Tropane Alkaloid Biosynthesis Research Articles

Assessment of metabolic changes in Acinetobacter johnsonii and Pseudomonas fluorescens co-culture from bigeye tuna (Thunnus obesus) spoilage by ultra-high-performance liquid chromatography-tandem mass spectrometry

Acinetobacter johnsonii and Pseudomonas fluorescens are specific spoilage microorganisms (SSO) of aquatic products, and their metabolites are effective indicators for analyzing these two SSOs. However, the metabolome of a coculture of A. johnsonii and P. fluorescens is still unclear. The aim of this study was to investigate the metabolomic changes in A. johnsonii, P. fluorescens, and their co-culture by screening for metabolic markers. PCA and PLS-DA revealed that A, P, and AP groups were different from each other, indicating a significantly varying metabolic profile among them. Based on UHPLC information, 540 metabolites with significant differences, were identified in the ESI+ and ESI- modes, which covered 79 metabolic pathways. The different metabolites were mainly related to the pathways, such as taurine and hypotaurine metabolism:M, bile secretion:OS, and arginine biosynthesis:M were in the A vs AP group, while PPAR signaling pathway:OS, tropane, piperidine, and pyridine alkaloid biosynthesis:M, longevity regulating pathway-worm:OS, choline metabolism in cancer:HD, biosynthesis of phenylpropanoids:M, and amoebiasis:HD were in the P vs AP group. Therefore, this study may provide significant information regarding the metabolic mechanisms of A. johnsonii, P. fluorescens, and their co-cultures, which may provide insights on their role in deteriorating the quality of aquatic products.

Engineering Tropane Alkaloid Production Based on Metabolic Characterization of Ornithine Decarboxylase in Atropa belladonna.

Ornithine decarboxylase (ODC) plays an important role in various biological processes; however, its role in plant secondary metabolism, especially in the biosynthesis of tropane alkaloids (TAs) such as pharmaceutical hyoscyamine, anisodamine, and scopolamine, remains largely unknown. In this study, we characterized the physiological and metabolic functions of the ODC gene of Atropa belladonna (AbODC) and determined its role in TA production using metabolic engineering approaches. Feeding assays with enzyme inhibitors indicated that ODC, rather than arginine decarboxylase (ADC), plays a major role in TA biosynthesis. Tissue-specific AbODC expression analysis and β-glucuronidase (GUS) staining assays showed that AbODC was highly expressed in secondary roots, especially in the cylinder tissue. Enzymatic assays indicated that AbODC was able to convert ornithine to putrescine, with the highest activity at pH 8.0 and 30 °C. Additionally, AbODC showed higher catalytic efficiency than other plant ODCs, as evident from the Km, Vmax, and Kcat values of AbODC using ornithine as the substrate. In A.belladonna root cultures, suppression of AbODC greatly reduced the production of putrescine, N-methylputrescine, and TAs, whereas overexpression of AbODC significantly increased the biosynthesis of putrescine, N-methylputrescine, hyoscyamine, and anisodamine. Moreover, transgenic A.belladonna plants overexpressing AbODC showed a significantly higher production of hyoscyamine and anisodamine compared with control plants. These findings indicate that AbODC plays a key role in TA biosynthesis and therefore is a valuable candidate for increasing TA production in A.belladonna.

Transcriptomic analysis of Eruca vesicaria subs. sativa lines with contrasting tolerance to polyethylene glycol-simulated drought stress

