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Anemarrhena asphodeloides Bunge (Liliaceae) is an important Traditional Chinese Medicine herb, which contains up to 6 % total steroidal saponins (timosaponins) and has multiple pharmacological properties. However, the timosaponin biosynthetic pathway has not been extensively investigated. Here we conducted de novo transcriptome sequencing and analysis of A. asphodeloides Bunge and screened for candidate genes involved in the timosaponin biosynthetic pathway. Targeted metabolite analysis showed that timosaponins primarily accumulated in rhizomes, while phytosterols (including cholesterol) were distributed throughout various organs. Most of the identified candidate genes of the timosaponin biosynthetic pathway were also highly expressed in the rhizome, consistent with the results of metabolic analysis. Based on the transcriptome results, two candidate 7-dehydrocholesterol reductase genes were cloned and heterologously expressed in the yeast Saccharomyces cerevisiae. The purified and identified products supported that Aa7DR1 possessed Δ 7 -reduction activity in yeast and therefore may be involved in the timosaponins biosynthetic pathway in A. asphodeloides Bunge. Phylogenetic analysis showed Aa7DR1 belongs to monocotyledonous Δ 7 reductase of phytosterol biosynthesis. These data expand our understanding of timosaponin biosynthesis., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Cellulases have been used to extract bioactive ingredients from medical plants; however, the poor enzymatic properties of current cellulases significantly limit their application. Two strategies are expected to address this concern: (1) new cellulase gene mining strategies have been promoted, optimized, and integrated, thanks to the improvement of gene sequencing, genomic data, and algorithm optimization, and (2) known cellulases are being modified, thanks to the development of protein engineering, crystal structure data, and computing power. Here, we focus on mining strategies and provide a systemic overview of two approaches based on sequencing and function. Strategies based on protein structure modification, such as introducing disulfide bonds, proline, salt bridges, N -glycosylation modification, and truncation of loop structures, have already been summarized. This review discusses four aspects of cellulase-assisted extraction. Initially, cellulase alone was used to extract bioactive substances, and later, mixed enzyme systems were developed. Physical methods such as ultrasound, microwave, and high hydrostatic pressure have assisted in improving extraction efficiency. Cellulase changes the structure of biomolecules during the extraction process to convert them into effective ingredients with better activity and bioavailability. The combination of cellulase with other enzymes and physical technologies is a promising strategy for future extraction applications.

Datura innoxia Mill., a traditional Chinese herbal medicine, produces tropane alkaloids such as hyoscyamine and scopolamine. Scopolamine has a larger demand than hyoscyamine due to its stronger pharmacological effects and fewer side reactions. It is extracted from solanaceous plants. However, the content of scopolamine is lower than hyoscyamine in D. innoxia. Hyoscyamine 6β-hydroxylase (H6H, EC1.14.11.11) is the key enzyme which can catalyze hyoscyamine to form scopolamine. In this study, a cDNA encoding H6H was cloned from D. innoxia roots and named Dih6h. The full-length cDNA is 1413 bp in length with a 1044-bp open reading frame encoding 347 amino acids. The deduced protein sequence of D. innoxia H6H (DiH6H) shared high identity with H6Hs from other plants. The DiH6H was heterologously expressed in Escherichia coli and purified via His-tag affinity technique. The recombinant DiH6H showed activity in transforming hyoscyamine to scopolamine. Despite Dih6h mRNA was detected in various tissues, its levels in roots were higher than that in other tissues. Indeed, scopolamine accumulation was low in roots, but it was very high in aerial parts, especially in flowers and seeds. These observations suggest that scopolamine may be synthesized in the roots and subsequently transported to the aerial parts. To further verify in vivo function of DiH6H, the cDNA of DiH6H was overexpressed in D. innoxia hairy roots. As expected, an increase of scopolamine production was observed in the positive transformants. The results provide a potential strategy for increasing scopolamine yield by metabolic engineering of its biosynthetic pathway in D. innoxia., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

Background and Objective: Flavonols in plants are catalyzed by flavonol synthase (FLS) enzyme. FLS was reported expressed in flowers and fruits, i.e., Dianthus caryophyllus L. (Caryophyllaceae), Petunia hybrida Hort. (Solanaceae), Arabidopsis thaliana L. (Brassicaceae), Citrus unshiu Marc. (Rutaceae). However, none reported about FLS in medicinal plants, particularly those which possess anti-inflammatory activity. This study was aimed to extract and identify FLS in the rhizome of Boesenbergia rotunda (Zingiberaceae) and to determine quercetin in the ethanol extract of the rhizome., Materials and Methods: The protein extraction of the rhizome was carried out by employing Laing and Christeller's (2004) and Wang's (2014) methods. The extracted-proteins were separated by using SDS-PAGE, followed by the measurement of FLS intensity by using Gel Analyzer. The FLS-1 of recombinant A. thaliana was employed as the standard. The determination of quercetin in the rhizome was carried out using LC-MS., Results: The FLS occurred as a thick band at 38 kDa with intensity 116-158. The LC chromatogram of the extract indicated a small peak at 7.94 min similar to that of quercetin standard. The MS spectra at 7.94 min indicated that quercetin is present in the B. rotunda rhizome (m/z = 303.0549). The concentration of quercetin in the extract is 0.022% w/v., Conclusion: The FLS, an enzyme which plays an important role in producing quercetin, was detected in B. rotunda rhizome planted in Indonesia. As a consequence, quercetin in a small amount, was also quantified in the rhizome of this plant. This report will add a scientific insight of B. rotunda for biological sciences.

Glycyrrhizin, a sweet triterpenoid saponin found in the roots and stolons of Glycyrrhiza species (licorice), is an important active ingredient in traditional herbal medicine. We previously identified two cytochrome P450 monooxygenases, CYP88D6 and CYP72A154, that produce an aglycone of glycyrrhizin, glycyrrhetinic acid, in Glycyrrhiza uralensis. The sugar moiety of glycyrrhizin, which is composed of two glucuronic acids, makes it sweet and reduces its side-effects. Here, we report that UDP-glycosyltransferase (UGT) 73P12 catalyzes the second glucuronosylation as the final step of glycyrrhizin biosynthesis in G. uralensis; the UGT73P12 produced glycyrrhizin by transferring a glucuronosyl moiety of UDP-glucuronic acid to glycyrrhetinic acid 3-O-monoglucuronide. We also obtained a natural variant of UGT73P12 from a glycyrrhizin-deficient (83-555) strain of G. uralensis. The natural variant showed loss of specificity for UDP-glucuronic acid and resulted in the production of an alternative saponin, glucoglycyrrhizin. These results are consistent with the chemical phenotype of the 83-555 strain, and suggest the contribution of UGT73P12 to glycyrrhizin biosynthesis in planta. Furthermore, we identified Arg32 as the essential residue of UGT73P12 that provides high specificity for UDP-glucuronic acid. These results strongly suggest the existence of an electrostatic interaction between the positively charged Arg32 and the negatively charged carboxy group of UDP-glucuronic acid. The functional arginine residue and resultant specificity for UDP-glucuronic acid are unique to UGT73P12 in the UGT73P subfamily. Our findings demonstrate the functional specialization of UGT73P12 for glycyrrhizin biosynthesis during divergent evolution, and provide mechanistic insights into UDP-sugar selectivity for the rational engineering of sweet triterpenoid saponins., (© 2019 The Authors The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Flavonoids are a group of secondary metabolites found in plants. They have many pharmacological functions and play an important role in Chinese sumac( Rhus chinensis),which is a well-known traditional Chinese medicinal plant. Chalcone isomerase( CHI,EC 5. 5. 1. 6) is one of the key enzymes in the flavonoids biosynthesis pathway. In this paper,the full-length c DNA sequence encoding the chalcone isomerase from R. chinensis( designated as Rc CHI) was cloned by RT-PCR and rapid-amplification of c DNA Ends( RACE). The Rc CHI c DNA sequence was 1 058 bp and the open reading frame( ORF) was 738 bp. The ORF predicted to encode a 245-amino acid polypeptide. Rc CHI gene contained an intron and two exons. The sequence alignments revealed Rc CHI shared47. 1%-71. 6% identity with the homologues in other plants. Real-time PCR analysis showed that the total flavonoid levels were positively correlated with tissue-specific expressions of Rc CHI mRNA in different tissues. The recombinant protein was successfully expressed in an Escherichia coli strain with the p GEX-6 P-1 vector. In this paper,the CHI gene was cloned and characterized in the family of Anacardiaceae and will help us to obtain better knowledge of the flavonoids biosynthesis of the flavonoid compounds in R. chinensis.

