Integrated phenotypic, transcriptomics and metabolomics: growth status and metabolite accumulation pattern of medicinal materials at different harvest periods of Astragalus Membranaceus Mongholicus.

BACKGROUND: Astragalus membranaceus var. mongholicus (Astragalus), acknowledged as a pivotal "One Root of Medicine and Food", boasts dual applications in both culinary and medicinal domains. The growth and metabolite accumulation of medicinal roots during the harvest period is intricately regulated by a transcriptional regulatory network. One key challenge is to accurately pinpoint the harvest date during the transition from conventional yield content of medicinal materials to high and to identify the core regulators governing such a critical transition. To solve this problem, we performed a correlation analysis of phenotypic, transcriptome, and metabolome dynamics during the harvesting of Astragalus roots. RESULTS: First, our analysis identified stage-specific expression patterns for a significant proportion of the Astragalus root genes and unraveled the chronology of events that happen at the early and later stages of root harvest. Then, the results showed that different root developmental stages can be depicted by co-expressed genes of Astragalus. Moreover, we identified the key components and transcriptional regulation processes that determine root development during harvest. Furthermore, through correlating phenotypes, transcriptomes, and metabolomes at different harvesting periods, period D (Nov.6) was identified as the critical period of yield and flavonoid content increase, which is consistent with morphological and metabolic changes. In particular, we identified a flavonoid biosynthesis metabolite, isoliquiritigenin, as a core regulator of the synthesis of associated secondary metabolites in Astragalus. Further analyses and experiments showed that HMGCR, 4CL, CHS, and SQLE, along with its associated differentially expressed genes, induced conversion of metabolism processes, including the biosynthesis of isoflavones and triterpenoid saponins substances, thus leading to the transition to higher medicinal materials yield and active ingredient content. CONCLUSIONS: The findings of this work will clarify the differences in the biosynthetic mechanism of astragaloside IV and calycosin 7-O-ß-D-glucopyranoside accumulation between the four harvesting periods, which will guide the harvesting and production of Astragalus.

Genome-wide identification and expression pattern analysis of the GRF transcription factor family in Astragalus mongholicus.

BACKGROUND: Astragalus membranaceus is a plant of the Astragalus genus, which is used as a traditional Chinese herbal medicine with extremely high medicinal and edible value. Astragalus mongholicus, as one of the representative medicinal materials with the same origin of medicine and food, has a rising market demand for its raw materials, but the quality is different in different production areas. Growth-regulating factors (GRF) are transcription factors unique to plants that play important roles in plant growth and development. Up to now, there is no report about GRF in A. mongholicus. METHODS AND RESULTS: This study conducted a genome-wide analysis of the AmGRF gene family, identifying a total of nine AmGRF genes that were classified into subfamily V based on phylogenetic relationships. In the promoter region of the AmGRF gene, we successfully predicted cis-elements that respond to abiotic stress, growth, development, and hormone production in plants. Based on transcriptomic data and real-time quantitative polymerase chain reaction (qPCR) validation, the results showed that AmGRFs were expressed in the roots, stems, and leaves, with overall higher expression in leaves, higher expression of AmGRF1 and AmGRF8 in roots, and high expression levels of AmGRF1 and AmGRF9 in stems. CONCLUSIONS: The results of this study provide a theoretical basis for the further exploration of the functions of AmGRFs in plant growth and development.

Characterization and protein engineering of glycosyltransferases for the biosynthesis of diverse hepatoprotective cycloartane-type saponins in Astragalus membranaceus.

