Overexpressing chalcone synthase (CHS) gene enhanced flavonoids accumulation in Glycyrrhiza uralensis hairy roots

Flavonoids are key bioactive ingredients in Glycyrrhizia uralensis, a widely used herbal medicine in China. Chalcone synthase (CHS) is the first rate-limiting enzyme involved in the flavonoid biosynthetic pathway. In this paper, we analyzed the function of CHS by inducing CHS overexpression in the hairy roots of G. uralensis. CHS was cloned from G. uralensis to construct a plant expression vector pCA-CHS, which was then transformed into Agrobacterium rhizogenes ACCC10060 by electrotransformation. The 7-day-old cotyledons and hypocotyls of G. uralensis were infected by the recombinant A. rhizogenes to induce transgenic hairy roots. Transgenic hairy root lines were confirmed by PCR and sequencing analysisIs. Copy number of CHS in these transgenic hairy root lines was determined by qRT-PCR as 9, 10, 11, 13, and 18, respectively. UPLC analysis showed that the contents of liquiritigenin, isoliquiritigenin, isoliquiritin, and total flavonoids, in transgenic hairy roots over expressing CHS were significantly higher than those in the wild type hairy roots. This result demonstrates that overexpressing CHS in hairy roots of G. uralensis is able to enhance flavonoids accumulation. A transgenic line (S20) with nine copies of CHS was found to contain the highest contents of flavonoids and was selected for further study. Our study demonstrates that CHS is a key gene in the flavonoid biosynthetic pathway and can be utilized to increase flavonoid production in G. uralensis.Abbreviations: CHS: Chalcone synthase; CHR: Chalcone reductase; CHI: Chalcone isomerase; GT: Glycosyl transferase; UPLC: Ultra Performance Liquid Chromatography; qRT-PCR: Quantitative real time polymerase chain reaction; PCR: Polymerase chain reaction; Kan: Kanamycin sulfate