Andrographis paniculata is a herbaceous dicot plant widely used for its anti-inflammatory and anti-viral properties across its distribution in China, India and other Southeast Asian countries. A. paniculata was used as a crucial therapeutic treatment during the influenza epidemic of 1919 in India, and is still used for the treatment of infectious disease in China. A. paniculata produces large quantities of the anti-inflammatory diterpenoid lactones andrographolide and neoandrographolide, and their analogs, which are touted to be the next generation of natural anti-inflammatory medicines for lung diseases, hepatitis, neurodegenerative disorders, autoimmune disorders and inflammatory skin diseases. Here, we report a chromosome-scale A. paniculata genome sequence of 269 Mb that was assembled by Illumina short reads, PacBio long reads and high-confidence (Hi-C) data. Gene annotation predicted 25 428 protein-coding genes. In order to decipher the genetic underpinning of diterpenoid biosynthesis, transcriptome data from seedlings elicited with methyl jasmonate were also obtained, which enabled the identification of genes encoding diterpenoid synthases, cytochrome P450 monooxygenases, 2-oxoglutarate-dependent dioxygenases and UDP-dependent glycosyltransferases potentially involved in diterpenoid lactone biosynthesis. We further carried out functional characterization of pairs of class-I and -II diterpene synthases, revealing the ability to produce diversified labdane-related diterpene scaffolds. In addition, a glycosyltransferase able to catalyze O-linked glucosylation of andrograpanin, yielding the major active product neoandrographolide, was also identified. Thus, our results demonstrate the utility of the combined genomic and transcriptomic data set generated here for the investigation of the production of the bioactive diterpenoid lactone constituents of the important medicinal herb A. paniculata., (© 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.)

Squalene epoxidase, thought to be one of the rate-limiting enzymes in the biosynthetic pathways of both membrane sterols and triterpenes (e.g., celastrol), catalyses the formation of oxidosqualene as the common precursor of sterols and triterpenoids. In this work, we first found five squalene epoxidase genes (TwSEs) from Tripterygium wilfordii. Tissue expression pattern, consistent with methyl jasmonate induction study, showed that TwSEs1-4 were involved in the production of special metabolites. In contrast, TwSE5 showed a different tissue expression pattern and was not induced by methyl jasmonate. To probe the functions of the TwSEs, we first tried using a prokaryotic system by constructing an engineered bacterium, but we failed to detect their products. Next, we used the CRISPR/Cas9 tool to construct an erg1 mutant yeast by knocking out the ERG1 gene of yeast strain BY4741 and then applied this mutant to identify the function of TwSEs. We found that only TwSEs1-4 can functionally complement the erg1 mutant yeast. This study laid the foundation for the heterologous biosynthesis of special metabolites in Tripterygium wilfordii., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Ethno-Pharmacological Relevance: The whole plant or the extracts obtained from them have long been used as medicine to treat various human diseases and disorders. Notably, those plants endowed with protease activity have been traditionally used as the agents for treating tumors, digestion disorders, swelling, blood coagulation, fibrinolysis and also for immune-modulation., Aim of the Study: Proteases occupy a pivotal position in enzyme based industries. Plant proteases have been increasingly exploited for pharmaceutical, food, leather and textile processing industries. Earlier investigations have focused on the occurrence of proteases in medicinally unimportant plants. Therefore it has been aimed to study the occurrence of proteolytic enzymes from medicinally important plants establish any correlation exists between protease activity and medicinal use of individual plants., Methods: Crude extract were obtained from the leaves of 80 different medicinal plants. Tris-HCl buffer was used as the extraction buffer and the supernatants obtained were used for determination of total protein and protease activity using spectrophotometric methods. Qualitative screening for the presence of protease was carried out with agar diffusion method by incorporating the substrate. SDS-PAGE was used to analyse the isoforms of protease and for determination of relative molecular mass., Results: Relatively higher protease activities were observed in the extracts of leaves of Pongamia pinnata (Fabaceae), Wrightia tinctoria (Apocyanaceae) Acalypha indica (Euphorbiaceae), Adhatoda vasica (Acanthaceae) and Curcuma longa (Zingiberaceae). No correlation was found between the total protein content and protease activity in individual plant species. SDS-PAGE analysis indicated the presence of multiple forms of protease of higher molecular weight range in several plant species. We found a strong correlation between the protease activity and medicinal application of the plant CONCLUSION: The present study has unequivocally revealed that the leaves of medicinal plants could serve as excellent sources of proteases which could be exploited for various industrial, food and pharmaceutical applications., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

Polygonatum sibiricum polysaccharides (PSPs) are used to improve immunity, alleviate dryness, promote the secretion of fluids, and quench thirst. However, the PSP biosynthetic pathway is largely unknown. Understanding the genetic background will help delineate that pathway at the molecular level so that researchers can develop better conservation strategies. After comparing the PSP contents among several different P. sibiricum germplasms, we selected two groups with the largest contrasts in contents and subjected them to HiSeq2500 transcriptome sequencing to identify the candidate genes involved in PSP biosynthesis. In all, 20 kinds of enzyme-encoding genes were related to PSP biosynthesis. The polysaccharide content was positively correlated with the expression patterns of β-fructofuranosidase ( sacA ), fructokinase ( scrK ), UDP-glucose 4-epimerase ( GALE ), Mannose-1-phosphate guanylyltransferase ( GMPP ), and UDP-glucose 6-dehydrogenase ( UGDH ), but negatively correlated with the expression of Hexokinase ( HK ). Through qRT-PCR validation and comprehensive analysis, we determined that sacA , HK , and GMPP are key genes for enzymes within the PSP metabolic pathway in P. sibiricum. Our results provide a public transcriptome dataset for this species and an outline of pathways for the production of polysaccharides in medicinal plants. They also present more information about the PSP biosynthesis pathway at the molecular level in P. sibiricum and lay the foundation for subsequent research of gene functions., Competing Interests: The authors declare no conflict of interest.

Triterpene saponins include bioactive compounds with structures consisting of triterpene aglycones (sapogenins) and one or more sugar moieties linked through acetal or ester glycosidic linkages at one or more sites. Centella asiatica (L.) Urban is a medicinal plant that contains bioactive ursane-type saponins, such as madecassoside and asiaticoside. In this work, glucosylation of triterpenoids in C. asiatica was investigated starting with plant extracts. An enzyme capable of glucosylating asiatic and madecassic acids was partially purified. Proteomics methods and cDNA sequence data were employed as tools to obtain a full-length cDNA clone encoding a glucosyltransferase. The recombinant gene product, UGT73AD1, was functionally expressed in Escherichia coli and purified by immobilized metal-affinity chromatography. Purified recombinant UGT73AD1 was found to have a narrow specificity, glucosylating asiatic and madecassic acids at the C28 carboxyl. mRNA accumulated in all tissues tested (leaves, stems, roots and flowers), with highest expression in leaves. Thus, UGT73AD1 was identified as a triterpenoid carboxylic acid: UDP-glucose 28-O-glucosyltransferase that appears to be involved in saponin biosynthesis in C. asiatica., (Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.)