Although plant secondary metabolites are important source of new drugs, obtaining these compounds is challenging due to their high structural diversity and low abundance. The roots of Astragalus membranaceus are a popular herbal medicine worldwide. It contains a series of cycloartane-type saponins (astragalosides) as hepatoprotective and antivirus components. However, astragalosides exhibit complex sugar substitution patterns which hindered their purification and bioactivity investigation. In this work, glycosyltransferases (GT) from A. membranaceus were studied to synthesize structurally diverse astragalosides. Three new GTs, AmGT1/5 and AmGT9, were characterized as 3-O-glycosyltransferase and 25-O-glycosyltransferase of cycloastragenol respectively. AmGT1G146V/I variants were obtained as specific 3-O-xylosyltransferases by sequence alignment, molecular modelling and site-directed mutagenesis. A combinatorial synthesis system was established using AmGT1/5/9, AmGT1G146V/S and the reported AmGT8 and AmGT8A394F . The system allowed the synthesis of 13 astragalosides in Astragalus root with conversion rates from 22.6% to 98.7%, covering most of the sugar-substitution patterns for astragalosides. In addition, AmGT1 exhibited remarkable sugar donor promiscuity to use 10 different donors, and was used to synthesize three novel astragalosides and ginsenosides. Glycosylation remarkably improved the hepatoprotective and SARS-CoV-2 inhibition activities for triterpenoids. This is one of the first attempts to produce a series of herbal constituents via combinatorial synthesis. The results provided new biocatalytic tools for saponin biosynthesis.

[Molecular mechanism underlying difference of astragaloside â £ content in imitating wild and cultivated Astragalus mongholicus].

The difference of astragaloside Ⅳ content and the expression of its biosynthesis related genes in imitating wild Astragalus mongolicus(IWA) and cultivated A.mongolicus(CA) under different growth years were systematically compared and analyzed.Then the key enzyme genes affected the difference of astragaloside Ⅳ content in the above two A.mongolicus were screened.High-perfo-rmance liquid chromatography(HPLC)was used to determine the content of astragaloside Ⅳ in A.mongolicusunderthe above two diffe-rent growth patterns.Based on the Illumina HiSeq and PacBio high-throughput sequencing platforms, thesecond-and third-generation transcriptome sequencing(RNA-Seq)databaseof the two A.mongolicuswas constructed.The related enzyme genes in the biosynthetic pathway of astragaloside Ⅳ were screened and verified byquantitative reverse transcriptase polymerase chain reaction(RT-qPCR).The RNA-sequencing(RNA-Seq) and RT-qPCR data of each gene were subjected to correlation analysis and trend analysis.The results showed that the variation trend of astragaloside Ⅳ contentby HPLC wasthe same as that of genes by RNA-Seq and RT-qPCR in 1-4 year IWA and 1-2 year CA.The trend level of astragaloside Ⅳ contentwas lower in 2-year IWA than 1-year IWA.Compared with 2-year IWA, 3-year IWA had an upward trend, while 4-year IWA hada downward trend versus 3-year IWA.Additionally, 1-year CA had increased trendthan 2-year CA.However, the content of astragaloside Ⅳ in 5-year IWA was higher than that of 6-year IWA, which wasinconsistent with the findings of RNA-Seq and RT-qPCR.This study preliminarily clarifiedthat the difference of astragaloside Ⅳ contentin 1-4 year IWA and 1-2 year CA wasclosely related to the expression of the upstream and midstream genes(MVK, CMK, PMK, MVD, SS) in the biosynthetic pathway.The results facilitate the production and planting of Radix Astragali seu Hedysari.

[Effects of water regulation on biosynthesis of calycosin-7-O-ß-D-glucoside in Astragalus membranaceus var. mongholicus].

