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ABSTRACT
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
Acknowledgments
We thank Dr. Yu Jiang at University of Pittsburgh for critically reading the manuscript.
Disclosure statement
No potential conflict of interest was reported by the authors.
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Notes on contributors
Yan-Chao Yin
Yan-Chao Yin, a third-year graduate student, and Jia-Ming Hou, a second-year graduate student, contributed equally to this paper and were responsible for the transformation, induction, selection, and identification of G. uralensis hairy roots.
Jia-Ming Hou
Jia-Ming Hou is a second-year graduate student and constructed the growth curves.
Shao-Kai Tian
Shao-Kai Tian and Zhi-Xin Zhang are first-year graduate students and analyzed the copy number of CHS gene in transgenic hairy roots by qRT-PCR.
Lin Yang
Lin Yang, an associate chief pharmacist, measured the flavonoids contents in wild and transgenic hairy root samples.
Zhi-Xin Zhang
Zhi-Xin Zhang, an associate professor at Beijing University of Chinese Medicine, designed all of the experiments and wrote the manuscript.