2-Alkylquinolone (2AQ) alkaloids are pharmaceutically and biologically important natural products produced by both bacteria and plants, with a wide range of biological effects, including antibacterial, cytotoxic, anticholinesterase, and quorum-sensing signaling activities. These diverse activities and 2AQ occurrence in vastly different phyla have raised much interest in the biosynthesis pathways leading to their production. Previous studies in plants have suggested that type III polyketide synthases (PKSs) might be involved in 2AQ biosynthesis, but this hypothesis is untested. To this end, we cloned two novel type III PKSs, alkyldiketide-CoA synthase (ADS) and alkylquinolone synthase (AQS), from the 2AQ-producing medicinal plant, Evodia rutaecarpa (Rutaceae). Functional analyses revealed that collaboration of ADS and AQS produces 2AQ via condensations of N -methylanthraniloyl-CoA, a fatty acyl-CoA, with malonyl-CoA. We show that ADS efficiently catalyzes the decarboxylative condensation of malonyl-CoA with a fatty acyl-CoA to produce an alkyldiketide-CoA, whereas AQS specifically catalyzes the decarboxylative condensation of an alkyldiketide acid with N -methylanthraniloyl-CoA to generate the 2AQ scaffold via C-C/C-N bond formations. Remarkably, the ADS and AQS crystal structures at 1.80 and 2.20 Å resolutions, respectively, indicated that the unique active-site architecture with Trp-332 and Cys-191 and the novel CoA-binding tunnel with Tyr-215 principally control the substrate and product specificities of ADS and AQS, respectively. These results provide additional insights into the catalytic versatility of the type III PKSs and their functional and evolutionary implications for 2AQ biosynthesis in plants and bacteria., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

The roots of Platycodon grandiflorus are widely used as a crude drug. The active components include a variety of triterpenoid saponins. Recent studies have revealed that Cyt P450 monooxygenases (P450s) function as triterpene oxidases in triterpenoid saponin biosynthesis in many plant species. However, there have been no reports regarding triterpene oxidases in P. grandiflorus. In this study, we performed transcriptome analysis of three different P. grandiflorus tissues (roots, leaves and petals) using RNA sequencing (RNA-Seq) technology. We cloned six P450 genes that were highly expressed in roots, and classified them as belonging to the CYP716A, CYP716D and CYP72A subfamilies. We heterologously expressed these P450s in an engineered yeast strain that produces β-amyrin, one of the most common triterpenes in plants. Two of the CYP716A subfamily P450s catalyzed oxidation reactions of the β-amyrin skeleton. One of these P450s, CYP716A140v2, catalyzed a three-step oxidation reaction at C-28 on β-amyrin to produce oleanolic acid, a reaction performed by CYP716A subfamily P450s in a variety of plant species. The other P450, CYP716A141, catalyzed the hydroxylation of β-amyrin at C-16β. This reaction is unique among triterpene oxidases isolated to date. These results enhance our knowledge of functional variation among CYP716A subfamily enzymes involved in triterpenoid biosynthesis, and provide novel molecular tools for use in synthetic biology to produce triterpenoid saponins with pre-defined structures., (© The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Triptolide and celastrol, two principal bioactive compounds in Tripterygium wilfordii, are produced from geranylgeranyl diphosphate (GGPP) and farnesyl diphosphate ((E,E)-FPP) through terpenoid biosynthesis pathway. However, little is known about T. wilfordii terpene synthases which could competitively utilize GGPP and (E,E)-FPP as substrates, producing C 15 and C 20 tertiary alcohols. Here we firstly cloned the genes encoding nerolidol synthase (NES) and geranyllinalool synthases (GES1, GES2), which are responsible for the biosynthesis of (E)-nerolidol and (E,E)-geranyllinalool. In vitro characterization of recombinant TwNES and TwGES1 revealed both were functional enzymes that could catalyze the conversion of (E,E)-FPP and GGPP to (E)-nerolidol and (E,E)-geranyllinalool, which were consistent with the results of yeast fermentation. Biochemical characterization revealed TwNES and TwGES1 had strong dependency for Mg 2+ , K m and K cat /K m values of TwNES for (E,E)-FPP were 12.700 μM and 0.029 s -1 /μM, and TwGES1 for GGPP were 2.039 μM and 0.019 s -1 /μM. Real-time PCR analysis showed the expression levels of NES and GES1 increased by several fold in the suspension cells treated with alamethicin, indicating TwNES and TwGES1 are likely to utilize GGPP and (E,E)-FPP to generate tertiary alcohols as precursor of plant volatiles, which play important roles in the ecological interactions between T. wilfordii and other organisms., Competing Interests: The authors declare no competing financial interests.

The effect of Lanthanum on the accumulation of active constituent and key enzymes expression of Salvia miltiorrhiza hairy root were studied and furthermore signaling molecules mediating the synthesis of secondary metabolism was also defined in order to provide references for the reveal of synthesis mechanism of active constituent of S. miltiorrhiza hairy root inducing by Lanthanum. The content of active constituents were detected by HPLC. RNA was extracted with RNA prep Pure RNA purification kit (Tiangen). The results shows that LaCl3 processing promoted the accumulation of tanshinones and phenolic acids in S. miltiorrhiza hairy root. The accumulation of phenolic acids reached the highest at 9 d after treatment, and tanshinones accumulation continued to increase in 15 days. Accumulation of active substance in S. miltiorrhiza may relate with FPPS, TAT, HPPR several key enzyme activation., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Chinese Pharmaceutical Association.)

Gynostemma pentaphyllum (Thunb.) Makino is a perennial medicinal herb widely distributed in China. This herb contains important medicinal components called gypenosides, which belong to dammarane-type triterpenoid saponins. Squalene epoxidase (SE, EC 1.14.99.7) catalyzes the epoxidation of squalene to form oxidosqualene and is a key regulatory enzyme in triterpenoid saponin biosynthesis. In this study, a SE gene designated as GpSE1 was isolated from G. pentaphyllum leaves. The deduced protein sequence of GpSE1 contained two conserved domains involved in the catalytic function of SE. GpSE1 was expressed as inclusion bodies in Escherichia coli cells, and the HIS-tagged recombinant protein was successfully purified and renatured in vitro. Immunofluorescence indicated that the polygonal reticular fluorescence signal of GpSE1 was significantly stronger in young leaves than in mature leaves and rhizomes. This finding is consistent with the tissue-specific expression pattern of GpSE1 and suggests that the young leaves of G. pentaphyllum mainly serve as the active site of gypenoside synthesis. Methyl jasmonate (MeJA) treatment upregulated GpSE1 expression in both the young and mature leaves of G. pentaphyllum, with greater upregulation in young leaves than in mature leaves. However, the expression of GpSE1 was not enhanced continually with the increase in MeJA concentration. Moreover, the GpSE1 expression was maximally regulated in response to 50 μM MeJA but not to 100 μM MeJA. This result indicates that MeJA exerts a concentration-dependent effect on GpSE1 expression., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

To investigate the molecular mechanism of quality formation of Pseudostellaria heterophylla, the carotenoid cleavage dioxygenases (CCDs) genes were cloned from the transcriptome database of P. heterophylla, and analyzed them with bioinformatics analysis and expression analysis. The sequence length of four new gene were 1 617, 1 461, 1 746, 1 875 bp, and subsequently, named as PhCCD1,PhNCED2,PhNCED3 and PhCCD4 according to its genetic relationship with Arabidopsis thaliana. The sequence analysis showed that four new gene were all containing REP65 domains and binding sites of ferrous ion, such as histidine, glutamates and aspartates. Analysis phylogeny showed that PhNCED2 and PhNCED3 were the cluster of NCEDs, PhCCD1 and PhCCD4 were the cluster of CCDs. In addition, PhCCD1 and AtCDD1 of Arabidopsis thaliana, PhCCD4 and AtCCD4 of A. thaliana,PhNCED2, PhNCED3 and AtNCED3 of A. thaliana have high similarities. Analysis of real-time fluorescence quantitative showed that PhNCED2 and PhNCED3 were expressed mainly in underground part, the expression quantity of PhNCED2 reached the highest in fibrous root, PhNCED3 keeps higher in phloem and xylem, it may be the key enzymes of ABA biosynthesis genes. Moreover,PhCCD1 and PhCCD4 were expressed mainly in aerial part,the expression quantity of PhCCD1 reached the highest in leaf,PhCCD4 keeps higher in stem and leaf.It may be involved in the biosynthesis of carotenoids for P. heterophylla. The study obtained CDDs gene of P. heterophylla for the first time,this would lay the foundation of developing the response mechanism of P. heterophylla about external stress further,and then exploring the biological approach of quality formation in P. heterophylla., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Chinese Pharmaceutical Association.)

