different developmental stages of HSK flowers were used for optimization of the flavonoid extraction and determining method. The antioxidant activities, flavonoid accumulation pattern, and synthesis regulatory network were analyzed using biochemistry, RNA-seq, and UPLC-MS/MS. The total content of flavonoids, vitexin rhamnoside, hyperoside, and rutin in HSK flowers at T3 stage (flower wilting) was significantly higher than in T2 (full flowering) and T1 (bud) stages. Compared with T1 and T2, the antioxidant capacity of the T3 flower alcohol extract was also the strongest, including the total reducing ability, DPPH clearance, OH clearance, O<sup>2−</sup> clearance, and total antioxidant capacity. A total of 156 flavonoids and 47,179 unigenes were detected by UPLC-MS/MS and RNA-Seq, respectively. The candidate genes and key metabolites involved in flavonoid biosynthesis were identified and the regulatory networks were also analyzed in this study. qRT-PCR test further proved that the gene expression level was consistent with the results of RNA sequence data. The relationship between the gene expression and flavonoid accumulation network provides a theoretical basis for the mining and regulation of functional genes related to the flavonoid biosynthesis and metabolism in <i>Abelmoschus manihot</i> L.
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