Transcriptome Level Reveals the Triterpenoid Saponin Biosynthesis Pathway of Bupleurum falcatum L.

Bupleurum falcatum L. is frequently used in traditional herbal medicine in Asia. Saikosaponins (SSs) are the main bioactive ingredients of B. falcatum, but the biosynthetic pathway of SSs is unclear, and the biosynthesis of species-specific phytometabolites is little known. Here we resolved the transcriptome profiles of B. falcatum to identify candidate genes that might be involved in the biosynthesis of SSs. By isoform sequencing (Iso-Seq) analyses of the whole plant, a total of 26.98 Gb of nucleotides were obtained and 124,188 unigenes were identified, and 81,594 unigenes were successfully annotated. A total of 1033 unigenes of 20 families related to the mevalonate (MVA) pathway and methylerythritol phosphate (MEP) pathway of the SS biosynthetic pathway were identified. The WGCNA (weighted gene co-expression network analysis) of these unigenes revealed that only the co-expression module of MEmagenta, which contained 343 unigenes, was highly correlated with the biosynthesis of SSs. Comparing differentially expressed gene analysis and the WGCNA indicated that 130 out of 343 genes of the MEmagenta module exhibited differential expression levels, and genes with the most "hubness" within this module were predicted. Manipulation of these genes might improve the biosynthesis of SSs.

Functional Characterization and Protein Engineering of a Triterpene 3-/6-/2'-O-Glycosyltransferase Reveal a Conserved Residue Critical for the Regiospecificity.

Engineering the function of triterpene glucosyltransferases (GTs) is challenging due to the large size of the sugar acceptors. In this work, we identified a multifunctional glycosyltransferase AmGT8 catalyzing triterpene 3-/6-/2'-O-glycosylation from the medicinal plant Astragalus membranaceus. To engineer its regiospecificity, a small mutant library was built based on semi-rational design. Variants A394F, A394D, and T131V were found to catalyze specific 6-O, 3-O, and 2'-O glycosylation, respectively. The origin of regioselectivity of AmGT8 and its A394F variant was studied by molecular dynamics and hydrogen deuterium exchange mass spectrometry. Residue 394 is highly conserved as A/G and is critical for the regiospecificity of the C- and O-GTs TcCGT1 and GuGT10/14. Finally, astragalosides III and IV were synthesized by mutants A394F, T131V and P192E. This work reports biocatalysts for saponin synthesis and gives new insights into protein engineering of regioselectivity in plant GTs.

Isolation and characterization of endophytic fungi having plant growth promotion traits that biosynthesizes bacosides and withanolides under in vitro conditions.

Endophytes are regarded with immense potentials in terms of plant growth promoting (PGP) elicitors and mimicking secondary metabolites of medicinal importance. Here in the present study, we explored Bacopa monnieri plants to isolate, identify fungal endophytes with PGP elicitation potentials, and investigate secretion of secondary metabolites such as bacoside and withanolide content under in vitro conditions. Three fungal endophytes isolated (out of 40 saponin producing isolates) from leaves of B. monnieri were examined for in vitro biosynthesis of bacosides. On morphological, biochemical, and molecular identification (ITS gene sequencing), the isolated strains SUBL33, SUBL51, and SUBL206 were identified as Nigrospora oryzae (MH071153), Alternaria alternata (MH071155), and Aspergillus terreus (MH071154) respectively. Among these strains, SUBL33 produced highest quantity of Bacoside A3 (4093 µg mL-1), Jujubogenin isomer of Bacopasaponin C (65,339 µg mL-1), and Bacopasaponin C (1325 µg mL-1) while Bacopaside II (13,030 µg mL-1) was produced by SUBL51 maximally. Moreover, these aforementioned strains also produced detectable concentration of withanolides-Withaferrin A, Withanolide A (480 µg mL-1), and Withanolide B (1024 µg mL-1) respectively. However, Withanolide A was not detected in the secondary metabolites of strain SUBL51. To best of our knowledge, the present study is first reports of Nigrospora oryzae as an endophyte in B. monnieri with potentials of biosynthesis of economically important phytomolecules under in vitro conditions.