Abscisic acid 8'-hydroxylase was one of key enzymes genes in the metabolism of abscisic acid (ABA). Seven menbers of abscisic acid 8'-hydroxylase were identified from Pseudostellaria heterophylla transcriptome sequencing results by using sequence homology. The expression profiles of these genes were analyzed by transcriptome data. The coding sequence of ABA8ox1 was cloned and analyzed by informational technology. The full-length cDNA of ABA8ox1 was 1 401 bp,with 480 encoded amino acids. The predicated isoelectric point (pI) and relative molecular mass (MW) were 8.55 and 53 kDa,respectively. Transmembrane structure analysis showed that there were 21 amino acids in-side and 445 amino acids out-side. High level of transcripts can detect in bark of root and fibrous root. Multi-alignment and phylogenetic analysis both show that ABA8ox1 had a high similarity with the CYP707As from other plants,especially with AtCYP707A1 and AtCYP707A3 in Arabidopsis thaliana. These results lay a foundation for molecular mechanism of tuberous root expanding and response to adversity stress., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Chinese Pharmaceutical Association.)

Background: Clinically important anti-cancer drugs vinblastine and vincristine are solely synthesized by the terpenoid indole alkaloid (TIA) pathway in Catharanthus roseus. Anthranilate synthase (AS) is a rate-limiting enzyme in the TIA pathway. The transgenic C. roseus hairy root line overexpressing a feedback insensitive ASα subunit under the control of an inducible promoter and the ASβ subunit constitutively was previously created for the overproduction of TIAs. However, both increases and decreases in TIAs were detected after overexpressing ASα. Although genetic modification is targeted to one gene in the TIA pathway, it could trigger global transcriptional changes that can directly or indirectly affect TIA biosynthesis. In this study, Illumina sequencing and RT-qPCR were used to detect the transcriptional responses to overexpressing AS, which can increase understanding of the complex regulation of the TIA pathway and further inspire rational metabolic engineering for enhanced TIA production in C. roseus hairy roots., Results: Overexpressing AS in C. roseus hairy roots altered the transcription of most known TIA pathway genes and regulators after 12, 24, and 48 h induction detected by RT-qPCR. Changes in the transcriptome of C. roseus hairy roots was further investigated 18 hours after ASα induction and compared to the control hairy roots using RNA-seq. A unigene set of 30,281 was obtained by de novo assembly of the sequencing reads. Comparison of the differentially expressed transcriptional profiles resulted in 2853 differentially expressed transcripts. Functional annotation of these transcripts revealed a complex and systematically transcriptome change in ASαβ hairy roots. Pathway analysis shows alterations in many pathways such as aromatic amino acid biosynthesis, jasmonic acid (JA) biosynthesis and other secondary metabolic pathways after perturbing AS. Moreover, many genes in overall stress response were differentially expressed after overexpressing ASα., Conclusion: The transcriptomic analysis illustrates overexpressing AS stimulates the overall stress response and affects the metabolic networks in C. roseus hairy roots. The up-regulation of endogenous JA biosynthesis pathway indicates the involvement of JA signal transduction to regulate TIA biosynthesis in ASαβ engineered roots and explained why many of the transcripts for TIA genes and regulators are seen to increase with AS overexpression.

The paper mainly studied the effects of enhanced UV-B radiation on the nutritional and active ingredient contents during the floral development of medicinal chrysanthemum. The experiment included two levels of UV-B radiation (0 and 400μWcm(-2)). The contents of hydrogen peroxide (H2O2), anthocyanin, UV-B absorbing compounds, total chlorophyll and carotenoids, and the activities of phenylalanine ammonia lyase enzyme (PAL) and cinnamic acid-4-hydroxylase enzyme (C4H) in flowers significantly decreased with the floral development. However, the contents of soluble sugar, amino acid and total vitamin C in flowers significantly increased with the floral development. The contents of flavonoid and chlorogenic acid were significantly different in the four stages of floral development, and their highest contents were found in the bud stage (stage 2). In the four stages of floral development, enhanced UV-B radiation significantly increased the contents of H2O2, UV-B absorbing compounds, chlorophyll, carotenoids, soluble sugar, amino acid, vitamin C, flavonoid and chlorogenic acid, and the activities of PLA and C4H in flowers. The results indicated that the highest contents of active and nutrient ingredients in flowers were found not to be in the same developmental stages of flowers. Comprehensive analysis revealed that the best harvest stage of chrysanthemum flowers was between the bud stage and the young flower stage (stage 2 and stage 3), which could simultaneously gain the higher contents of active and nutritional ingredients in flowers., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Members of the Apocynaceae plant family produce a large number of monoterpenoid indole alkaloids (MIAs) with different substitution patterns that are responsible for their various biological activities. A novel N-methyltransferase involved in the vindoline pathway in Catharanthus roseus showing distinct similarity to γ-tocopherol C-methyltransferases was used in a bioinformatic screen of transcriptomes from Vinca minor, Rauvolfia serpentina, and C. roseus to identify 10 γ-tocopherol-like N-methyltransferases from a large annotated transcriptome database of different MIA-producing plant species (www.phytometasyn.ca). The biochemical function of two members of this group cloned from V. minor (VmPiNMT) and R. serpentina (RsPiNMT) have been characterized by screening their biochemical activities against potential MIA substrates harvested from the leaf surfaces of MIA-accumulating plants. The approach was validated by identifying the MIA picrinine from leaf surfaces of Amsonia hubrichtii as a substrate of VmPiNMT and RsPiNMT. Recombinant proteins were shown to have high substrate specificity and affinity for picrinine, converting it to N-methylpicrinine (ervincine). Developmental studies with V. minor and R. serpentina showed that RsPiNMT and VmPiNMT gene expression and biochemical activities were highest in younger leaf tissues. The assembly of at least 150 known N-methylated MIAs within members of the Apocynaceae family may have occurred as a result of the evolution of the γ-tocopherol-like N-methyltransferase family from γ-tocopherol methyltransferases., (© 2016 American Society of Plant Biologists. All Rights Reserved.)

Diosgenin is a steroid derived from cholesterol in plants and used as a typical initial intermediate for synthesis of numerous steroidal drugs in the world. Commercially, this compound is extracted mainly from the rhizomes or tubers of some Dioscorea species. Squalene synthase (SQS: EC 2.5.1.21) catalyzes the condensation of two molecules of farnesyl diphosphate to form squalene, the first committed step for biosynthesis of plant sterols including cholesterol, and is thought to play an important role in diosgenin biosynthesis. A full-length cDNA of a putative squalene synthase gene was cloned from D. zingiberensis and designated as DzSQS (Genbank Accession Number KC960673). DzSQS was contained an open reading frame of 1,230 bp encoding a polypeptide of 409 amino acids with a predicted molecular weight of 46 kDa and an isoelectric point of 6.2. The deduced amino acid sequence of DzSQS shared over 70 % sequence identity with those of SQSs from other plants. The truncated DzSQS in which 24 amino acids were deleted from the carboxy terminus was expressed in Escherichia coli, and the resultant bacterial crude extract was incubated with farnesyl diphosphate and NADPH. GC-MS analysis showed that squalene was detected in the in vitro reaction mixture. Quantitative real-time PCR analysis revealed that DzSQS was expressed from highest to lowest order in mature leaves, newly-formed rhizomes, young leaves, young stems, and two-year-old rhizomes of D. zingiberensis.