The effects of water regulation on the biosynthesis of calycosin-7-O-ß-D-glucoside in 2-year-old Astragalus membranaceus var. mongholicus were studied,and the mechanism was explained from the aspects of key enzyme gene expression and antioxidant enzyme system. The content of calycosin-7-O-ß-D-glucoside was determined by HPLC,and the expression levels of six key enzyme genes( PAL,4 CL,CHS,CHI,IFS,13'H) in the synthesis pathway were analyzed by q RT-PCR. The activities of protective enzymes and contents of osmoregulation substances and malondialdehyde were also determined. In the water deficit group,the maximum concentration of calycosin-7-O-ß-D-glucoside was 0. 49 mg·g-1 on the 24 th day of treatment. In the whole water regulation,the water deficit group outweighed the water adequate group in osmoregulation substance and MDA contents. The activities of A. membranaceus var.mongholicus antioxidant enzymes SOD,POD,and CAT increased during the initial period of water regulation,but decreased with time.The expression of PAL,CHS,and 13'H in the water deficit group was at a low level,and the 4 CL had active expression,slightly lower than that in the water adequate group. The expression of CHI and IFS elevated rapidly when water deficit occurred. Correlation analysis showed that the content of calycosin-7-O-ß-D-glucoside was positively correlated with CHI expression( P<0. 01) and IFS expression( P<0. 05). Therefore,water regulation can change the accumulation pattern of calycosin-7-O-ß-D-glucoside,and water deficit may be an effective way to increase its content. CHI and IFS are the key genes in response to water deficit.

Integrated metabolomics and transcriptomics study of traditional herb Astragalus membranaceus Bge. var. mongolicus (Bge.) Hsiao reveals global metabolic profile and novel phytochemical ingredients.

BACKGROUND: Astragalus membranaceus Bge. var. mongolicus (Bge.) Hsiao is one of the most common herbs widely used in South and East Asia, to enhance people's health and reinforce vital energy. Despite its prevalence, however, the knowledge about phytochemical compositions and metabolite biosynthesis in Astragalus membranaceus Bge. var. mongolicus (Bge.) Hsiao is very limited. RESULTS: An integrated metabolomics and transcriptomics analysis using state-of-the-art UPLC-Q-Orbitrap mass spectrometer and advanced bioinformatics pipeline were conducted to study global metabolic profiles and phytochemical ingredients/biosynthesis in Astragalus membranaceus Bge. var. mongolicus (Bge.) Hsiao. A total of 5435 metabolites were detected, from which 2190 were annotated, representing an order of magnitude increase over previously known. Metabolic profiling of Astragalus membranaceus Bge. var. mongolicus (Bge.) Hsiao tissues found contents and synthetic enzymes for phytochemicals were significantly higher in leaf and stem in general, whereas the contents of the main bioactive ingredients were significantly enriched in root, underlying the value of root in herbal remedies. Using integrated metabolomics and transcriptomics data, we illustrated the complete pathways of phenylpropanoid biosynthesis, flavonoid biosynthesis, and isoflavonoid biosynthesis, in which some were first reported in the herb. More importantly, we discovered novel flavonoid derivatives using informatics method for neutral loss scan, in addition to inferring their likely synthesis pathways in Astragalus membranaceus Bge. var. mongolicus (Bge.) Hsiao. CONCLUSIONS: The current study represents the most comprehensive metabolomics and transcriptomics analysis on traditional herb Astragalus membranaceus Bge. var. mongolicus (Bge.) Hsiao. We demonstrated our integrated metabolomics and transcriptomics approach offers great potentials in discovering novel metabolite structure and associated synthesis pathways. This study provides novel insights into the phytochemical ingredients, metabolite biosynthesis, and complex metabolic network in herbs, highlighting the rich natural resource and nutritional value of traditional herbal plants.

Integrating transcriptomics and metabolomics to characterise the response of Astragalus membranaceus Bge. var. mongolicus (Bge.) to progressive drought stress.