De Novo Biosynthesis of the Oleanane-Type Triterpenoids of Tunicosaponins in Yeast.

Tunicosaponins are natural products extracted from Psammosilene tunicoides, which is an important ingredient of Yunnan Baiyao Powder, an ancient and famous Asian herbal medicine. The representative aglycones of tunicosaponins are the oleanane-type triterpenoids of gypsogenin and quillaic acid, which were found to manipulate a broad range of virus-host fusion via wrapping the heptad repeat-2 (HR2) domain prevalent in viral envelopes. However, the unknown biosynthetic pathway and difficulty in chemical synthesis hinder the therapeutic use of tunicosaponins. Here, two novel cytochrome P450-dependent monooxygenases that take part in the biosynthesis of tunicosaponins, CYP716A262 (CYP091) and CYP72A567 (CYP099), were identified from P. tunicoides. In addition, the whole biosynthesis pathway of the tunicosaponin aglycones was reconstituted in yeast by transforming the platform strain BY-bAS with the CYP716A262 and CYP716A567 genes, the resulting strain could produce 146.84 and 314.01 mg/L of gypsogenin and quillaic acid, respectively. This synthetic biology platform for complicated metabolic pathways elucidation and microbial cell factories construction can provide alternative sources of important natural products, helping conserve natural plant resources.

Characterization of C-26 aminotransferase, indispensable for steroidal glycoalkaloid biosynthesis.

Steroidal glycoalkaloids (SGAs) are toxic specialized metabolites found in members of the Solanaceae, such as Solanum tuberosum (potato) and Solanum lycopersicum (tomato). The major potato SGAs are α-solanine and α-chaconine, which are biosynthesized from cholesterol. Previously, we have characterized two cytochrome P450 monooxygenases and a 2-oxoglutarate-dependent dioxygenase that function in hydroxylation at the C-22, C-26 and C-16α positions, but the aminotransferase responsible for the introduction of a nitrogen moiety into the steroidal skeleton remains uncharacterized. Here, we show that PGA4 encoding a putative γ-aminobutyrate aminotransferase is involved in SGA biosynthesis in potatoes. The PGA4 transcript was expressed at high levels in tuber sprouts, in which SGAs are abundant. Silencing the PGA4 gene decreased potato SGA levels and instead caused the accumulation of furostanol saponins. Analysis of the tomato PGA4 ortholog, GAME12, essentially provided the same results. Recombinant PGA4 protein exhibited catalysis of transamination at the C-26 position of 22-hydroxy-26-oxocholesterol using γ-aminobutyric acid as an amino donor. Solanum stipuloideum (PI 498120), a tuber-bearing wild potato species lacking SGA, was found to have a defective PGA4 gene expressing the truncated transcripts, and transformation of PI 498120 with functional PGA4 resulted in the complementation of SGA production. These findings indicate that PGA4 is a key enzyme for transamination in SGA biosynthesis. The disruption of PGA4 function by genome editing will be a viable approach for accumulating valuable steroidal saponins in SGA-free potatoes.

Melatonin increases leaf disease resistance and saponin biosynthesis in Panax notogiseng.

Panax notoginseng (Bruk.) FH Chen is a valuable traditional herb in China, with saponins being the main medicinal components in its roots. However, leaf diseases are a major factor limiting growth and production of P. notoginseng. Melatonin is a ubiquitous signaling molecule associated with abiotic stress resistance. In this study, we investigated the role of melatonin in leaf disease resistance of P. notoginseng in field conditions. Additionally, saponin concentrations were analyzed to evaluate the suitability of melatonin use in agricultural practice. Our results showed that exogenous application of melatonin promoted the endogenous phytomelatonin accumulation via upregulation of genes involved in its biosynthesis. The application of 10 µM melatonin decreased the incidence of leaf diseases (gray mold, round spot, and black spot) by about 40% compared with the solvent control, which might have been due to the increased expression of genes associated with immunity and disease resistance. Furthermore, concentrations of saponins and expression of their biosynthesis-related genes were significantly increased by melatonin. Taken together, the data presented here suggested that melatonin could be used in agricultural management of P. notoginseng because it increased leaf disease resistance and biosynthesis of saponins.