BackgroundEruca vesicaria subsp. sativa is one of the Cruciferae species most tolerant to drought stress. In our previous study some extremely drought-tolerant/sensitive Eruca lines were obtained. However little is known about the mechanism for drought tolerance in Eruca.MethodsIn this study two E. vesicaria subs. sativa lines with contrasting drought tolerance were treated with liquid MS/PEG solution. Total RNA was isolated from 7-day old whole seedlings and then applied to Illumina sequencing platform for high-throughput transcriptional sequencing.ResultsKEGG pathway analysis indicated that differentially expressed genes (DEGs) involved in alpha-Linolenic acid metabolism, Tyrosine metabolism, Phenylalanine, Tyrosine and tryptophan biosynthesis, Galactose metabolism, Isoquinoline alkaloid biosynthesis, Tropane, Piperidine and pyridine alkaloid biosynthesis, Mineral absorption, were all up-regulated specifically in drought-tolerant (DT) Eruca line under drought stress, while DEGs involved in ribosome, ribosome biogenesis, Pyrimidine metabolism, RNA degradation, Glyoxylate and dicarboxylate metabolism, Aminoacyl-tRNA biosynthesis, Citrate cycle, Methane metabolism, Carbon fixation in photosynthetic organisms, were all down-regulated. 51 DEGs were found to be most significantly up-regulated (log2 ratio ≥ 8) specifically in the DT line under PEG treatment, including those for ethylene-responsive transcription factors, WRKY and bHLH transcription factors, calmodulin-binding transcription activator, cysteine-rich receptor-like protein kinase, mitogen-activated protein kinase kinase, WD repeat-containing protein, OPDA reductase, allene oxide cyclase, aquaporin, O-acyltransferase WSD1, C-5 sterol desaturase, sugar transporter ERD6-like 12, trehalose-phosphate phosphatase and galactinol synthase 4. Eight of these 51 DEGs wre enriched in 8 COG and 17 KEGG pathways.ConclusionsDEGs that were found to be most significantly up-regulated specifically in the DT line under PEG treatment, up-regulation of DEGs involved in Arginine and proline metabolism, alpha-linolenic acid metabolism and down-regulation of carbon fixation and protein synthesis might be critical for the drought tolerance in Eruca. These results will be valuable for revealing mechanism of drought tolerance in Eruca and also for genetic engineering to improve drought tolerance in crops.

Tropane alkaloids biosynthesis involves an unusual type III polyketide synthase and non-enzymatic condensation

The skeleton of tropane alkaloids is derived from ornithine-derived N-methylpyrrolinium and two malonyl-CoA units. The enzymatic mechanism that connects N-methylpyrrolinium and malonyl-CoA units remains unknown. Here, we report the characterization of three pyrrolidine ketide synthases (PYKS), AaPYKS, DsPYKS, and AbPYKS, from three different hyoscyamine- and scopolamine-producing plants. By examining the crystal structure and biochemical activity of AaPYKS, we show that the reaction mechanism involves PYKS-mediated malonyl-CoA condensation to generate a 3-oxo-glutaric acid intermediate that can undergo non-enzymatic Mannich-like condensation with N-methylpyrrolinium to yield the racemic 4-(1-methyl-2-pyrrolidinyl)-3-oxobutanoic acid. This study therefore provides a long sought-after biosynthetic mechanism to explain condensation between N-methylpyrrolinium and acetate units and, more importantly, identifies an unusual plant type III polyketide synthase that can only catalyze one round of malonyl-CoA condensation.

Overexpression of the AbSAUR1 gene enhanced biomass production and alkaloid yield in Atropa belladonna

Tropane alkaloids (TAs), including hyoscyamine, anisodamine and scopolamine, are widely used as anticholinergic drugs in the clinic. At present, the production of TAs is completely dependent on extraction from medicinal plants of Solanaceae. Atropa belladonna, commonly known as deadly nightshade, is a major commercial source of TAs. Previous studies have focused on increasing TA content by overexpressing TA biosynthesis genes. However, research on increasing TA yield by increasing the biomass of A. belladonna has not been reported. In this study, a Small Auxin Up-regulated RNA (SAUR) gene from A. belladonna (AbSAUR1) had been cloned for the first time. Based on heat map and tissue expression analysis, it was demonstrated that AbSAUR1 was specifically expressed in roots. Compared to control plants, transgenic lines of A. belladonna overexpressing AbSAUR1 showed a number of phenotypic changes, including increased plant height, thickened stems, increased number of branches, leaves and roots. Meanwhile, the biomass of AbSAUR1-overexpressing plants also increased significantly, with fresh weight and dry weight being 3.79- and 3.20-fold higher than control plants, respectively. On average, there was no significant change in TA content, but the production of hyoscyamine, anisodamine and scopolamine was slightly enhanced. The overall yield of TAs was significantly improved, reaching 3.55 times that of the control. In summary, the AbSAUR1 gene can be employed as an effective molecular tool to increase the yield of TAs by increasing the biomass of A. belladonna.

Biochemical characterization reveals the functional divergence of two tropinone reductases from Przewalskia tangutica.