BACKGROUND: Astragalus membranaceus Bge. var. mongolicus (Bge.) Hsiao (A. mongolicus) is an important traditional Chinese herb that is cultivated on a large scale in northwestern China. Understanding plant responses to drought has important effects on ecological environment recovery and local economic development. Here, we combined transcriptomics (Illumina Hiseq 2000) and metabolomics ((1)H-NMR) to investigate how the roots of two-year-old A. mongolicus responded to 14 days of progressive drought stress. RESULTS: The dried soil reduced the relative water content (RWC) of the leaves and biomass, induced the differential expression of a large fraction of the transcriptome and significantly altered the metabolic processes. PCA analysis demonstrated that the sucrose, proline, and malate metabolites contributed greatly to the separation. Strikingly, proline was increased by almost 60-fold under severe stress compared to the control. Some backbone pathways, including glycolysis, tricarboxylic acid (TCA) cycle, glutamate-mediated proline biosynthesis, aspartate family metabolism and starch and sucrose metabolism, were significantly affected by drought. An integrated analysis of the interaction between key genes and the altered metabolites involved in these pathways was performed. CONCLUSIONS: Our findings demonstrated that the expression of drought-responsive genes showed a strong stress-dose dependency. Analysis of backbone pathways of the transcriptome and metabolome revealed specific genotypic responses to different levels of drought. The variation in molecular strategies to the drought may play an important role in how A. mongolicus and other legume crops adapt to drought stress.

[Study on quality evaluation of sequence and SSR information in transcriptome of Astragalus membranacus].

In this study, 454/Roche GS FLX sequencing technology was used to obtain the data of the Astragalus membranaceus. Four hundred and fifty-four Sequencing System Software was applied to carry out the transcription of the group from scratch. Using MISA tools, 9 893 unigenes were selected for the sequence of the genome of A. membranaceus, and the information of SSR locus was analyzed. According to the result, the average length of reads was 413 bp, about 86% of the reads was involved in the splicing, the length of the N50 was 1 205 bp, the number of unigenes was measured by the whole transcript. 1 729 SSR loci in the A. membranaceus transcriptome were searched, the occurrence frequency of SSR was 9.24%, the frequency of SSR in the whole transcriptome was 13.42%, the average length of SSR was 7.97 kb. One hundred and twenty-seven kinds of core repeat sequences were found, the dominant type was TG/AC type of dinucleotide, it appeared to account for 4.25% of the total SSR locus. The results of the sequence of the transcription of the A. membranaceus transcriptome revealed the overall expression, and a large number of unigenessequence was obtained, and the SSR locus in the genome of the A. membranaceus is high, and the type is diverse, and the polymorphism of the gene is high.

Global transcriptome analysis profiles metabolic pathways in traditional herb Astragalus membranaceus Bge. var. mongolicus (Bge.) Hsiao.

BACKGROUND: Astragalus membranaceus Bge. var. mongolicus (Bge.) Hsiao (A. mongolicus, family Leguminosae) is one of the most important traditional Chinese herbs. Among many secondary metabolites it produces, the effective bioactive constituents include isoflavonoids and triterpene saponins. The genomic resources regarding the biosynthesis of these metabolites in A. mongolicus are limited. Although roots are the primary material harvested for medical use, the biosynthesis of the bioactive compounds and its regulation in A. mongolicus are not well understood. Therefore, a global transcriptome analysis on A. mongolicus tissues was performed to identify the genes essential for the metabolism and to profile their expression patterns in greater details. RESULTS: RNA-sequencing was performed for three different A. mongolicus tissues: leaf, stem, and root, using the Illumina Hiseq2000 platform. A total of 159.5 million raw sequence reads were generated, and assembled into 186,324 unigenes with an N50 of 1,524bp. Among them, 129,966 unigenes (~69.7%) were annotated using four public databases (Swiss-Prot, TrEMBL, CDD, Pfam), and 90,202, 63,946, and 78,326 unigenes were found to express in leaves, roots, and stems, respectively. A total of 8,025 transcription factors (TFs) were identified, in which the four largest families, bHLH, MYB, C3H, and WRKY, were implicated in regulation of tissue development, metabolisms, stress response, etc. Unigenes associated with secondary metabolism, especially those with isolavonoids and triterpene saponins biosynthesis were characterized and profiled. Most genes involved in the isoflavonoids biosynthesis had the lowest expression in the leaves, and the highest in the stems. For triterpene saponin biosynthesis, we found the genes in MVA and non-MVA pathways were differentially expressed among three examined tissues, indicating the parallel but compartmentally separated biosynthesis pathways of IPP and DMAPP in A. mongolicus. The first committed enzyme in triterpene saponin biosynthesis from A. mongolicus, cycloartenol synthase (AmCAS), which belongs to the oxidosqualene cyclase family, was cloned by us to study the astragalosides biosynthesis. Further co-expression analysis indicated the candidate CYP450s and glycosyltransferases (GTs) in the cascade of triterpene saponins biosynthesis. The presence of the large CYP450 families in A. mongolicus was further compared with those from Medicago truncatula and Arabidopsis thaliana, and the diversity and phylegenetic relationships of the CYP450 families were established. CONCLUSION: A transcriptome study was performed for A. mongolicus tissues to construct and profile their metabolic pathways, especially for the important bioactive molecules. The results revealed a comprehensive profile for metabolic activities among tissues, pointing to the equal importance of leaf, stem, and root in A. mongolicus for the production of bioactive compounds. This work provides valuable resources for bioengineering and in vitro synthesis of the natural compounds for medical research and for potential drug development.

