[Show abstract][Hide abstract] ABSTRACT: Magnolia officinalis Rehder et Wilson is a traditional Chinese herbal medicine that is used to treat various diseases such as neurosis, anxiety, and stroke. The main secondary metabolites in magnolia bark are phenolic compounds and terpenoids. Squalene synthase plays a significant role in catalyzing two farnesyl diphosphate molecules to form squalene, the first precursor of triterpenoid, phytosterol, and cholesterol biosynthesis. In this study, a full-length cDNA of squalene synthase was cloned from M. officinalis and designated MoSQS (GenBank accession no.KT223496). The gene contains a 1240-bp open reading frame and it encodes a protein with 409 amino acids. Bioinformatic and phylogenetic analysis clearly suggested that MoSQS shared high similarity with squalene synthases among other plants. Prokaryotic expression showed that a transmembrane domain-deleted (385-409 aa) MoSQS mutant (MoSQSΔTM) could be expressed in its soluble form in Escherichia coli Transetta (DE3). GC-MS analysis showed that squalene was detected in an in vitro reaction mixture. These results indicated that MoSQSΔTM was functional, thereby establishing an important foundation for the study of triterpenoid biosynthesis in M. officinalis.
No preview · Article · Dec 2015 · Protein Expression and Purification
[Show abstract][Hide abstract] ABSTRACT: Cytochromes P450 (CYPs) play a key role in generating the structural diversity of terpenoids, the largest group of plant natural products. However, functional characterization of CYPs has been challenging because of the expansive families found in plant genomes, diverse reactivity and inaccessibility of their substrates and products. Here we present the characterization of two CYPs, CYP76AH3 and CYP76AK1, which act sequentially to form a bifurcating pathway for the biosynthesis of tanshinones, the oxygenated diterpenoids from the Chinese medicinal plant Danshen (Salvia miltiorrhiza). These CYPs had similar transcription profiles to that of the known gene responsible for tanshinone production in elicited Danshen hairy roots. Biochemical and RNA interference studies demonstrated that both CYPs are promiscuous. CYP76AH3 oxidizes ferruginol at two different carbon centers, and CYP76AK1 hydroxylates C-20 of two of the resulting intermediates. Together, these convert ferruginol into 11,20-dihydroxy ferruginol and 11,20-dihydroxy sugiol en route to tanshinones. Moreover, we demonstrated the utility of these CYPs by engineering yeast for heterologous production of six oxygenated diterpenoids, which in turn enabled structural characterization of three novel compounds produced by CYP-mediated oxidation. Our results highlight the incorporation of multiple CYPs into diterpenoid metabolic engineering, and a continuing trend of CYP promiscuity generating complex networks in terpenoid biosynthesis.
[Show abstract][Hide abstract] ABSTRACT: Specific molecularly imprinted polymers for dencichine were developed for the first time in this study by the bulk polymerization using phenylpyruvic acid and dl-tyrosine as multi-templates. The photographs confirmed that molecularly imprinted polymers prepared using N,. N'-methylene diacrylamide as cross-linker and glycol dimethyl ether as porogen displayed excellent hydrophilicity. Selectivity, adsorption isotherm and adsorption kinetics were investigated. The sample loading-washing-eluting solvent was optimized to evaluate the property of molecularly imprinted solid phase extract. Compared with LC/WCX-SPE, water-compatible molecularly imprinted solid phase extraction displayed more excellent specific adsorption performance. The extracted dencichine from Panax notoginseng with the purity of 98.5% and the average recovery of 85.6% (n = 3) was obtained.
No preview · Article · Dec 2015 · Journal of chromatography. B, Analytical technologies in the biomedical and life sciences
[Show abstract][Hide abstract] ABSTRACT: Physicochemical and bio-functional properties of three starches including native starch (NS), the dual enzyme-treated starch (DES) and the cross-linked carboxymethyl starch (CCS) were investigated in the current study. The resistant starch (RS) contents ranged from 33.91 to 43.71%, which were decreased by modification. For the first time, the anti-constipation and hypolipidemic effect of yam starches are demonstrated. Three starches were characterized by scanning electron microscopy (SEM), X-ray powder diffraction (XRD) and FT-IR spectroscopy. DES's crystalline structure collapsed and lost its granular shape during modification, indicating that DES had a high water binding capacity and high solubility. However, only minor changes of CCS's granular surface and crystallinity were observed. Furthermore, anti-constipation effect of the three yam starches was evaluated by intestine propulsion and defecation in constipated mice. There was more short chain fatty acids (SCFAs) produced in colon with DES and CCS, than that with native starches. Therefore, the modified starches were proved to be better for improving the small intestine propulsion function by generating more SCFAs. Hypolipidemic effect was also evaluated by monitoring the triglyceride (TG) and total cholesterol (TC) levels in mice. This study reports new bioactive starches, with potential applications in the pharmaceutical and food industries.