Przewalskia tangutica is a traditional medicinal plant from Tibet used for the analgesic effect from the tropane alkaloids (TAs) produced by the plant. Its roots have the highest yield of hyoscyamine in all plant species and so have been overharvested becoming an endangered medicinal plant species. Metabolic engineering is a good way to improve the yield of TAs in plants. In our study, two functionally distinct tropinone reductases genes, PtTRI and PtTRII, were cloned from P. tangutica and the functional divergence were characterized. The enzyme kinetics of PtTRI and PtTRII were investigated. The phylogenetic analysis classified them into different clades: PtTRI and PtTRII were in the clade of tropine-forming reductase and pseudotropine-forming reductase, respectively. We found PtTRI to be expressed in the roots but less in leaves, whereas PtTRII was expressed in the roots at higher levels than in the leaves. The kinetic parameters (Km , Vmax , and Kcat ) were analyzed using purified recombinant enzymes at their optimum pH. Enzymatic analysis results showed that tropinone is a better substrate for PtTRII compared with PtTRI, suggesting that PtTRII might be a potential gene target for TA biosynthesis engineering. Compared with the reported TRIs, PtTRI exhibited a higher affinity for tropinone.

Efficient Synthesis of Methyl (S)-4-(1-Methylpyrrolidin-2-yl)-3-oxobutanoate as the Key Intermediate for Tropane Alkaloid Biosynthesis with Optically Active Form

Efficient Synthesis of Methyl (S)-4-(1-Methylpyrrolidin-2-yl)-3-oxobutanoate as the Key Intermediate for Tropane Alkaloid Biosynthesis with Optically Active Form

A Phenylpyruvic Acid Reductase Is Required for Biosynthesis of Tropane Alkaloids.

Solanaceous medicinal plants produce tropane alkaloids (TAs). We discovered a novel gene from Atropa belladonna, AbPPAR, which encodes a phenylpyruvic acid reductase required for TA biosynthesis. AbPPAR was specifically expressed in root pericycles and endodermis. AbPPAR was shown to catalyze reduction of phenylpyruvic acid to phenyllactic acid, a precursor of TAs. Suppression of AbPPAR disrupted TA biosynthesis through reduction of phenyllactic acid levels. In summary, we identified a novel enzyme involved in TA biosynthesis.

Targeted expression of Vitreoscilla hemoglobin improves the production of tropane alkaloids in Hyoscyamus niger hairy roots

Under hypoxic conditions, the expression of Vitreoscilla hemoglobin (VHb) in plants is proposed to increase the productivity of certain oxygen-requiring metabolic pathways by promoting the delivery of oxygen. Tropane alkaloids (TAs) are a class of important plant secondary metabolites with significant medicinal value; the final step in their biosynthesis requires oxygen. Whether heterologous expression of VHb, especially in different subcellular compartments, can accelerate the accumulation of TAs is not known. Herein, the effect of heterologous expression of VHb in different subcellular locations on the TA profile of H. niger hairy roots was investigated. The targeted expression of VHb in the plastids (using pVHb-RecA construct), led to the accumulation of 197.68 μg/g hyoscyamine in the transgenic H. niger hairy roots, which was 1.25-fold of the content present in the lines in which VHb expression was not targeted, and 3.66-fold of that present in the wild type (WT) lines. The content of scopolamine was increased by 2.20- and 4.70-fold in the pVHb-RecA transgenic lines compared to that in the VHb transgenic and WT lines. Our results demonstrate that VHb could stimulate the accumulation of TAs in the transgenic H. niger hairy roots. Quantitative RT-PCR analysis revealed that the expression of key genes involved in TA biosynthesis increased significantly in the VHb transgenic lines. We present the first description of a highly efficient strategy to increase TA content in H. niger. Moreover, our results also shed light on how the production of desired metabolites can be efficiently enhanced by using more accurate and appropriate genetic engineering strategies.

Re-engineering the tropane alkaloid biosynthesis pathway in potato

Re-engineering the tropane alkaloid biosynthesis pathway in potato

Isolation, spectral characterization, molecular docking, and cytotoxic activity of alkaloids from Erythroxylum pungens O. E. Shulz

