Molecular cloning and functional characterization of UGTs from Glycyrrhiza uralensis flavonoid pathway.

Glycyrrhiza uralensis Fisch., a well-known medicinal plant, contains flavonoids including liquiritigenin and isoliquiritigenin, and their corresponding glycoside liquiritin and isoliquiritin. Although some genes encoding UDP-glycosyltransferases (UGTs) have been functionally characterized in G. uralensis, other UGTs mechanisms of glycosylation remain to be elucidated. Against this background the aim of the present study included cloning and characterization of two full-length cDNA clones of GuUGT isoforms from the UGT multigene family. These included GuUGT2 (NCBI acc. MK341791) and GuUGT3 (NCBI acc. MK341793) with an ORF of 1473 and 1332 bp, respectively. Multiple alignments and phylogenetic analysis revealed GuUGTs protein of Glycine max had a high homology to that of other plants. Meanwhile, quantitative real-time PCR was performed to detect the transcript levels of GuUGTs in different tissues. The results indicated that GuUGTs was more expressed in roots as compared to the leaves, and significantly up-regulated upon NaCl stress. The recombinant protein was heterologous expressed in Escherichia coli and exhibited a high level of UGT activity, catalyzing formation of isoliquiritin and liquiritin from isoliquiritigenin and liquiritigenin. The key residues of GuUGT2 for liquiritigenin glycosylation (Asn223), isoliquiritigenin (Asp272) were predicted by molecular docking and residue scanning based on simulated mutations. These results could serve as an important reference to understand the function of the UGT family. In addition, the identification of GuUGT2 and GuUGT3 provides a foundation for future studies of flavonoid biosynthesis in G. uralensis.

[Mechanism of genuineness of Glycyrrhiza uralensis based on SNP of ß-Amyrin synthase gene].

ß-Amyrin synthase (ß-AS) genes of Glycyrrhiza uralensis from 6 different regions were analyzed by PCR-SSCP and sequenced, then the correlationship between ß-AS SNP and regions of Glycyrrhiza uralensis were determined. According to the 1 coding single nucleotide polymorphism on the first exon of ß-AS gene at 94 bp site, Glycyrrhiza uralensis could be divided into 3 genotypes. In these genotypes, the percentage of 94A type in genuine regions was much higher, and it had significant differences with the percentage in non-genuine regions (P < 0.001). The results of the experiment proved that different ß-AS genotypes at 94 bp site from different regions may be one of the important reasons to result in the genuineness of Glycyrrhiza uralensis.

[Relationship Between Seedling Grade of Glycyrrhiza uralensis and Plant Biomass Accumulation, Yield and Quality of Product].

OBJECTIVE: To research the relationship between the seedling grade of Glycyrrhiza uralensis and the biomass accumulation, yield and quality of product, so as to provide basis for establishing seedling standard. METHODS: The weight of single seedling root was measured and the seedlings were divided into three grades by the clustering analysis. The different grade treatments of seedlings were made to conduct field trials and laboratory experiments. RESULTS: The weight of the whole plant and dry root in growth period of grades 1 and 2 (the weight of single root greater than 10.0 g) were larger than grade 3, and the yield was also the case. The root of Glycyrrhiza uralensis, growing for three years, of grades 1 and 2 contained higher contents of active compounds than grade 3 and the content of glycyrrhizin and liquiritin in the root of Glycyrrhiza uralensis of all treatments were higher than the standard of Chinese Pharmacopoeia (2010 edition). CONCLUSION: The plant from the grades 1 and 2 seedlings with larger growth increment,higher output and better quality is the best seedling in cultivation.

Application of mixture analysis to crude materials from natural resources (IV)[1(a-c)]: identification of Glycyrrhiza species by direct analysis in real time mass spectrometry (II).

In order to identify Glycyrrhiza species by chemical fingerprinting, the bark of the roots and stolons of Glycyrrhiza uralensis Fischer and G. glabra Linné were analyzed using DART (Direct Analysis in Real Time)-MS. The characteristic peaks of each species were determined statistically by volcano plot. This summarizes the relationship between the p-values of a statistical test and the magnitude of the difference in values of the samples in the groups. In this experiment, peaks that had a p value <0.05 in the t test and Z2 absolute difference were defined as characteristic. As a result, characteristic peaks of G. uralensis were found at m/z 299, 315, 341, and 369. In contrast, characteristic peaks of G. glabra were found at m/z 323, 325, 337, 339, and 391. In conclusion, we found several characteristic peaks to distinguish G. uralensis and G. glabra by DART-MS using volcano plot. This method can be applied to identify the Glycyrrhiza species.

[Cloning and characterization of 3-hydroxy-3-methylglutary CoA reductase cDNA of Glycyrrhiza uralensis].

OBJECTIVE: To clone and analysis the sequence of 3-hydroxy-3-methylglutary CoA reductase (HMGR) cDNA from Glycyrrhiza uralensis. METHOD: The primers were designed based on the conservative region of HMGR nucleic acids sequence from public database. The target gene was obtained from root of G. uralensis by use of homologous cDNA amplificati on and RACE technologies. The sequence alignment was performed using BLAST. The open reading frame was identified by use of the ORF Finder. The protein domains were defined by use of Prosite software. Clustal was used to conduct multiple amino acid sequence alignment and MEGA 5.0 was used to conduct the phylogenetic tree. RESULT: The GuHMGR cDNA sequence was obtained contains 1 842 bp contains a 1 722 bp ORF, encoding 573 amino acids with 3-hydroxy-3-methylglutary CoA reductases family profile. Deduced amino acid sequence had 84% and 76% homology to the amino acid sequence of Pisum sativum, Medicago truncatula. CONCLUSION: The cloning of 3-hydroxy-3-methylglutary CoA reductase (HMGR) cDNA will provide a foundation for exploring the function of HMGR in glycyrrhizin biosynthesis.

