The role of licorice DXS knockout and overexpression in glycyrrhizic acid biosynthesis / 药学学报

1-Deoxy-D-xylulose-5-phosphate synthase (DXS) is a rate-limiting enzyme involved in the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for terpenoid precursor biosynthesis. DXS plays an essential role in glycyrrhizic acid (GA) biosynthesis. Based on our previous transcriptome study, there was a negative correlation between DXS expression and GA content. Therefore, we explored the regulatory role of DXS in GA biosynthesis using both gene overexpression and gene knockout in a hairy root culture system. DXS was cloned from Glycyrrhiza glabra L. (GenBank Accession No. MN158121). A plant binary expression vector pCA-DXS was constructed by a gene fusion method. The sgRNA sequence was designed based on the first exon of DXS to construct the gene editing vector pHSE-DXS. Hairy roots overexpressing or knocking out DXS were generated through an Agrobacterium-mediated method with licorice hypocotyls as explants. Wild-type hairy roots and negative control hairy roots containing empty plasmids were also evaluated. UPLC was used to determine the GA content in each licorice hairy root line. Results showed that the content of GA in the hairy root group knocking out DXS was significantly higher than that in the wild-type and negative control groups, while in the hairy root group overexpressing DXS was significantly lower, suggesting that DXS plays a negative role in GA biosynthesis. This study provides a foundation for determining the function of DXS in terpenoid metabolism and for further establishment of a molecular regulatory network of GA biosynthesis.

Overexpressing of chalcone isomerase (CHI) gene enhances flavonoid accumulation in Glycyrrhiza uralensis hairy roots / 药学学报

Chalcone isomerase (CHI) is the second rate-limiting enzyme involved in the biosynthetic pathway of flavonoids in Glycyrrhiza uralensis. Based on our previous studies, we selected the specific CHI haplotype (GenBank Accession No. KY115232) to maximize flavonoid accumulation. We constructed a plant binary expression vector for overexpression of this CHI gene by the gene fusion method and transfected the plasmid into Agrobacterium tumefaciens ACCC10060 by electroporation. The recombinant A. tumefaciens ACCC10060 subsequently was used to infect cotyledons and hypocotyls of G. uralensis to obtain transgenic hairy roots. A qRT-PCR method was used to determine the copy number of CHI and a UPLC method was used to assay the content of four flavonoids in different hairy root lines. The qRT-PCR results showed that the copy number of CHI in hairy roots was 1 or 5. UPLC results showed that the content of total flavonoids, liquiritin, liquiritigenin, and isoliquiritigenin in transgenic hairy root samples was significantly higher than that in wild-type samples. This study demonstrates that overexpression of CHI significantly increases the content of flavonoids in hairy roots of G. uralensis. This work provides a theoretical basis for clarifying the function of CHI. Three transgenic hairy root lines of G. uralensis were isolated which can be used to increase the accumulation of licorice flavonoids in vitro.

Cloning and Bioinformatic Analysis of ARPI Gene from Glycyrrhiza glabra / 中国实验方剂学杂志

Objective::To clone the complementary deoxyribonucleic acid (cDNA) of auxin/indole acetic acid protein (Aux/IAA) from Glycyrrhiza glabra (GgARPI) and analyze its sequence by bioinformatics. Method::RNA was extracted from fresh root of G. glabra, the cDNA sequence of GgARPI gene was cloned by reverse transcription polymerase chain reaction (RT-PCR), then sequencing and bioinformatic analysis were performed. Result::The GgARPI cDNA sequence with the full length of 686 bp was obtained from G. glabra. The full open reading frame (ORF) was 585 bp, encoding 194 amino acid residues. The bioinformatic analysis showed that the protein coded by GgARPI was a stable hydrophilic protein, with a relative molecular weight of 21.95 kDa and isoelectric point of 6.85.It contained no signal peptides or transmembrane domain. Its secondary structure mainly consisted of random coil. An Aux/IAA superfamily was included in the conversed domain. Homology analysis indicated that it had a close evolutionary relationship with leguminous plants, and a distant evolutionary relationship with monocotyledon, such as Setaria italica. Conclusion::GgARPI cDNA sequence is successfully cloned from G. glabra for the first time, which will lay a foundation for studying the function of GgARPI and explaining the molecular regulatory mechanism of biosynthesis of glycyrrhizic acid in G. glabra.

Molecular Cloning and Sequence Analysis of GgUGE Gene in Glycyrrhiza glabra / 中国实验方剂学杂志

Objective::To clone the cDNA sequence of UDP-glucose 4-epimerase (UGE) in Glycyrrhiza glabra and analyze its sequence, so as to explore the potential relationship between the UGE gene and the molecular regulatory mechanisms of glycyrrhizic acid biosynthesis. Method::The cDNA sequence of UGE was cloned from the root of G. glabra by reverse transcription polymerase chain reaction (RT-PCR), then sequenced and analyzed by bioinformatics software. Results::A GgUGE cDNA sequence with the full length of 1 121 bp was obtained. The open reading fame (ORF) of GgUGE was 1 053 bp, encoding 350 amino acid residues. The GgUGE cDNA sequence was submitted to GenBank, and the accession No. was MK638908. Sequence analysis showed that GgUGE was an unstable hydrophilic protein, its average relative molecular weight was 39.02 kDa, and isoelectric point was 6.13. It contained no signal peptides or transmembrane domains. Its secondary structure mainly constituted of α-helix and had a conversed domain of UDP-glucose 4-epimerase superfamily. The homologoue analysis showed that the cDNA and amino acid sequences of GgUGE had the closest evolutionary relationship to Leguminosae and relatively distant evolutionary relationship to Salicaceae. Conclusion::In this study, GgUGE cDNA sequence is successfully cloned from G. glabra for the very first time, which will provide reference for studying the function of GgUGE and explaining the molecular regulatory mechanisms of glycyrrhizic acid biosynthesis in G. glabra.