Glycyrrhiza glabra is an important medicinal plant throughout the world. Glycyrrhizin is a triterpenoid that is among the most important secondary metabolites produced by liquorice. Drought stress is proposed to enhance the levels of secondary metabolites. In this study, the effect of drought stress on the expression of important genes involved in the glycyrrhizin biosynthetic pathway was examined. Drought stress at the seedling stage was applied to 8-day-old plants using polyethylene glycol. Subsequently, the samples were collected 0, 4, 8 or 24 h post-treatment. At the adult plant stage, 10-month-old plants were subjected to drought stress by discontinuing irrigation. Subsequently, samples were collected at 2, 16 and 28 days after drought imposition (S(2d), S(16d) and S(28d), respectively). We performed semi-quantitative RT-PCR assays to evaluate the gene expression levels of sequalene synthase (SQS), β-amyrin synthase (bAS), lupeol synthase (LUS) and cycloartenol synthase (CAS) during stress. Finally, the glycyrrhizin content of stolons was determined via HPLC. The results revealed that due to osmotic stress, the gene expression levels of SQS and bAS were increased, whereas those of CAS were relatively unchanged at the seedling stage. At the adult plant stage, the expression levels of SQS and bAS were increased under drought stress conditions, whereas the gene expression level of CAS remained relatively constant. The glycyrrhizin content in stolons was increased only under severe drought stress conditions (S(28d)). Our results indicate that application of controlled drought stress up-regulates the expression of key genes involved in the biosynthesis of triterpenoid saponins and directly enhances the production of secondary metabolites, including glycyrrhizin, in liquorice plants., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Cell suspension cultures of Arnebia euchroma were established from the friable callus on liquid Murashige and Skoog medium supplemented with 6-benzylaminopurine (10.0 μM) and indole-3-butyric acid (5.0 μM). Salicylic acid was used to study its effect on the enzymes which participate in shikonin biosynthesis with respect to metabolite (shikonin) content in the cell suspension culture of A. euchroma. In our study, phenylalanine ammonia lyase and PHB geranyltransferase were selected from the entire biosynthetic pathway. Results showed that phenylalanine ammonia lyase is responsible for growth and PHB geranyltransferase for metabolite production. Salicylic acid exhibited an inverse relationship with the metabolite content (shikonin); salicylic acid (100 μM) completely inhibited shikonin biosynthesis. The results presented in the current study can be successfully employed for the metabolic engineering of its biosynthetic pathway for the enhancement of shikonin, which will not only help in meeting its industrial demand but also lead to the conservation of species in its natural habitat.

Terpenoids are an extensive and diverse group of plant secondary metabolites. To date, they have been applied in many fields including industry, medicine and health. The wide variety of terpenoid compounds cannot arise solely from simple cyclisations of a precursor molecule or from a single-step reaction; their structural diversity depends on the modification of many specific chemical groups, rearrangements of their skeletal structures and on the post-modification reactions. Most of the post-modification enzymes that catalyse these reactions are cytochrome P450 monooxygenases. Therefore, the discovery and identification of plant P450 genes plays a vital role in the exploration of terpenoid biosynthesis pathways. This review summarises recent research progress relating to the function of plant cytochrome P450 enzymes, describes P450 genes that have been cloned from full-length cDNA and identifies the function of P450 enzymes in the terpenoid biosynthesis pathways of several medicinal plants.

The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes the conversion of HMG-CoA to mevalonate in mavalonic acid pathway, which is the first committed step for isoprenoid biosynthesis in plants. However, it still remains unclear whether HGMR gene plays a role in the isoprenoid biosynthesis in Dendrobium officinale, an endangered epiphytic orchid species. In the present study, a HMGR encoding gene, designed as DoHMGR1 (GenBank accession JX272632), was identified from D. officinale using the reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) methods, for the first time. The full length cDNA of DoHMGR1 was 2 071 bp in length and encoded a 562-aa protein with a molecular weight of 59.73 kD and an isoelectric point (pI) of 6.18. The deduced DoHMGR1 protein, like other HMGR proteins, constituted four conserved domains (63-561, 147-551, 268-383 and 124-541) and two transmembrane motifs (42-64 and 85-107). Multiple sequence alignment and phylogenetic analyses demonstrated that DoHMGR1 had high identity (67%-89%) to a number of HMGR genes from various plants and was closely related to Vanda hybrid cultivar, rice and maize monocots. Real time quantitative PCR (qPCR) analysis revealed that DoHMGR1 was expressed in the three included organs. The transcripts were the most abundant in the roots with 2.13 fold over that in the leaves, followed by that in the stems with 1.98 fold. Molecular characterization of DoHMGR1 will be useful for further functional elucidation of the gene involving in isoprenoid biosynthesis pathway in D. officinale.

There exists many kinds and a huge number of terpenoid in medicinal plants, which show a wide range of pharmacological activities. 3-Hydroxy-3-metllylglutaryl coenzyme A reductase(HMGR) is a key rate-limiting enzyme in terpenoid biosynthetic pathway . HMGR plays an important role in the regulation of secondary metabolism of the terpenoid. The paper summarized the biological function and the catalytic mechanism of HMGR, the cloning and the structure of the gene as well as its research progress in some medicinal plants.

Dendrobium officinale Kimura et Migo (Orchidaceae) is a traditional Chinese medicinal plant. The stem contains an alkaloid that is the primary bioactive component. However, the details of alkaloid biosynthesis have not been effectively explored because of the limited number of expressed sequence tags (ESTs) available in GenBank. In this study, we analyzed RNA isolated from the stem of D. officinale using a single half-run on the Roche 454 GS FLX Titanium platform to generate 553,084 ESTs with an average length of 417 bases. The ESTs were assembled into 36,407 unique putative transcripts. A total of 69.97% of the unique sequences were annotated, and a detailed view of alkaloid biosynthesis was obtained. Functional assignment based on Kyoto Encyclopedia of Genes and Genomes (KEGG) terms revealed 69 unique sequences representing 25 genes involved in alkaloid backbone biosynthesis. A series of qRT-PCR experiments confirmed that the expression levels of 5 key enzyme-encoding genes involved in alkaloid biosynthesis are greater in the leaves of D. officinale than in the stems. Cytochrome P450s, aminotransferases, methyltransferases, multidrug resistance protein (MDR) transporters and transcription factors were screened for possible involvement in alkaloid biosynthesis. Furthermore, a total of 1061 simple sequence repeat motifs (SSR) were detected from 36,407 unigenes. Dinucleotide repeats were the most abundant repeat type. Of these, 179 genes were associated with a metabolic pathway in KEGG. This study is the first to produce a large volume of transcriptome data from D. officinale. It extends the foundation to facilitate gene discovery in D. officinale and provides an important resource for the molecular genetic and functional genomic studies in this species., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Background: Argonaute (AGO) is the core component of RNA-induced silencing complex. The AGO gene family has been analyzed in various plant species; however, there is no report about AGOs in the well-known Traditional Chinese Medicine (TCM) plant, Salvia miltiorrhiza., Results: Through a genome-wide analysis, we identified ten SmAGO genes in S. miltiorrhiza. Full-length cDNAs of all SmAGOs were subsequently cloned and sequenced. These SmAGOs were characterized using a comprehensive approach. Sequence features, gene structures and conserved domains were analyzed by the comparison of SmAGOs and AtAGOs. Phylogenetic relationships among AGO proteins from S. miltiorrhiza, Arabidopsis and rice were revealed. The expression levels of SmAGO genes in various tissues of S. miltiorrhiza were investigated. The results implied that some SmAGOs, such as SmAGO1, SmAGO2, SmAGO3, SmAGO7 and SmAGO10, probably played similar roles as their counterparts in Arabidopsis; whereas the others could be more species-specialized. It suggests the conservation and diversity of AGOs in plants. Additionally, we identified a total of 24 hairpin structures, representing six miRNA gene families, to be miRNA precursors. Using the modified 5'-RACE method, we confirmed that SmAGO1 and SmAGO2 were targeted by S. miltiorrhiza miR168a/b and miR403, respectively. It suggests the conservation of AGO1-miR168 and AGO2-miR403 regulatory modules in S. miltiorrhiza and Arabidopsis., Conclusions: This is the first attempt to explore SmAGOs and miRNAs in S. miltiorrhiza. The results provide useful information for further elucidation of gene silencing pathways in S. miltiorrhiza.