Cloning, expression and purification of isoflavone-2'-hydroxylase from Astragalus membranaceus Bge. Var. mongolicus (Bge.) Hsiao.

Plant cytochrome P450 enzymes play vital roles in the biosynthesis of plant secondary metabolites, including phenylpropanoids and phytoalexins. Isoflavone-2'-hydroxylase (AmI2'H) from Astragalus membranaceus Bge. Var. mongolicus (Bge.) Hsiao is a membrane protein and an eukaryotic cytochrome P450 enzyme involved in isoflavonoid biosynthesis. We cloned the AmI2'H gene by employing RACE methods and modified the gene sequence to facilitate protein expression and increase protein solubility. Two vectors, pET-28a(+) and pCW ori(+), were used to express AmI2'H in Escherichia coli. The expression efficiency and purity of target protein were analyzed and demonstrated that pET-28a(+) vector containing the AmI2'H gene could produce larger amounts of target proteins with higher purity than pCWori(+). The purified proteins were identified as AmI2'H by LC-ESI-MS/MS analysis and their proper folding was assessed by CO difference spectrum.

[Comparison between Astragalus membranaceus var. mongholicus and Hedysarum polybotrys based on ITS sequences and metabolomics].

Astragalus membranaceus var. mongholicus and Hedysarum polybotrys belong to different genera, but have similar drug efficacy in traditional Chinese medicine theory, and H. polybotrys was used as the legal A. membranaceus var. mongholicus previously. In this study, similarities and differences between them were analyzed via their ITS/ITS2 fragments information. The ITS (internal transcribed spacer) regions were amplified using polymerase chain reaction and then sequenced in two-way. The alignment lengths of ITS regions were 616 bp, in which 508 loci were consistent, and 103 loci were different, accounting for 82.47% and 16.72% of the total ITS nucleotides in length, respectively. As genotype determines phenotype, 1HNMR-based metabolomic approach was further used to reveal the chemical similarities and differences between them. Thirty-four metabolites were identified in the 1H NMR spectra, and twenty-seven metabolites were the common components. Amino acids, carbohydrates and other primary metabolites were similar, while a large difference existed in the flavonoids and astragalosides. This study suggests that A. membranaceus var. mongholicus and H. polybotrys show similarities and differences from molecular and chemical perspectives, which has laid a foundation for elucidating the effective material basis of drug with similar efficacy and resources utilization.

Accumulation of astragalosides and related gene expression in different organs of Astragalus membranaceus Bge. var mongholicus (Bge.).