No preview · Article · Dec 2015 · Food Hydrocolloids
[Show abstract][Hide abstract] ABSTRACT: Isopentenyl diphosphate isomerase (IPI) catalyzes the isomerization between the common terpene precursor substances isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) during the terpenoid biosynthesis process. In this study, tissue expression analysis revealed that the expression level of the Salvia miltiorrhiza IPI1 gene (SmIPI1) was higher in the leaves than in the roots and stems. Furthermore, color complementation and RNA interference methods were used to verify the function of the SmIPI1 gene from two aspects. A recombinant SmIPI1 plasmid was successfully constructed and transferred into engineered E. coli for validating the function of SmIPI1 through the color difference in comparison to the control group; the observed color difference indicated that SmIPI1 served in promoting the accumulation of lycopene. Transformant hairy root lines with RNA interference of SmIPI1 were successfully constructed mediated by Agrobacterium rhizogenes ACCC 10060. RNA interference hairy roots had a severe phenotype characterized by withering, deformity or even death. The mRNA expression level of SmIPI1 in the RSi3 root line was only 8.4% of that of the wild type. Furthermore the tanshinone content was too low to be detected in the RNA interference lines. These results suggest that SmIPI1 plays a critical role in terpenoid metabolic pathways. Addition of an exogenous SmIPI1 gene promoted metabolic flow toward the biosynthesis of carotenoids in E. coli, and SmIPI1 interference in S. miltiorrhiza hairy roots may cause interruption of the 2-C-methyl-D-erythritol-4-phosphate metabolic pathway.
[Show abstract][Hide abstract] ABSTRACT: A label-free, homogenous and sensitive one-step method for the molecular authentication of medicinal snakes has been developed by combining a rapid PCR technique with water-soluble cationic conjugated polyelectrolytes (CCPs). Three medicinal snake materials (Deinagkistrodon acutus, Zaocys dhumnades and Bungarus multicinctus; a total of 35 specimens) and 48 snake specimens with similar morphologies and textures were clearly distinguished by the naked eye by utilizing a CCP-based assay in a high-throughput manner. The identification of medicinal snakes in patented Chinese drugs was successfully performed using this detection system. In contrast to previous fluorescence-labeled oligonucleotide detection and direct DNA stain hybridization assays, this method does not require designing dye-labeled primers, and unfavorable dimer fluorescence is avoided in this homogenous method.
[Show abstract][Hide abstract] ABSTRACT: In this study, the first successfully developed superhydrophilic molecularly imprinted polymers (MIPs) for gastrodin recognition have been described. MIPs were prepared via the bulk polymerization process in an aqueous solution using alkenyl glycosides glucose (AGG) as the water-soluble functional monomer. The non-imprinted polymers (NIPs) were also synthesized using the same method without the use of the template. The dynamic water contact angles and photographs of the dispersion properties confirmed that the molecularly imprinted polymers displayed excellent superhydrophilicity. The results demonstrated that the MIPs exhibited high selectivity and an excellent imprinting effect. A molecularly imprinted solid phase extraction (MISPE) method was established. Optimization of various parameters affecting MISPE was investigated. Under the optimized conditions, a wide linear range (0.001-100.0μgmL(-1)) and low limits of detection (LOD) and quantification (LOQ) (0.03 and 0.09ngmL(-1), respectively) were achieved. When compared with the NIPs, higher recoveries (90.5% to 97.6%) of gastrodin with lower relative standard deviations values (below 6.4%) using high performance liquid chromatography were obtained at three spiked levels in three blank samples. These results demonstrated one efficient, highly selective and environmentally-friendly MISPE technique with excellent reproducibility for the purification and pre-concentration of gastrodin from an aqueous extract of Gastrodia elata roots.