Saponins: Extraction, bio-medicinal properties and way forward to anti-viral representatives.

Medicinal or herbal plants are widely used for their many favourable properties and are generally safe without any side effects. Saponins are sugar conjugated natural compounds which possess a multitude of biological activities such as medicinal properties, antimicrobial activity, antiviral activity, etc. Saponin production is a part of the normal growth and development process in a lot of plants and plant extracts such as liquorice and ginseng which are exploited as potential drug sources. Herbal compounds have shown a great potential against a wide variety of infectious agents, including viruses such as the SARS-CoV; these are all-natural products and do not show any adverse side effects. This article reviews the various aspects of saponin biosynthesis and extraction, the need for their integration into more mainstream medicinal therapies and how they could be potentially useful in treating viral diseases such as COVID-19, HIV, HSV, rotavirus etc. The literature presents a close review on the saponin efficacy in targeting mentioned viral diseases that occupy a high mortality rate worldwide. This manuscript indicates the role of saponins as a source of dynamic plant based anti-viral remedies and their various methods for extraction from different sources.

Recent Progress in Saikosaponin Biosynthesis in Bupleurum.

BACKGROUND: Chaihu is a popular traditional Chinese medicine that has been used for centuries. It is traditionally used to treat cold fever and liver-related diseases. Saikosaponins (SSs) are one of the main active components of chaihu, in addition to essential oils, flavonoids, and polysaccharides. Considerable effort is needed to reveal the biosynthesis and regulation of SSs on the basis of current progress. OBJECTIVE: The aim of this study is to provide a reference for further studies and arouse attention by summarizing the recent achievements of SS biosynthesis. METHODS: All the data compiled and presented here were obtained from various online resources, such as PubMed Scopus and Baidu Scholar in Chinese, up to October 2019. RESULTS: A few genes of the enzymes of SSs participating in the biosynthesis of SSs were isolated. Among these genes, only the P450 gene was verified to catalyze the SS skeleton ß-amyrin synthase. Several UDP-glycosyltransferase genes were predicted to be involved in the biosynthesis of SSs. SSs could be largely biosynthesized in the phloem and then transported from the protoplasm, which is the biosynthetic site, to the vacuoles to avoid self-poisoning. As for the other secondary metabolites, the biosynthesis of SSs was strongly affected by environmental factors and the different species belonging to the genus of Bupleurum. Transcriptional regulation was studied at the molecular level. CONCLUSION: Profound discoveries in SSs may elucidate the mechanism of diverse the monomer formation of SSs and provide a reference for maintaining the stability of SS content in Radix Bupleuri.

Yeast extract improved biosynthesis of astragalosides in hairy root cultures of Astragalus membranaceus.

The dried root of Astragalus membranaceus is a well-known herbal medicine, and it is useful in treating chronic diseases and weakness, as well as for improving overall health and vitality. Astragalosides, which are root quality indicators of A. membranaceus, are natural triterpenoid saponins that are used in the treatment of diabetes and cardiovascular diseases. Currently, there is an urgent need to improve their production because of their low quantity in plants and the difficulty of chemical synthesis. In this study, yeast extract was added to facilitate elicitation in Agrobacterium-mediated hairy root cultures, thereby enhancing astragaloside production in A. membranaceus. Results showed that yeast extract could stimulate astragaloside content effectively in the hairy roots of A. membranaceus. Moreover, astragaloside accumulation was positively correlated with the upregulation of mevalonate biosynthetic gene expression in the presence of yeast extract. Our study demonstrated that pretreatment with yeast extract (3.65 mM) for 72 h serves as an effective strategy to enhance astragaloside levels in A. membranaceus hairy root cultures. Thus, these optimal conditions can provide valuable information for the improvement of astragaloside industrial production in A. membranaceus.