Astragalus membranaceus is one of the most important traditional Korean and Chinese medicinal herbs because it contains triterpenoid saponins (astragaloside I, II, III, and IV), which have beneficial and pharmacological effects on health. In this study, we analyzed 10 mevalonate pathway genes that are involved in astragaloside biosynthesis using the Illumina/Solexa HiSeq2000 platform. We determined the expression levels of the 10 genes using quantitative real-time PCR, and analyzed the accumulation of astragalosides in different organs using high-performance liquid chromatography. Genes related to the mevalonate pathway were expressed in different levels in different organs. Almost all genes showed high transcript levels in the stem and leaf, with the lowest transcript levels being recorded in the root. In contrast, most astragalosides accumulated in the root. In particular, the astragaloside IV content was distributed in the following order: root (0.58 mg/g DW) > flower (0.27 mg/g DW) > stem (0.23 mg/g DW) > leaf (0.04 mg/g DW). In the root, astragaloside II exhibited the highest content (2.09 mg/g DW) compared to astragaloside I, III, and IV. Notably, gene expression did not follow the same pattern as astragaloside accumulation. We suggest carefully that astragalosides are synthesized in the leaves and stem and then translocated to the root. This study contributes towards improving our understanding of astragaloside biosynthesis in A. membranaceus.

Chromosome-scale genome assembly of Astragalus membranaceus using PacBio and Hi-C technologies.

Astragalus membranaceus (Fisch.) Bge (AM) is a medicinal herb plant belonging to the Leguminosae family. In this study, we present a chromosome-scale genome assembly of AM, aiming to enhance the molecular biology and functional studies of Astragali Radix. The genome size of AM is about 1.43 Gb, with a contig N50 value of 1.67 Mb. A total of 98.16% of the assembly anchored to 9 pseudochromosomes using Hi-C technology. The assembly completeness was estimated to be 97.27% using BUSCO with the long terminal repeat assembly index (LAI) of 16.22 and quality value (QV) of 48.58. Additionally, the genome contained 67.98% repetitive sequences. Genome annotation predicted 29,914 protein-coding genes, including 73 genes involved in the flavonoid biosynthetic pathway and 2,048 transcription factors. The high-quality genome assembly and gene annotation resources will greatly facilitate future functional genomic studies in Leguminosae species.

The effect of different LED wavelengths on the components and biosynthesis of isoflavonoid in sprout Astragalus membranaceus.

An artificial light source is the optimal element for studying the usability of the medicinal plant Astragalus membranaceus as a sprout vegetable. Based on artificial light source conditions, formononetin (FO) level was the highest (2.6 mg/L) in A. membranaceus exposed to white light emitting diode (LED) light, and calycosin (CA) level was the highest (3.09 mg/L) in the plant exposed to red LED light. According to the publicly available transcriptome data of LED-exposed sprout A. membranaceus LED, reference genes related to the content enhancement of FO, an isoflavone compound, and those related to the content enhancement of CA were selected. The expression patterns of these genes were assayed using qPCR. Among the genes related to FO enhancement, Gene-225190T showed the highest mRNA levels in cells of LED-white light-exposed sprout A. membranaceus; among the genes related to CA enhancement, Gene_042770T showed the highest expression under red LED light. Most genes related to the overall biosynthesis regulation of flavonoids of the upper concept of isoflavone were highly expressed in response to red LED light, and the transcriptional level of 4CL in response to red LED light was the highest. Based on these results, the artificial light sources that regulated the FO and CA contents in sprouts A. membranaceus were white and red LED lights, and the selected reference genes were capable of regulating isoflavone biosynthesis.

[Between Hengshanhuangqi and Chuanhuangqi based on metabolomics and ITS2 sequences].