Objective: Erigeron breviscapus is a medicinal plant with the most developmental potential in Yunnan province, which is belongs to Erigeron genus of Compositae family. Scutellarin, the main active component of Erigeron breviscapus is one of flavone 7-O-glucuronide derivatives, its biosynthesis pathway is still not clear., Method: Full length cDNA encoding flavone syhthase II gene in E. breviscapus was cloned in this study using R-PCR, 3'-RACE and 5'-RACE., Result: The opening reading frame of FS II cDNA of E. breviscapus is 1 557 bp long and encoding 518 amino acids, designed as EbFS II, which is highly homologous with FS II of Compositae species, like Callistephus chinensis, Cynara cardunculus var. scolymus, Gerbera hybrida, Dahlia pinnata and Lobelia erinus., Conclusion: Phylogenetic analysis showed that EbFS II might has the function of directly converting flavanone to flavone.

Objective: Scutellarin from Erigeron breviscapus is a flavonoid with remarkable pharmacological activity, whose route of biosynthesis is still fully clear. Chalcone synthase (CHS) is the key enzyme regulating flavonoids biosynthesis, and the aim of this study is to explain the relationship between patterns of the gene expression and scutellarin content through studying CHS gene expression patterns combined with scutellarin content in various parts of E. breviscapus., Method: Through RT-PCR and RACE, the full length of CHS was cloned and analyzed by fluorescent quantitative PCR. The scutellarin content in tissues was analyzed by HPLC., Result: The full-length gene sequence was 1 270 bp, encoding 405 amino acids. Software analysis found that the DNA sequence was 80% similarity with Compositae plant homeo-box gene. Fluorescence quantitative analysis showed that CHS had the highest expression level in leaves, far higher than that in root, stem and flower. HPLC analysis showed that the scutellarin was the highest in leaves, followed by the flowers and stems, scutellarin was not detected in root., Conclusion: Correlation analysis showed that CHS expression amount and scutellarin content in different parts of E. breviscapus is positive correlation (r = 0.761, P < 0.05), it suggests that CHS gene expression level has important effect on biosynthesis of scutellarin.

S-Adenosyl-L-methionine decarboxylase (SAMDC) is a key enzyme in the polyamines biosynthesis, thus is essential for basic physiological and biochemical processes in plant. In the present study, a full length cDNA of DoSAMDC1 gene was obtained from symbiotic germinated seeds of an endangered medicinal orchid species Dendrobium officinale, using the rapid amplification of cDNA ends (RACE)-PCR technique for the first time. The full length cDNA was 1 979 bp, with three open reading frames, i.e. tiny-uORF, small-uORF and main ORF (mORF). The mORF was deduced to encode a 368 amino acid (aa) protein with a molecular mass of 40.7 kD and a theoretical isoelectric point of 5.2. The deduced DoSAMDC1 protein, without signal peptide, had two highly conserved function domains (proenzyme cleavage site and PEST domain) and a 22-aa transmembrane domain (89-110). Multiple sequence alignments and phylogenetic relationship analyses revealed DoSAMDC1 had a higher level of sequence similarity to monocot SAMDCs than those of dicot. Expression patterns using qRT-PCR analyses showed that DoSAMDC1 transcripts were expressed constitutively without significant change in the five tissues (not infected with fungi). While in the symbiotic germinated seeds, the expression level was enhanced by 2.74 fold over that in the none-germinated seeds, indicating possible involvement of the gene in symbiotic seed germination of D. officinale.

3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) is the first rate-limiting enzyme for sesquiterpene synthesis in the mevalonate (MVA) pathway. The specific primers were designed according to the transcript sequence of AsHMGR2 from the Aquilaria sinensis (Lour.) Gilg transcriptome database. The full-length cDNA of AsHMGR2 was cloned by RT-PCR and rapid amplification of cDNA ends (RACE) technology, and was analyzed at bioinformatics levels; AsHMGR2 expression profiles in different tissues and in responds to different treatments were analyzed by real-time PCR. The length of AsHMGR2 Open Reading Frame (ORF) was 1 749 bp, encoding 582 amino acids. The GenBank accession number is KC140287. Tissue expression analysis indicated that AsHMGR2 was mainly expressed in root and shoot tips, followed by stem, and was lowest in leaves. Inducible-experiments showed that the genes were induced by mechanical wound as well as chemical liquid induction, and reached the highest expression level at 6 h and 8 h, separately. The full-length cDNA of AsHMGR2 and its expression patterns will provide a foundation for further research on its function in agarwood sesquiterpene biosynthesis.

Disaccharide intolerance is the inability to digest certain carbohydrates due to a lack of one or more intestinal disaccharidases (e.g., lactase, maltase, isomaltase and sucrase). Symptoms include diarrhea, abdominal distention and flatulence. Management of the disorder by external enzymes supplementation has not yet been attempted. We report that the medicinal plant Tinospora cordifolia contains substantial amounts of all disaccharidases required for intestinal digestion of carbohydrates. The plant is also a rich source of saccharifying amylase. We recovered (units/100 g fresh stem) amylase: 49,000+500, maltase: 400+50, isomaltase: 130+50, sucrase: 4500+500, acid lactase: 350+30, cellobiase: 35+10 and trehalase: 40+10 by buffer extraction of the blended stem. Crude enzymes in the forms of stem powder, lyophilized aqueous extract and ethanol precipitated protein were found to be stable. Disaccharidases were optimally active at 50 (0) C in the pH range of 4-5. Lactase was an acid lactase similar to the type linked with human lactose intolerance. Enzymes were catalytically stable in the pH range of 2-7 and temperature range of up to 40 (0) C. T. cordifolia enzyme was non-toxic up to a dose of 200 mg protein/kg body weight., (Copyright © 2012 John Wiley & Sons, Ltd.)

The roots of Glycyrrhiza uralensis are widely used in Chinese medicine for their action of clearing heat, detoxicating, relieving cough, dispelling sputum and tonifying spleen and stomach. The reason why Glycyrrhiza uralensis has potent and significant actions is that it contains various active secondary metabolites, especially glycyrrhizic acid. In the present study, we cloned the cDNA coding 3-hydroxy-3-methylglutary CoA reductase (HMGR) involved in glycyrrhizic acid biosynthesis in Glycyrrhiza uralensis. The corresponding cDNA was expressed in Escherichia coli as fusion proteins. Recombinant HMGR exhibited catalysis activity in reduction of HMG-CoA to mevalonic acid (MVA) just as HMGR isolated from other species. Because HMGR gene is very important in the biosynthesis of glycyrrhizic acid in Glycyrrhiza uralensis, this work is significant for further studies concerned with strengthening the efficacy of Glycyrrhiza uralensis by means of increasing glycyrrhizic acid content and exploring the biosynthesis of glycyrrhizic acid in vitro.

Withania somnifera (L.) Dunal is one of the most valuable medicinal plants synthesizing a large number of pharmacologically active secondary metabolites known as withanolides, the C28-steroidal lactones derived from triterpenoids. Though the plant has been well characterized in terms of phytochemical profiles as well as pharmaceutical activities, not much is known about the biosynthetic pathway and genes responsible for biosynthesis of these compounds. In this study, we have characterized the gene encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR; EC 1.1.1.34) catalyzing the key regulatory step of the isoprenoid biosynthesis. The 1,728-bp full-length cDNA of Withania HMGR (WsHMGR) encodes a polypeptide of 575 amino acids. The amino acid sequence homology and phylogenetic analysis suggest that WsHMGR has typical structural features of other known plant HMGRs. The relative expression analysis suggests that WsHMGR expression varies in different tissues as well as chemotypes and is significantly elevated in response to exposure to salicylic acid, methyl jasmonate, and mechanical injury. The functional color assay in Escherichia coli showed that WsHMGR could accelerate the biosynthesis of carotenoids, establishing that WsHMGR encoded a functional protein and may play a catalytic role by its positive influence in isoprenoid biosynthesis.