Stem bark, root bark, and leaf extracts of Erythroxylum pungens were subjected to phytochemical analysis. N,N-dimethyltryptamine (DMT) was isolated and characterized from E. pungens roots. This unprecedented result is remarkable since no indole alkaloid has been previously reported from Erythroxylaceae so far. Eleven known tropane alkaloids were identified by their mass spectra and 3-(2-methylbutyryloxy)tropan-6,7-diol as well as 3-(2-methylbutyryloxy)nortropan-6,7-diol were isolated and characterized based on mass spectrometry, 1H, 13C, COSY, and NOESY NMR analysis. The complete NMR data are reported for the first time. Inverse Structure-based and Ligand-Based virtual screening were carried out to identify possible targets for 3-(2-methylbutyryloxy)tropan-6,7-diol. The level of cytotoxicity of this tropane alkaloid aliphatic ester was discrete with potencies on the order of 0.3–1.0 mg/mL and better results against HeLa (50% cell viability reduction). Otherwise, atropine (0.3 mg/mL), a Solanaceae tropane alkaloid, and DMT (0.5 mg/mL) from E. pungens roots impaired at 50% the cell viability against HeLa, SiHa, PC3, and 786-0. This study stimulates scientific investigation of the impact of edaphoclimatic features in a semi-arid environment on tropane alkaloid biosynthesis.

Type III Polyketide Synthase Involved in Tropane and Granatane Alkaloid Biosynthesis

The lack of data concerning the enzymes and genes responsible for the production of tropane and granatane alkaloids has hindered endeavors to metabolically engineer important pharmacologically active specialized metabolites. It is important to understand how biochemical pathways perform when they originate from evolutionarily diverse families. It is hypothesized that a type III polyketide synthase enzyme is involved in the extension and subsequent cyclization of the second ring in the bicyclic tropane and grantane core structure. This represents a new class of type III polyketide synthases in which the heterocyclic starter molecule is charged and also lacks a Coenzyme A thioester. Putative polyketide synthase enzymes were identified in the Erythroxylum coca and Punica granatum cDNA transcriptome database and heterologously expressed in Pichia pastoris. LC‐MS/MS analysis has revealed the formation of a 4‐(1‐methyl‐2‐pyrrolidinyl)‐3‐oxobutanoate intermediate that is hypothesized to lead to the second ring formation. Understanding how evolutionary changes have occurred in type III polyketide synthases from different metabolic pathways that determine the development and expansion of chemical diversity to produce high valued compounds will be a powerful tool for bioengineering pharmaceuticals.Support or Funding InformationNational Science FoundationThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Metabolic characterization of Hyoscyamus niger root-specific putrescine N-methyltransferase

N-methylputrescine is the precursor of nicotine and pharmaceutical tropane alkaloids such as hyoscyamine. Putrescine N-methyltransferase (PMT) catalyzes the N-methylation of putrescine to form N-methylputrescine. While the role of PMT in nicotine biosynthesis is clear, knowledge of PMT in the biosynthesis of tropane alkaloids (TAs) and the regulation of polyamines remains limited. We characterized a PMT gene from Hyoscyamus niger, designated HnPMT that was specifically expressed in roots, especially in the secondary roots and dramatically induced by methyl jasmonate (MeJA). The GUS gene was specifically expressed in Arabidopsis roots or in the vascular tissues, including pericycles and endodermis, of the H. niger hairy root cultures, when it was driven by the 5′-flanking promoter region of HnPMT. The recombinant HnPMT was purified for enzymatic assays. HnPMT converted putrescine to form N-methylputrescine, as confirmed by LC-MS. The kinetics analysis revealed that HnPMT had high affinity with putrescine but low catalytic activity, suggesting that it was a rate-limiting enzyme. When HnPMT was suppressed in the H. niger plants by using the VIGS approach, the contents of N-methylputrescine and hyoscyamine were markedly decreased, but the contents of putrescine, spermidine and a mixture of spermine and thermospermine were significantly increased; this suggested that HnPMT was involved in the biosynthesis of tropane alkaloids and played a competent role in regulating the biosynthesis of polyamines. Functional identification of HnPMT facilitated the understanding of TA biosynthesis and thus implied that the HnPMT-catalyzed step might be a target for metabolic engineering of the TA production in H. niger.

The Desirability Optimization Methodology; a Tool to Predict Two Antagonist Responses in Biotechnological Systems: Case of Biomass Growth and Hyoscyamine Content in Elicited Datura starmonium Hairy Roots.