To compare the differences between Hengshanhuangqi (HH) and Chuanhuangqi (CH) at molecular level, 1H NMR based plant metabolomics approach was used to reveal the chemical difference between HH and CH. Then, the contents of astragaloside IV and calycosin-7-O-beta-D-glucoside, the marker compounds specified in China Pharmacopoeia, were determined. In addition, the ITS2 fragments of HH and CH were sequenced. Twenty-three metabolites were identified in the 1H NMR spectrum, and the principal component analysis showed CH and HH could be separated clearly. HH contained more aspartic acid, GABA, citric acid, astragaloside IV and calycosin-7-O-beta-D-glucoside, while CH contained more threonine, alanine, acetic acid, choline, arginine, fructose and sucrose. And the astragaloside IV is almost undetectable in CH. In addition, the ITS2 fragment sequences of HH and CH were different at eight bases. Thus, the HH and CH showed significant differences chemically and genetically.

A reference-grade genome assembly for Astragalus mongholicus and insights into the biosynthesis and high accumulation of triterpenoids and flavonoids in its roots.

Astragalus membranaceus var. mongholicus (AMM), a member of the Leguminosae, is one of the most important medicinal plants worldwide. The dried roots of AMM have a wide range of pharmacological effects and are a traditional Chinese medicine. Here, we report the first chromosome-level reference genome of AMM, comprising nine pseudochromosomes with a total size of 1.47 Gb and 27 868 protein-encoding genes. Comparative genomic analysis reveals that AMM has not experienced an independent whole-genome duplication (WGD) event after the WGD event shared by the Papilionoideae species. Analysis of long terminal repeat retrotransposons suggests a recent burst of these elements at approximately 0.13 million years ago, which may explain the large size of the AMM genome. Multiple gene families involved in the biosynthesis of triterpenoids and flavonoids were expanded, and our data indicate that tandem duplication has been the main driver for expansion of these families. Among the expanded families, the phenylalanine ammonia-lyase gene family was primarily expressed in the roots of AMM, suggesting their roles in the biosynthesis of phenylpropanoid compounds. The functional versatility of 2,3-oxidosqualene cyclase genes in cluster III may play a critical role in the diversification of triterpenoids in AMM. Our findings provide novel insights into triterpenoid and flavonoid biosynthesis and can facilitate future research on the genetics and medical applications of AMM.

Identification and Functional Characterization of UDP-Glycosyltransferases Involved in Isoflavone Biosynthesis in Astragalus membranaceus.

Isoflavones are rich natural compounds present in legumes and are essential for plant growth and development. Moreover, they are beneficial for animals and humans. Isoflavones are primarily found as glycoconjugates, including calycosin-7-O-ß-d-glucoside (CG) in Astragalus membranaceus, a legume. However, the glycosylation mechanism of isoflavones in A. membranaceus remains unclear. In the present study, three uridine diphosphate (UDP)-glycosyltransferases (UGTs) that may be involved in the biosynthesis of isoflavone were identified in the transcriptome of A. membranaceus. Enzymatic analysis revealed that AmUGT88E29 and AmUGT88E30 had high catalytic activity toward isoflavones in vitro. In addition, AmUGT88E29 and AmUGT88E30 could accept various flavones, flavanones, flavonols, dihydroflavonols, and dihydrochalcones as substrates. AmUGT71G10 was only active against phloretin and dihydroresveratrol. Overexpression of AmUGT88E29 significantly increased the contents of CG, an isoflavone glucoside, in the hairy roots of A. membranaceus. This study provided candidate AmUGT genes for the potential metabolic engineering of flavonoid compounds in plants and a valuable resource for studying the calycosin glycosides biosynthesis pathway.

[Molecular identification of astragali radix and its adulterants by ITS sequences].