Triterpenoid saponins in tea (Camellia sinensis) plants: biosynthetic gene expression, content variations, chemical identification and cytotoxicity.

Very little is known about saponins in tea and their biosynthesis in tea plants despite of the importance. Here, we studied tea saponins and their biosynthesis genes. Saponins were promptly recovered in tea infusions. Cytotoxicity of tea saponin extracts on human tongue squamous and hepatocellular carcinoma lines showed respective IC50 values of 29.2 and 17.5 µg/mL, which may be attributable to over 40 saponins identified in green tea. Saponin contents varied in shoot tips of 42 tea plant varieties but did not change drastically during tea processing. Saponin biosynthetic gene expression was consistent with its contents in plant tissues. Thus, plant tips produce significant amounts of saponins, which are stable during tea processing, and ready to be recovered to tea infusions to provide potent health benefits to consumers. This study paves a road towards clarifying the biosynthesis and genetic improvement of saponins in tea plants.

Cloning, Yeast Expression, and Characterization of a ß-Amyrin C-28 Oxidase (CYP716A249) Involved in Triterpenoid Biosynthesis in Polygala tenuifolia.

Polygala tenuifolia Willd. is a traditional Chinese herbal medicine that is widely used in treating nervous system disorders. Triterpene saponins in P. tenuifolia (polygala saponins) have excellent biological activity. As a precursor for the synthesis of presenegin, oleanolic acid (OA) plays an important role in the biosynthesis of polygala saponins. However, the mechanism behind the biosynthesis of polygala saponins remains to be elucidated. In this study, we found that CYP716A249 (GenBank: ASB17946) oxidized the C-28 position of ß-amyrin to produce OA. Using quantitative real-time PCR, we observed that CYP716A249 had the highest expression in the roots of 2-year-old P. tenuifolia, which provided a basis for the selection of samples for gene cloning. To identify the function of CYP716A249, the strain R-BE-20 was constructed by expressing ß-amyrin synthase in yeast. Then, CYP716A249 was co-expressed with ß-amyrin synthase to construct the strain R-BPE-20 by using the lithium acetate method. Finally, we detected ß-amyrin and OA by ultra-HPLC-Q Exactive hybrid quadrupole-Orbitrap high-resolution accurate mass spectrometry and GC-MS. The results of this study provide insights into the biosynthesis pathway of polygala saponins.

Comparative transcriptome analysis of root, stem, and leaf tissues of Entada phaseoloides reveals potential genes involved in triterpenoid saponin biosynthesis.

BACKGROUND: Entada phaseoloides (L.) Merr. is an important traditional medicinal plant. The stem of Entada phaseoloides is popularly used as traditional medicine because of its significance in dispelling wind and dampness and remarkable anti-inflammatory activities. Triterpenoid saponins are the major bioactive compounds of Entada phaseoloides. However, genomic or transcriptomic technologies have not been used to study the triterpenoid saponin biosynthetic pathway in this plant. RESULTS: We performed comparative transcriptome analysis of the root, stem, and leaf tissues of Entada phaseoloides with three independent biological replicates and obtained a total of 53.26 Gb clean data and 116,910 unigenes, with an average N50 length of 1218 bp. Putative functions could be annotated to 42,191 unigenes (36.1%) based on BLASTx searches against the Non-redundant, Uniprot, KEGG, Pfam, GO, KEGG and COG databases. Most of the unigenes related to triterpenoid saponin backbone biosynthesis were specifically upregulated in the stem. A total of 26 cytochrome P450 and 17 uridine diphosphate glycosyltransferase candidate genes related to triterpenoid saponin biosynthesis were identified. The differential expressions of selected genes were further verified by qPT-PCR. CONCLUSIONS: The dataset reported here will facilitate the research about the functional genomics of triterpenoid saponin biosynthesis and genetic engineering of Entada phaseoloides.