No preview · Article · Nov 2015 · Journal of Chromatography A
[Show abstract][Hide abstract] ABSTRACT: 1-Deoxy-d-xylulose-5-phosphate synthase (DXS) and 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR) genes are the key enzyme genes of terpenoid biosynthesis but still unknown in Tripterygium wilfordii Hook. f. Here, three full-length cDNA encoding DXS1, DXS2 and DXR were cloned from suspension cells of T. wilfordii with ORF sizes of 2154 bp (TwDXS1, GenBank accession no.KM879187), 2148 bp (TwDXS2, GenBank accession no.KM879186), 1410 bp (TwDXR, GenBank accession no.KM879185). And, the TwDXS1, TwDXS2 and TwDXR were characterized by color complementation in lycopene accumulating strains of Escherichia coli, which indicated that they encoded functional proteins and promoted lycopene pathway flux. TwDXS1 and TwDXS2 are constitutively expressed in the roots, stems and leaves and the expression level showed an order of roots > stems > leaves. After the suspension cells were induced by methyl jasmonate, the mRNA expression level of TwDXS1, TwDXS2, and TwDXR increased, and triptophenolide was rapidly accumulated to 149.52 µg·g(-1), a 5.88-fold increase compared with the control. So the TwDXS1, TwDXS2, and TwDXR could be important genes involved in terpenoid biosynthesis in Tripterygium wilfordii Hook. f.
Preview · Article · Oct 2015 · International Journal of Molecular Sciences
[Show abstract][Hide abstract] ABSTRACT: The water-soluble phenolic acids of S. miltiorrhiza Bge. f. alba can protect against herbivores or pathogenic bacteria and have numerous pharmacological activities that are beneficial to human health. The main phenolic acids in S. miltiorrhiza, including rosmarinic acid and salvianolic acid B, are derived from the rosmarinic acid biosynthetic pathway, which has been partially characterized. However, little is known about the endogenous bHLH and MYB transcription factors that function in this pathway. In this paper, we describe the cloning and functional characterization of SmPAP1, a cDNA of the R2R3-MYB transcription factor isolated from S. miltiorrhiza Bge. f. alba. SmPAP1 contains an open reading frame of 645 bp in length and encodes a putative 214-amino acid protein. The deduced amino acid sequence of the N-terminal R2R3 repeat sequence of SmPAP1 shares high identity with other DNA-binding domains of plant MYB-type proteins. Expression studies have indicated that SmPAP1 is mainly expressed in leaves. The expression of SmPAP1 was induced by 0.1 mM methyl jasmonate, salicylic acid and abscisic acid in the hairy roots of S. miltiorrhiza Bge. f. alba. Yeast two-hybrid assays have demonstrated that SmPAP1 can interact with the bHLH factor SmMYC2 and heterologous AtMYC2, which are TFs known to induce anthocyanin synthesis in Arabidopsis. A transient co-expression experiment in tobacco leaves showed that SmPAP1 activated the promoters of two key rosmarinic acid biosynthetic pathway genes of S. miltiorrhiza, SmPAL1 (phenylalanine ammonia lyase) and SmC4H (cinnamic acid 4-hydroxylase). The overexpression of SmPAP1 induced the substantial accumulation of rosmarinic acid, salvianolic acid B, total phenolics and total flavonoids in the roots of transgenic S. miltiorrhiza Bge. f. alba. These data suggest that SmPAP1 is involved in the regulation of the phenolic acid biosynthetic pathway by interacting with bHLH TFs and activating genes encoding key enzymes in S. miltiorrhiza Bge. f. alba.
No preview · Article · Sep 2015 · Plant Cell Tissue and Organ Culture
[Show abstract][Hide abstract] ABSTRACT: Litsea cubeba is characterized by the presence of aporphine alkaloids. But few recent reports about the preparative separation of alkaloids from L. cubeba are found. The traditional separation method is time consuming and solvent consuming and irreversible adsorption is inevitable. In this research, pH-zone-refining counter-current chromatography and high-speed counter-current chromatography are applied to separate the alkaloids from a chloroform extract of L. cubeba. The crude extract was fractionated using the solvent system: chloroform-methanol-water (4:3:3, v/v) with different concentrations of hydrochloric acid (retainer) in the aqueous stationary phase and triethylamine (eluter) in the organic mobile phase to determine the ideal conditions for screening for the aporphine alkaloids. Using 1.5 g of the chloroform extract, 68.1 mg of norisocorydine (93.5% purity), 215.5 mg of isoboldine (96.3% purity), 612.3 mg of the mixture of boldine and laurotetanine, 108.8 mg of reticuline (97.4% purity) and 92.6 mg of laurolitsine (97.6% purity) were obtained with the selected conditions where 60 mM of hydrochloric acid was added to the stationary phase and 10 mM of triethylamine was used in the mobile phase. The mixture of boldine and laurotetanine was further separated using high-speed counter-current chromatography with a two-phase solvent system composed of ethyl acetate-methanol-water (4:1:5, v/v). Two alkaloids, laurotetanine (285.7 mg) and boldine (112.3 mg), were obtained from 500 mg of the mixture, in a one-step separation, with the relative purity of 94.8% and 96.2%, respectively. The purities of the isolated alkaloids were determined using high performance liquid chromatography and the chemical structures were confirmed using electrospray ionization-mass spectrometry, proton nuclear magnetic resonance (1H-NMR) and carbon-13 (13C)-NMR.