[Study on the inducement and influential factors of callus with different organs of Glycyrrhiza uralensis].

OBJECTIVE: To investigate the influential factors and establish culture method for G. uralensis callus. METHODS: To study the possible effective factors of culture condition by comparing with different explants, light, plant hormones and its ratio. RESULTS: The hypocotyl was the best among different explants, its inducing ratios was 94%, and the callus occurred earliest. When the calluses were inoculated on MS + 6 - BA (1.0-2.0) mg/L + NAA (0.5-1.0) mg/L, adventitious buds formed. 2,4-D could induce non-embryogenic callus, the compounding proportions of 6 - BA and NAA could induce embryogenic callus. Light influenced induction and growth of callus. CONCLUSION: Different explants, components of hormone and light are the influencial factors of callus induction and growth of G. uralensis.

[Cloning and characterization of open reading frame encoding beta-amyrin synthase in Glycyrrhiza uralensis].

OBJECTIVE: To clone and sequence the open reading frame of beta-amyrin synthase (bAS) from Glycyrrhiza uralensis. METHOD: The primers were designed according to the cDNA sequence of beta-amyrin synthase from G. glabra reported by Hiroaki HAYASHI, and the open reading frame of beta-amyrin synthase was cloned by RT-PCR strategy with the template of total RNA extracted from roots of G. uralensis. RESULT: The GubAS (GenBank Accession number: FJ627179) was 2 289 bp in length encoding one pelypeptide of 762 amino acid. Deduced amino acid sequence had 99%, 92%, 90%, 90% and 89% homology to the amino acid sequence of G. glabra, Lotus japonicus, Pisum sativum, Medicago truncatula, Glycine max, respectively. CONCLUSION: The open reading frame of bAS from G. uralensis is cloned and reported for the first time. The conclusion will provide a foundation for exploring the mechanism of triterpenes biosynthesis.

[Research of seed quality grading of Glycyrrhiza uralensis].

OBJECTIVE: To formulate the seed quality grading standard of Glycyrrhiza uralensis. METHOD: Thousand-grain weight, seed moisture, germination rate, purity of G. uralensis seed samples from 24 regions were tested. Through statistical analysis, the key indicator and the reference indicators for seed quality grading were defined. RESULT: Germination percentage was the primary indicator of seed quality grading, thousand-grain weight, cleanliness and moisture content were important reference indicators. CONCLUSION: The seed quality of each grade should reach the following requirements: for first grade seeds, germination percentage > or = 85% , purity > or = 92%, thousand-grain weight > or = 13 g, seed moisture < or = 11%; for second grade seeds, germination percentage 75%-85%, purity 83%-92%, thousand-grain weight 11-13 g, seed moisture < or = 11%; for third grade seeds, germination percentage 65%-75%, purity 74%-83%, thousand-grain weight 9-11 g, seed moisture < or = 11%.

[Study on geographical variation of morphologic and germination characteristic of different Glycyrrhiza uralensis provenance seeds].

OBJECTIVE: To study the geographical variation of morphologic and germination characteristic of different Glycyrrhiza uralensis provenance seeds, approach the geographical variation mode and ecology mechanism, and laid theoretical foundation for districting and allocating of G. uralensis seeds. METHOD: Field investigation and laboratory analysis were applied. Seed shape and kilosseed weight were sampled randomly, germination rate germination force by general methods. RESULT: The morphologic characteristic of G. uralensis seeds showed roughly longitude variation tendency that the seeds increased gradually from west to east. While the germination characteristic showed roughly altitude variation tendency that the seeds germination rate and germination force increased with the increase of the altitude, and the average germination rate was the same with the seeds morphologic characteristic. The results of analysis correlated with the climatic factors show that the morphologic characteristic of G. uralensis was positive correlated with annual rain-fall of the habitat, and the germination rate was quickened by drought, high temperature and strong sunshine. CONCLUSION: The morphologic and germination characteristic and of G. uralensis seeds present distinguished geographical variation, and the formation of the variation was related to the ecological environment in which the seed provenance adapted.

[Study on the identification of standard and false Gancao by Fourier transform infrared spectroscopy].

Standard Gancao (Glycyrrhiza uralensis Fisch) and false Ciguogancao (Glycyrrhiza pallidiflata Batal) were identified fast, nondestructively by Fourier transform infrared spectroscopy (FTIR) combined with derivative spectra and two-dimensional correlation spectroscopy (2D) in the present article. The result shows that although the two kinds of Gancao belong to one genus, there are some certain differences in their chemical components that are reflected in the IR spectra, but with some similarity and dissimilarity in the IR spectra. The two kinds of Gancao are quite different from each other in second derivative spectra and 2D spectra. Based on the differences reflected in the IR and 2D IR, the standard gancao can be identified from the false easily and clearly. The result also proved that there is a relationship between the IR spectra and the chemical components of the herbs. This method is fast, accurate and nondestructive, and the wastage of sample is less. The fast, accurate property of 2D spectroscopy makes it a powerful and new approach to evaluating medicinal herbs impersonally.

[Identification on Chinese materia medica liquoric root by X-ray diffraction Fourier fingerprint pattern method].

OBJECTIVE: To develop a new identification and analysis method of Chinese medicinal materia Liquoric Root. METHODS: Powder X-ray diffraction Fourier fingerprint pattern. RESULTS: Experiments and analysis were carried out on ten samples. The standard X-ray diffraction Fourier fingerprint pattern and characteristic diffraction peaks of Liquoric Root were obtained. CONCLUSION: This method can be used for identification of Chinese medicinal materia Liquoric Root.