Full-length transcriptome sequencing and modular organization analysis of oleanolic acid- and dammarane-type saponins related gene expression patterns in Panax japonicus.

The saponins found in Panax japonicus, a traditional medicinal herb in Asia, exhibit high degrees of structural and functional similarity. In this study, metabolite analysis revealed that oleanolic acid-type and dammarane-type saponins were distributed unevenly in three tissues (rhizome_Y, rhizome_O, and secRoot) of P. japonicus. Single-molecule real-time (SMRT) sequencing and next generation sequencing (NGS) data revealed distinct and tissue-specific transcriptomic patterns relating to the production of these two types of saponins. In the co-expression network and hierarchical clustering analyses, one 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) and two 1-deoxy-D-xylulose-5-phosphate synthase (DXS) etc. transcripts were found to be key genes associated with the biosynthesis of oleanolic acid and dammarane-type saponins in P. japonicus, respectively. In addition, cytochrome p450 (CYP) and UDP-glucuronosyltransferase (UGT) family proteins that serve as regulators of saponin biosynthesis-related genes were also found to exhibit tissue-specific expression patterns. Together these results offer a comprehensive metabolomic and transcriptomic overview of P. japonicus.

Advances in biosynthesis of triterpenoid saponins in medicinal plants.

In recent years, biosynthesis of triterpenoid saponins in medicinal plants has been widely studied because of their active ingredients with diverse pharmacological activities. Various oxidosqualene cyclases, cytochrome P450 monooxygenases, uridine diphosphate glucuronosyltransferases, and transcription factors related to triterpenoid saponins biosynthesis have been explored and identified. In the biosynthesis of triterpenoid saponins, the progress of gene mining by omics-based sequencing, gene screening, gene function verification, catalyzing mechanism of key enzymes and gene regulation are summarized and discussed. By the progress of the biosynthesis pathway of triterpenoid saponins, the large-scale production of some triterpenoid saponins and aglycones has been achieved through plant tissue culture, transgenic plants and engineered yeast cells. However, the complex biosynthetic pathway and structural diversity limit the biosynthesis of triterpenoid saponins in different system. Special focus can further be placed on the systematic botany information of medicinal plants obtained from omics large dataset, and triterpenoid saponins produced by synthetic biology strategies, gene mutations and gene editing technology.

Plant terpenoid metabolism co-opts a component of the cell wall biosynthesis machinery.

Glycosylation is one of the most prevalent molecular modifications in nature. Single or multiple sugars can decorate a wide range of acceptors from proteins to lipids, cell wall glycans and small molecules, dramatically affecting their activity. Here, we discovered that by 'hijacking' an enzyme of the cellulose synthesis machinery involved in cell wall assembly, plants evolved cellulose synthase-like enzymes (Csls) and acquired the capacity to glucuronidate specialized metabolites, that is, triterpenoid saponins. Apparently, endoplasmic reticulum-membrane localization of Csls and of other pathway proteins was part of evolving a new glycosyltransferase function, as plant metabolite glycosyltransferases typically act in the cytosol. Discovery of glucuronic acid transferases across several plant orders uncovered the long-pursued enzymatic reaction in the production of a low-calorie sweetener from licorice roots. Our work opens the way for engineering potent saponins through microbial fermentation and plant-based systems.

Identification and analysis of CYP450 and UGT supergene family members from the transcriptome of Aralia elata (Miq.) seem reveal candidate genes for triterpenoid saponin biosynthesis.