[Show abstract][Hide abstract] ABSTRACT: Rhizosphere and root-associated microbial communities are known to be related to soil-borne disease and plant health. In the present study, the microbial communities in rhizosphere soils and roots of both healthy and diseased Panax notoginseng were analyzed by high-throughput sequencing of 16S rRNA for bacteria and 18S rRNA internal transcribed spacer for fungi, to reveal the relationship of microbial community structure with plant health status. In total, 5593 bacterial operational taxonomic units (OTUs) and 963 fungal OTUs were identified in rhizosphere soils, while 1794 bacterial and 314 fungal OTUs were identified from root samples respectively. Principal coordinate analysis separated the microbial communities both in the rhizosphere soils and roots of diseased P. notoginseng from healthy plants. Compared to those of healthy P. notoginseng, microbial communities in rhizosphere soils and roots of diseased plants showed a decrease in alpha diversity. By contrast, bacterial community dissimilarity increased and fungal community dissimilarity decreased in rhizosphere soils of diseased plants, while both bacterial and fungal community dissimilarity in roots showed no significant difference between healthy and diseased plants. Redundancy analysis at the phylum level showed that mycorrhizal colonization and soil texture significantly affected microbial community composition in rhizosphere soils, whereas shoot nutrition status had a significant effect on microbial community composition in root samples. Our study provided strong evidence for the hypothesis that microbial diversity could potentially serve as an indicator for disease outbreak of medicinal plants, and supported the ecological significance of microbial communities in maintaining plant healthy and soil fertility.
Full-text · Article · Aug 2015 · Antonie van Leeuwenhoek
[Show abstract][Hide abstract] ABSTRACT: Tripterygium wilfordii Hook.F. is one of the most valuable medicinal plants because it contains a large variety of active terpenoid compounds, including triptolide, celastrol and wilforlide. All of the pharmacologically active secondary metabolites are synthesized from the 2-C-methyl-D-erythritol 4-phosphateand mevalonate pathway in the isoprenoid biosynthetic system. The key step in this pathway is the isomerization of dimethylallyl diphosphate and isopentenyl diphosphate, which is catalyzed by isopentenyl-diphosphate-isomerase (IPP). In the present study, a full-length cDNA encoding IPI (designate as TwIPI, GenBank accession no.KT279355) was cloned from a suspension of cultured cells from T. wilfordii. The full-length cDNA of TwIPI was 1564 bp and encoded a polypeptide of 288 amino acids. The bioinformatics analysis showed that the deduced TwIPI sequence contained the TNTCCSHPL and WGEHELDY motif. The transcription level of the TwIPI in the suspension cells increased almost five-fold after treatment with methyl jasmonate as an elicitor. A functional color assay in Escherichia coli indicated that TwIPI could promote the accumulation of lycopene and encoded a functional protein. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
No preview · Article · Aug 2015 · Biotechnology and Applied Biochemistry
[Show abstract][Hide abstract] ABSTRACT: Angelica sinensis (Apiaceae) is an endangered alpine herb that is widely used as a medicinal plant in traditional Chinese medicine (TCM). Wild populations of A. sinensis have become quite rare in China. Thus, population genetics studies of this species are urgently needed for its effective conservation and sustainable use. However, to date, no microsatellite loci have been isolated in A. sinensis. To address this issue, we isolated 18 polymorphic loci and genotyped 120 individuals collected from 6 populations. The number of alleles per locus ranged from 1.2 to 5.5, and the average was 2.4. The observed and expected heterozygosity per locus for a population varied, respectively, from 0.000 to 0.983 (averaged at 0.198) and from 0.066 to 0.661 (averaged at 0.333). Deviation from the HardyeWeinberg equilibrium (p < 0.01) was observed for 4 to 14 loci in various populations. These microsatellite markers were cross-amplified in 10 species affinis, and 7 loci were successfully amplified in all species. These microsatellite markers are useful for genetic studies, the conservation management of A. sinensis, and identification of A. sinensis.