OBJECTIVE: To explore a new method for identification Astragali Radix from its adulterants by using ITS sequence. METHOD: Thirteen samples of the different Astragali Radix materials and 6 samples of the adulterants of the roots of Hedysarum polybotrys, Medicago sativa and Althaea rosea were collected. ITS sequence was amplified by PCR and sequenced unidirectionally. The interspecific K-2-P distances of Astragali Radix and its adulterants were calculated, and NJ tree and UPGMA tree were constructed by MEGA 4. RESULT: ITS sequences were obtained from 19 samples respectively, there were Astragali Radix 646-650 bp, H. polybotrys 664 bp, Medicago sativa 659 bp, Althaea rosea 728 bp, which were registered in the GenBank. Phylogeny trees reconstruction using NJ and UPGMA analysis based on ITS nucleotide sequences can effectively distinguish Astragali Radix from adulterants. CONCLUSION: ITS sequence can be used to identify Astragali Radix from its adulterants successfully and is an efficient molecular marker for authentication of Astragali Radix and its adulterants.

Differential Expression of Calycosin-7-O-ß-D-glucoside Biosynthesis Genes and Accumulation of Related Metabolites in Different Organs of Astragalus membranaceus Bge. var. mongholicus (Bge.) Hsiao Under Drought Stress.

Calycosin-7-O-ß-D-glycoside (CG), as a flavonoid, plays an important role in the abiotic stress response of Astragalus membranaceus Bge. var. mongholicus (Bge.) Hsiao (A. mongholicus). CG is also an active ingredient in A. mongholicus with high medicinal value. However, the response mechanism of the CG biosynthetic pathway of drought stress is not clear. In this research, drought stress was inflicted upon A. mongholicus, and the variations in flavonoid metabolites and the correlating gene expression in CG biosynthesis were studied in roots, stems, and leaves of A. mongholicus by UHPLC-MRM-MS/MS and qRT-PCR. Drought stress reduced the dry weight and increased the content of malondialdehyde (MDA) and proline. Drought was beneficial to the accumulation of L-phenylalanine and 4-coumaric acid in leaves and promoted the accumulation of all target compounds in the roots, except calycosin. Overexpression of AmIOMT was observed in the leaves, but the content of formononetin which is the product of isoflavone O-methyltransferase (IOMT) catalysis was higher in stems than in leaves. This research aims to further understand the acclimation of abiotic stress and the regulation mechanism of flavonoid accumulation in A. mongholicus.

Ultraviolet irradiation induces accumulation of isoflavonoids and transcription of genes of enzymes involved in the calycosin-7-O-ß-d-glucoside pathway in Astragalus membranaceus Bge. var. mongholicus (Bge.) Hsiao.

Isoflavonoids are a group of phenolic secondary metabolites found almost exclusively in leguminous plants. Formononetin, calycosin and calycosin-7-O-ß-d-glucoside (CG) are isoflavonoid products in the CG pathway. Accumulation of the three isoflavonoids plus daidzein and expression of six genes of enzymes involved in the CG pathway were studied in Astragalus membranaceus Bge. var. mongholicus (Bge.) Hsiao with ultraviolet (UV) irradiation. Our results showed that (1) main isoflavonoids in roots, stems and leaves were CG, daidzein and calycosin, respectively; they accumulated significantly under the induction of UV irradiation during 8 days but their content declined later; (2) expression of six genes of enzymes involved in the CG pathway was inhibited slightly at early stage but the expression was increased greatly afterward; (3) chalcone synthase, chalcone reductase and chalcone isomerase were expressed to their individual maximum level within shorter hours than were cinnamate 4-hydroxylase, isoflavone synthase (IFS) and isoflavone 3'-hydroxylase and (4) more calycosin but less daidzein accumulated in leaves. IFS was highly expressed in leaves, which might lead to high accumulation of the common precursor of daidzein and 2,7-dihydroxy-4'-O-methoxy-isoflavanone, the latter of which would be converted to formononetin, calycosin and CG via a series of reactions. Little daidzein accumulated in leaves, which suggested that rather than be converted to daidzein, the 2,7,4'-trihydroxyisoflavanone was probably more easily caught by 2-hydroxyisoflavanone 4'-O-methyltransferase and hence provided more precursors for formononetin. The findings were discussed in terms of the influence of UV irradiation in the accumulation of isoflavonoids.