The intermolecular coupling of 2,5-dimethoxyaniline (DMA) as mediated electron transfer reaction in presence of H(2)O(2) and peroxidase in acetate buffer of pH 4.2 resulting green colored product having maximum absorption at λ(max)=740 nm was investigated by spectrophotometer. Under optimum conditions, linearity range for the quantification of H(2)O(2) was 2.0-288.0 μM and for peroxidase were 0.59-9.46 and 0.443-9.46 nM by kinetic and fixed-time method, respectively. The catalytic efficiency and catalytic power were K(eff)(D)=2.354 × 10(5)M(-1)min(-1) and K(pow)(D)=4.59 × 10(-4)min(-1), respectively. From the plot of d(1/D(o)) vs d(1/V(o)) and d(1/H(o)) vs d(1/V(o)), Michaelis-Menten constants for DMA and H(2)O(2)were found that K(m)(D)=1,458 μM and [Formula: see text] =301 μM. Applicability of the method was tested for peroxidase activity in some plant extracts and compared with guaiacol/peroxidase system. Regarding superiority of the method, it is suggested that DMA/peroxidase system can be a better hydrogen donor for HRP assay than guaiacol system as evident from kinetic data., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Atropa belladonna L. is the officially medicinal plant species and the main commercial source of scopolamine and hyoscyamine in China. In this study, we reported the simultaneous overexpression of two functional genes involved in biosynthesis of scopolamine, which respectively encoded the upstream key enzyme putrescine N-methyltransferase (PMT; EC 2.1.1.53) and the downstream key enzyme hyoscyamine 6beta-hydroxylase (H6H; EC 1.14.11.11) in transgenic hair root cultures of Atropa belladonna L. HPLC results suggested that four transgenic hair root lines produced higher content of scopolamine at different levels compared with nontransgenic hair root cultures. And scopolamine content increased to 8.2 fold in transgenic line PH2 compared with that of control line; and the other four transgenic lines showed an increase of scopolamine compared with the control. Two of the transgenic hair root lines produced higher levels of tropane alkaloids, and the content increased to 2.7 fold in transgenic line PH2 compared with the control. The gene expression profile indicated that both PMT and H6H expressed at a different levels in different transgenic hair root lines, which would be helpful for biosynthesis of scopolamine. Our studies suggested that overexpression of A. belladonna endogenous genes PMT and H6H could enhance tropane alkaloid biosynthesis.

The iridoids comprise a large family of distinctive bicyclic monoterpenes that possess a wide range of pharmacological activities, including anticancer, anti-inflammatory, antifungal and antibacterial activities. Additionally, certain iridoids are used as sex pheromones in agriculturally important species of aphids, a fact that has underpinned innovative and integrated pest management strategies. To harness the biotechnological potential of this natural product class, the enzymes involved in the biosynthetic pathway must be elucidated. Here we report the discovery of iridoid synthase, a plant-derived enzyme that generates the iridoid ring scaffold, as evidenced by biochemical assays, gene silencing, co-expression analysis and localization studies. In contrast to all known monoterpene cyclases, which use geranyl diphosphate as substrate and invoke a cationic intermediate, iridoid synthase uses the linear monoterpene 10-oxogeranial as substrate and probably couples an initial NAD(P)H-dependent reduction step with a subsequent cyclization step via a Diels-Alder cycloaddition or a Michael addition. Our results illustrate how a short-chain reductase was recruited as cyclase for the production of iridoids in medicinal plants. Furthermore, we highlight the prospects of using unrelated reductases to generate artificial cyclic scaffolds. Beyond the recognition of an alternative biochemical mechanism for the biosynthesis of cyclic terpenes, we anticipate that our work will enable the large-scale heterologous production of iridoids in plants and microorganisms for agricultural and pharmaceutical applications.

The mitogen-activated protein kinase (MAPK) cascade, composed of MAPK kinase kinase (MAP3K), MAPK kinase (MAP2K), and MAPK, is abundantly conserved in all eukaryotes. MAPK along with MAPK cascade plays a vital regulatory role in the plant-arbuscular mycorrhiza/rhizobium nodule symbioses. However, the biological function of MAPK in orchid mycorrhiza (OM) symbiosis remains elusive. In the present study, a MAPK gene, designated as DoMPK1 (GenBank accession No. JX297594), was identified from D. officinale roots infected by an OM fungus-Mycena sp. using the reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) methods. The full length cDNA of DoMPK1 was 1 263 bp and encoded a 372 aa protein with a molecular weight of 42.61 kD and an isoelectric point (pI) of 6.07. The deduced DoMPK1 protein contained the conserved serine/threonine-protein kinase catalytic domain (39-325) and MAP kinase signature (77-177). Multiple sequence alignment and phylogenetic analysis demonstrated that DoMPK1 was highly homologous (71%-85%) to MAPK genes from various plant species and was closely related to those from monocots. Real time quantitative PCR (qPCR) analysis revealed that DoMPK1 was constitutively expressed in leaves, stems, roots and seeds, and the transcript abundance was not significantly different in the four included tissues. Furthermore, DoMPK1 transcript was markedly induced in roots at 30 d after fungal infection, with 7.91 fold compared to that of the mock inoculated roots, suggesting implication of DoMPK1 in the early D. officinale and Mycena sp. interaction and an essential role in the symbiosis. Our study characterized a MAPK gene associated with OM symbiosis for the first time, and will be helpful for further functional elucidation of DoMPK1 involving in D. officinale and Mycena sp. symbiotic interaction.

Calcium-dependent protein kinases (CDPKs) play an important regulatory role in the plantarbuscular mycorrhiza/rhizobium nodule symbiosis. However, the biological action of CDPKs in orchid mycorrhiza (OM) symbiosis remains unclear. In the present study, a CDPK encoding gene, designated as DoCPK1 (GenBank accession No. JX193703), was identified from D. officinale roots infected by an OM fungus-Mycena sp. using the reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) methods, for the first time. The full length cDNA of DoCPK1 was 2137 bp in length and encoded a 534 aa protein with a molecular weight of 59.61 kD and an isoelectric point (pI) of 6.03. The deduced DoCPK1 protein contained the conserved serine/threonine-protein kinase catalytic domain and four Ca2+ binding EF hand motifs. Multiple sequence alignment demonstrated that DoCPK1 was highly homologous (85%) to the Panax ginseng PgCPK1 (ACY78680), followed by CDPKs genes from wheat, rice, and Arabidopsis (ABD98803, ADM14342, Q9ZSA2, respectively). Phylogenetic analysis showed that DoCPK1 was closely related to CDPKs genes from monocots, such as wheat, maize and rice. Real time quantitative PCR (qPCR) analysis revealed that DoCPK1 was constitutively expressed in the included tissues and the transcript levels were in the order of roots > stems > seeds > leaves. Furthermore, DoCPK1 transcripts were significantly accumulated in roots 30 d after fungal infection, with 5.16 fold compared to that of the mock roots, indicating involvement of DoCPK1 during the early interaction between D. officinale and Mycena sp., and a possible role in the symbiosis process. This study firstly provided important clues of a CDPK gene associated with OM symbiosis, and will be useful for further functional determination of the gene involving in D. officinale and Mycena sp. symbiosis.

Aralia elata is an important medicinal plant in China; it produces large amounts of oleanane type triterpene saponins. A full-length cDNA encoding β-amyrin synthase (designated as AeAS) was isolated from young leaves of A. elata by reverse transcription-PCR. The full-length cDNA of AeAS was found to have a 2292-bp open reading frame, encoding a protein with 763 amino acid residues. The deduced amino acid sequence of AeAS showed the highest identity (97%) to Panax ginseng β-amyrin synthase. When AeAS cDNA was expressed in Escherichia coli, an 87.8-kDa recombinant protein was detected by SDS-PAGE and Western blotting. The sequence was also heterologously expressed in the yeast Pichia pastoris, and production of β-amyrin was detected by HPLC. Tissue expression pattern analysis by real-time reverse transcription-PCR revealed that AeAS is strongly expressed in leaves and stems, and weakly expressed in roots and flowers.