BACKGROUND: Members of the cytochrome P450 (CYP450) and UDP-glycosyltransferase (UGT) gene superfamily have been shown to play essential roles in regulating secondary metabolite biosynthesis. However, the systematic identification of CYP450s and UGTs has not been reported in Aralia elata (Miq.) Seem, a highly valued medicinal plant. RESULTS: In the present study, we conducted the RNA-sequencing (RNA-seq) analysis of the leaves, stems, and roots of A. elata, yielding 66,713 total unigenes. Following annotation and KEGG pathway analysis, we were able to identify 64 unigenes related to triterpenoid skeleton biosynthesis, 254 CYP450s and 122 UGTs, respectively. A total of 150 CYP450s and 92 UGTs encoding > 300 amino acid proteins were utilized for phylogenetic and tissue-specific expression analyses. This allowed us to cluster 150 CYP450s into 9 clans and 40 families, and then these CYP450 proteins were further grouped into two primary branches: A-type (53%) and non-A-type (47%). A phylogenetic analysis of 92 UGTs and other plant UGTs led to clustering into 16 groups (A-P). We further assessed the expression patterns of these CYP450 and UGT genes across A. elata tissues, with 23 CYP450 and 16 UGT members being selected for qRT-PCR validation, respectively. From these data, we identified CYP716A295 and CYP716A296 as the candidate genes most likely to be associated with oleanolic acid synthesis, while CYP72A763 and CYP72A776 were identified as being the most likely to play roles in hederagenin biosynthesis. We also selected five unigenes as the best candidates for oleanolic acid 3-O-glucosyltransferase. Finally, we assessed the subcellular localization of three CYP450 proteins within Arabidopsis protoplasts, highlighting the fact that they localize to the endoplasmic reticulum. CONCLUSIONS: This study presents a systematic analysis of the CYP450 and UGT gene family in A. elata and provides a foundation for further functional characterization of these two multigene families.

Exploring plant metabolic genomics: chemical diversity, metabolic complexity in the biosynthesis and transport of specialized metabolites with the tea plant as a model.

The diversity and complexity of secondary metabolites in tea plants contribute substantially to the popularity of tea, by determining tea flavors and their numerous health benefits. The most significant characteristics of tea plants are that they concentrate the complex plant secondary metabolites into one leaf: flavonoids, alkaloids, theanine, volatiles, and saponins. Many fundamental questions regarding tea plant secondary metabolism remain unanswered. This includes how tea plants accumulate high levels of monomeric galloylated catechins, unlike the polymerized flavan-3-ols in most other plants, as well as how they are evolved to selectively synthesize theanine and caffeine, and how tea plants properly transport and store these cytotoxic products and then reuse them in defense. Tea plants coordinate many metabolic pathways that simultaneously take place in young tea leaves in response to both developmental and environmental cues. With the available genome sequences of tea plants and high-throughput metabolomic tools as great platforms, it is of particular interest to launch metabolic genomics studies using tea plants as a model system. Plant metabolic genomics are to investigate all aspects of plant secondary metabolism at the genetic, genome, and molecular levels. This includes plant domestication and adaptation, divergence and convergence of secondary metaboloic pathways. The biosynthesis, transport, storage, and transcriptional regulation mechanisms of all metabolites are of core interest in the plant as a whole. This review highlights relevant contexts of metabolic genomics, outstanding questions, and strategies for answering them, with aim to guide future research for genetic improvement of nutrition quality for healthier plant foods.

Transcriptome Analysis of Clinopodium chinense (Benth.) O. Kuntze and Identification of Genes Involved in Triterpenoid Saponin Biosynthesis.

Clinopodium chinense (Benth.) O. Kuntze (C. chinense) is an important herb in traditional Chinese medicine. Triterpenoid saponins are a major class of active compounds in C. chinense with broad pharmacological activities and hemostatic, antitumor, and anti-hyperglycemic effects. To identify genes involved in triterpenoid saponin biosynthesis, transcriptomic analyses of leaves, stems, and roots from C. chinense were performed. A total of 135,968 unigenes were obtained by assembling the leaf, stem, and root transcripts, of which 102,154 were annotated in public databases. Differentially expressed genes were determined based on expression profile analysis and analyzed for differential expression of unique genes related to triterpenoid saponin biosynthesis. Multiple unigenes encoding crucial enzymes or transcription factors involved in triterpenoid saponin synthesis were identified and analyzed. The expression levels of unigenes encoding enzymes were experimentally validated using quantitative real-time PCR. This study greatly broadens the public transcriptome database for this species and provides a valuable resource for identifying candidate genes involved in the biosynthesis of triterpenoid saponins and other secondary metabolites.