[Show abstract][Hide abstract] ABSTRACT: DNA barcoding is a promising species identification method, but it has proved difficult to find a standardized DNA marker in plant. Although the ITS/ITS2 RNA transcript has been proposed as the core barcode for seed plants, it has been criticized for being too conserved in some species to provide enough information or too variable in some species to align it within the different taxa ranks. We selected 30 individuals, representing 16 species and four families, to explore whether ITS2 can successfully resolve species in terms of secondary structure. Secondary structure was predicted using Mfold software and sequence-structure was aligned by MARNA. RNAstat software transformed the secondary structures into 28 symbol code data for maximum parsimony (MP) analysis. The results showed that the ITS2 structures in our samples had a common four-helix folding type with some shared motifs. This conserved structure facilitated the alignment of ambiguous sequences from divergent families. The structure alignment yielded a MP tree, in which most topological relationships were congruent with the tree constructed using nucleotide sequence data. When the data was combined, we obtained a well-resolved and highly supported phylogeny, in which individuals of a same species were clustered together into a monophyletic group. As a result, the different species that are often referred to as the herb "Mu tong" were successfully identified using short fragments of 250 bp ITS2 sequences, together with their secondary structure. Thus our analysis strengthens the potential of ITS2 as a promising DNA barcode because it incorporates valuable secondary structure information that will help improve discrimination between species.
[Show abstract][Hide abstract] ABSTRACT: Tetrabutylphosphonium hydroxide (TBPH) aqueous solution, a novel ionic liquid that could dissolve cellulose rapidly at ambient temperature (25 °C), was used for the first time to develop an extraction method for salidroside from Rhodiola crenulata, used as the model sample, with infrared-assisted extraction (IRAE) in this paper. IRAE-TBPH procedures were optimized using a series of single-factor experiments and under the optimal conditions, the IRAE-TBPH technique not only took a shorter time (from 1.0 h to 8 min) but also afforded a higher extraction rate of salidroside from the herbs (increased by 15.41-38.65%) compared with other extraction techniques, such as TBPH-based heat reflux extraction (HRE-TBPH), ultrasound-assisted extraction (UAE-TBPH) and conventional solvent (methanol, ethanol and pure water) based IRAE. The results indicated IRAE-TBPH to be a fast and efficient extraction technique. Furthermore, the mechanism of IRAE-TBPH was preliminarily studied by means of surface structures and chemical compositions of samples before and after different extraction techniques. On the basis of the destruction of herb surface microstructures, cellulose dissolving property of TBPH and high efficiency heating of infrared irradiation in IRAE-TBPH process, the IRAE-TBPH technique eventually got the maximum yield value. Therefore, TBPH solution as a novel, effective and alternative solvent with higher extraction efficiency in the IRAE of active compounds from medicinal plants showed a great promising prospect.
[Show abstract][Hide abstract] ABSTRACT: Color variation in sea cucumber is one of the most crucial traits affecting price and taste in East Asian countries. However, the relationship and taxonomic status of the three color variants are still unclear. We used 14 samples that covered all three color variants and their geographic distributions, to construct the first phylogeny for the color variants based on the complete mitochondrial genome sequence and a number of tree-building methods (maximum parsimony (MP), maximum likelihood (ML), and Bayesian inference (BI)). The divergence times within color variants were estimated by the Bayesian molecular clock approach using the BEAST program. Our results showed that the color variants were not monophyletic in the well-resolved phylogenetic tree, which strongly refuted their separate species status. The molecular dating estimate revealed that the sea cucumber was a young group, which originated in the early Miocene period (22.03 mya) and rapidly diverged after the late Miocene period. It is interesting that individuals within each variant or geographic distribution were not always closely related and thus did not share a common origin. We propose that although they differ in body color, the three color morphs all belong to a single species of Apostichopus japonicus and the historical marine climate and the hydrographic complexity of the ocean currents could be responsible for their present distribution patterns.
No preview · Article · May 2015 · Mitochondrial DNA