Background: Hawthorn is the common name of all plant species in the genus Crataegus, which belongs to the Rosaceae family. Crataegus are considered useful medicinal plants because of their high content of proanthocyanidins (PAs) and other related compounds. To improve PAs production in Crataegus tissues, the sequences of genes encoding PAs biosynthetic enzymes are required., Findings: Different bioinformatics tools, including BLAST, multiple sequence alignment and alignment PCR analysis were used to design primers suitable for the amplification of DNA fragments from 10 candidate genes encoding enzymes involved in PAs biosynthesis in C. aronia. DNA sequencing results proved the utility of the designed primers. The primers were used successfully to amplify DNA fragments of different PAs biosynthesis genes in different Rosaceae plants., Conclusion: To the best of our knowledge, this is the first use of the alignment PCR approach to isolate DNA sequences encoding PAs biosynthetic enzymes in Rosaceae plants.

Squalene synthase (SQS) is a key enzyme in plant terpenoid biosynthetic pathway. This study focused on cloning and analysis of Huperzia serrata SQS (HsSQS1) gene. After searching the transcriptome dataset of H serrata, one unique sequence encoding SQS was discovered. The primers were designed according to the transcript sequence of HsSQS1 from the H. serrata transcriptome dataset. The open reading frame of HsSQS1 was cloned using RT-PCR strategy. The bioinformatic analysis of this gene and its corresponding protein were performed. The cDNA (named as HsSQS1) contains a 1263 bp open reading frame and encodes a predicted protein of 420 amino acids. The GenBank accession number for this gene is JQ004938. HsSQS1 contains two transmembrane regions, without signal peptide. The conserved domain of squalene synthase was presented in HsSQS1. HsSQS1 was more abundant in H. serrata root than in leaf and stem. This study cloned and analyzed squalene synthase gene from H. serrata for the first time. The result will provide a foundation for exploring the mechanism ofterpenoid biosynthesis in H. serrata plants.

Mevalonate kinase (MVK) is a key enzyme in plant terpenoid biosynthetic pathway. This study focused on cloning and analysis of Panax notoginseng (Burk.) F. H. Chen MVK (PnMVK1) gene. After searching the transcriptome dataset of P. notoginseng, one unique sequence encoding MVK was discovered. The primers were designed according to the transcript sequence of PnMVK1 from the P. notoginseng transcriptome dataset. And then, the open reading frame of PnMVK1 was cloned from P. notoginseng by using RT-PCR strategy. The physical and chemical properties, secondary structure and three-dimensional structure of the PnMVK1 protein were forecasted and analyzed, and its structure and function were predicted. The cDNA (named as PnMVK1) contains a 1164 bp open reading frame and encodes a predicted protein of 387 amino acids. The GenBank accession number for this gene is JQ957844. No transmembrane region and signal peptide were present in PnMVK1. The conserved domain of mevalonate kinase was present in PnMVK1. PnMVK1 was more abundant in P. notoginseng root than other organisms. This study cloned and analyzed PnMVK1 gene from P. notoginseng for the first time. The result will provide a foundation for exploring the mechanism of terpenoid biosynthesis in P. notoginseng plants.

After searching the transcriptome dataset of Panax notoginseng, one unique sequence Pn02086 encoding UDP-glucosyltransferase (UGT), which may be involved in triterpene saponin biosynthesis, was discovered. The open reading frame of the UGT gene, named as PnUGT1, was cloned by 5'-RACE and RT-PCR method from P. notoginseng. The GenBank accession number for this gene is JX018210. The bioinformatic analysis of this gene and its corresponding protein was performed. The PnUGT1 gene contains a 1488 bp open reading frame and encodes a predicted protein of 495 amino acids. The molecular weight is 55.453 kD and the protein is unstable. In the secondary structure, the percentage of alpha helix, beta turn, random coil were 36.16%, 11.31%, 52.53%, respectively. The PnUGT1 contains 7 conserved domains predicted by InterProScan, including PSPG-box which is a unique consensus sequence of glycosyltransferases involved in plant secondary metabolism. The PnUGT1 was most likely to be located in the cytoplasm, without signal peptide and transmembrane region. Sequence alignment and phylogenetic analysis demonstrated that PnUGT1 had relative close relationship to the triterpene UDP-glucosyltransferase of Medicago truncatula (AAW56092), with the 66% similarity of conserved domain PSPG-box. PnUGT1 was more abundant in P. notoginseng leaf than in flower, stem and root. Therefore, PnUGT1 gene may be involved in notoginsenoside biosynthesis.

The leucoanthocyantin reducase (LAR) gene, an important functional gene of catechins biosynthesis pathway, was cloned from Fagopyrum dibotrys (D.Don) Hara by degenerate PCR and rapid amplification of cDNA ends (RACE). The full-length cDNA of FdLAR is 1 581 bp (GenBank accession: JN793953), containing a 1 176 bp ORF encoding a 391 amino acids protein, and its 3'-untranslated region has an obvious polyadenylation signal. The recombinant plasmid containing FdLAR completed ORF was transformed into E. coli BL21 (DE3). The target fusion peptide with molecular weight of 66 kD was expressed under the condition of 16 degrees C and induced by IPTG at final concentration of 1.0 mmol x L(-1). Bioinformation analysis indicated that the amino acid sequence of FdLAR showed great homology to other LAR with the NADB-Rossmann conversed domain in the N-terminus. Real-time quantitative PCR was used to detect the expression levels of FdLAR gene during different development periods. The determination of flavonoids contents in appropriate rhizomes showed that the relationship between FdLAR gene expression and the accumulation of flavonoids displayed different trends during vegetative growth and reproductive growth stages, suggesting that the FdLAR gene may be involved in the pathway of flavonoid metabolisms in Fagopyrum dibotrys.

Objective: To reveal the temporal and spatial specificity of the expression of beta-AS gene of Glycyrriza uralensis., Methods: Used PCR to obtain the cDNA of beta-AS gene of Glycyrriza uralensis at different time and from different part and Gel-Pro to carry out the densitometric analysis of the electrophoretic band, then calculate the relative expression., Results: The spatial specificity experiment showed that beta-AS gene didn't express in the overground part of Glycyrriza uralensis,while in the underground part,the expression of beta-AS in root tip was higher than that of rootstock. And the temporal specificity experiment showed that the expression of beta-AS gene of Glycyrriza uralensis could be divided into 4 stages. From December to February, the expression of beta-AS gene was under the detection limit. From March to May, beta-AS gene began to express. From May to September,the expression of beta-AS gene kept at a high level. And in October and November the expression of beta-AS gene began to decrease., Conclusion: When the beta-AS gene of Glycyrriza uralensis is researched, the root tip is the suitable plant material and May, June, August and September are the right acquisition time.

A 34 kDa serine protease, designated as hirtin, with fibrinolytic activity was purified to homogeneity from the latex of Euphorbia hirta by the combination of ion exchange and gel filtration chromatography. The N-terminal sequence of hirtin was found to be YAVYIGLILETAA/NNE. Hirtin exhibited esterase and amidase activities along with azocaseinolytic, gelatinolytic, fibrinogenolytic and fibrinolytic activities. It preferentially hydrolyzed Aα and α-chains, followed by Bβ and β, and γ and γ-γ chains of fibrinogen and fibrin clot respectively. The optimum pH and temperature for enzyme activity was found to be pH 7.2 and 50 °C respectively. Enzymatic activity of hirtin was significantly inhibited by PMSF and AEBSF. It showed higher specificity for synthetic substrate p-tos-GPRNA for thrombin. The CD spectra of hirtin showed a high content of β-sheets as compared to α-helix. The results indicate that hirtin is a thrombin-like serine protease and may have potential industrial and therapeutic applications., (Copyright © 2011 Elsevier Masson SAS. All rights reserved.)