Effects of drought-re-watering-drought on the photosynthesis physiology and secondary metabolite production of Bupleurum chinense DC.

KEY MESSAGE: Drastic changes in soil water content can activate the short-term high expression of key enzyme-encoding genes involved in secondary metabolite synthesis thereby increasing the content of secondary metabolites. Bupleurum chinense DC. is a traditional medicinal herb that is famous for its abundant saikosaponins. In the current study, the effects of drought-re-watering-drought on the photosynthesis physiology and biosynthesis of saikosaponins were investigated in 1-year-old B. chinense. The results showed that alterations in soil moisture altered the photosynthesis physiological process of B. chinense. The dry weight and fresh weight of the roots, photosynthesis capacity, chlorophyll fluorescence parameters, and SOD, POD and CAT activities were significantly reduced, and the contents of SP, soluble sugars, PRO and MDA increased. There were strong correlations between different physiological stress indices. All indices promoted and restricted each other, responded to soil moisture changes synergistically, maintained plant homeostasis and guaranteed normal biological activities. It was found that RW and RD_1 were the key stages of the water-control experiment affecting the expression of saikosaponin-related genes. At these two stages, the expression of multiple genes was affected by changes in soil moisture, with their expression levels reaching several-fold higher than those at the previous stage. We noticed that the expression of saikosaponin synthesis genes (which were rapidly upregulated at the RW and RD_1 stages) did not coincide with the rapid accumulation of saikosaponins (at the RD-2 stage), which were found to correspond to each other at the later stages of the water-control experiment. This finding indicates that there is a time lag between gene expression and the final product synthesis. Rapid changes in the external environment (RW to RD_1) have a short-term promoting effect on gene expression. This study reveals that short-term stress regulation may be an effective way to improve the quality of medicinal materials.

[Short-term water changes response of saponin biosynthesis process in Astragalus membranceus].

The study is aimed to explore the effect of different water on the content of total saponins,astragaloside Ⅳ and gene expression in the growth of Astragalus membranceus. In this study, one-year-old A. membranaceus was used as the experimental material, by pot culture different water treatments were simulated at herbal garden in Jilin Agricultural University. The content of astragaloside Ⅳ was determined by HPLC and the total saponins by UV spectrophotometry. With 18 S RNA as a reference gene, fluorescent quantitative PCR was applied to analyze the eight key enzymes in astragalus saponin synthesis pathway AACT,HMGS,HMGR,IDI,FPS,SS,SE,CAS expression. With the decrease of soil water, the content of astragaloside Ⅳ in the root tissue of A. membranaceus showed an increasing trend, up to 1.46 mg·g~(-1). The total saponin content tended to increase, up to 6.80 mg·g~(-1). The results of relative expression of genes showed that the eight genes showed different effects at different water. With the change of soil water content, the amount of(AACT,IDI,SS) relative expression in drought stress group firstly increased and then decreased, then increased, and then decreased. The amount of(HMGS,HMGR,FPS) relative expression in drought stress group increased firstly and then decreased. The amount of(SE,CAS) relative expression in drought stress group increased firstly and then decreased, and continued to decrease after rehydration. The expression of key enzyme genes involved in the synthesis of astragaloside was influenced by each other, and the expression of key enzyme in roots showed a correlation with the content of astragaloside. Correlation analysis showed that there was a very significant positive correlation between HMGR gene and total saponins content in drought stress group and a significant negative correlation between content of CAS and total saponins. The contents of FPS,SE,CAS and astragaloside Ⅳ were very significantly and negative correlated. The relationship between other genes and quality was positive. Therefore, HMGR, FPS, SE and CAS genes have significant effects on the regulation of saponin content under water control. On the 15 th day after water regulation, the total amount of astragaloside and total saponins reached the highest value and could be harvested.