BackgroundThe use of the desirability function approach combined with the response surface methodology (RSM), also called Desirability Optimization Methodology (DOM), has been successfully applied to solve medical, chemical, and technological questions. It is particularly efficient for the determination of the optimal conditions in natural or industrial processes involving different factors leading to the antagonist responses.ObjectivesSurprisingly, DOM has never been applied to the research programs devoted to the study of plant responses to the complex environmental changes, and thus to biotechnological questions.Materials and MethodsIn this article, DOM is used to study the response of Datura stramonium hairy roots (HRs), obtained by genetic transformation with Agrobacterium rhizogenes A4 strain, subjected to the jasmonate treatments.ResultsAntagonist effects on the growth and tropane alkaloid biosynthesis are confirmed. With a limited number of experimental conditions, it is shown that 0.06 mM jasmonic acid (JA) applied for 24 h leads to an optimal compromise. Hyoscyamine levels increase by up to 290% after 24 h and this treatment does not significantly inhibit biomass growth.ConclusionsIt is thus demonstrated that the use of DOM can efficiently - with a minimized number of replicates - leads to the optimization of the biotechnological processes.

Localization and Organization of Scopolamine Biosynthesis in Duboisia myoporoides R. Br.

Tropane alkaloids (TAs), especially hyoscyamine and scopolamine, are important precursors for anticholinergic and antispasmodic drugs. Hyoscyamine and scopolamine are currently obtained at commercial scale from hybrid crosses of Duboisia myoporoides × Duboisia leichhardtii plants. In this study, we present a global investigation of the localization and organization of TA biosynthesis in a Duboisia myoporoides R. Br. wild-type line. The tissue-specific spatial distribution of TAs within D. myoporoides is presented, including quantification of the TAs littorine, 6-hydroxy hyoscyamine, hyoscyamine, scopolamine and, additionally, hyoscyamine aldehyde as well as scopolamine glucoside. Scopolamine (14.77 ± 5.03 mg g-1), and to a lesser extent hyoscyamine (3.01 ± 1.54 mg g-1) as well as 6-hydroxy hyoscyamine (4.35 ± 1.18 mg g-1), are accumulated in leaves during plant development, with the highest concentration of total TAs detected in 6-month-old plants. Littorine, an early precursor in TA biosynthesis, was present only in the roots (0.46 ± 0.07 mg g-1). During development, the spatial distribution of all investigated alkaloids changed due to secondary growth in the roots. Transcripts of pmt, tr-I and cyp80f1 genes, involved in early stages of TA biosynthesis, were found to be most abundant in the roots. In contrast, the transcript encoding hyoscyamine 6β-hydroxylase (h6h) was highest in the leaves of 3-month-old plants. This investigation presents the spatial distribution of biochemical components as well as gene expression profiles of genetic factors known to participate in TA biosynthesis in D. myoporoides. The results of this investigation may aid in future breeding or genetic enhancement strategies aimed at increasing the yields of TAs in these medicinally valuable plant species.

Enhancing Tropane Alkaloid Production Based on the Functional Identification of Tropine-Forming Reductase in Scopolia lurida, a Tibetan Medicinal Plant.

Scopolia lurida, a native herbal plant species in Tibet, is one of the most effective producers of tropane alkaloids. However, the tropane alkaloid biosynthesis in this plant species of interest has yet to be studied at the molecular, biochemical, and biotechnological level. Here, we report on the isolation and characterization of a putative short chain dehydrogenase (SDR) gene. Sequence analysis showed that SlTRI belonged to the SDR family. Phylogenetic analysis revealed that SlTRI was clustered with the tropine-forming reductases. SlTRI and the other TA-biosynthesis genes, including putrescine N-methyltransferase (SlPMT) and hyoscyamine 6β-hydroxylase (SlH6H), were preferably or exclusively expressed in the S. lurida roots. The tissue profile of SlTRI suggested that this gene might be involved in tropane alkaloid biosynthesis. By using GC-MS, SlTRI was shown to catalyze the tropinone reduction to yield tropine, the key intermediate of tropane alkaloids. With the purified recombinant SlTRI from Escherichia coli, an enzymatic assay was carried out; its result indicated that SlTRI was a tropine-forming reductase. Finally, the role of SlTRI in promoting the tropane alkaloid biosynthesis was confirmed through metabolic engineering in S. lurida. Specifically, hairy root cultures of S. lurida were established to investigate the effects of SlTRI overexpression on tropane alkaloid accumulation. In the SlTRI-overexpressing root cultures, the hyoscyamine contents were 1.7- to 2.9-fold higher than those in control while their corresponding scopolamine contents were likewise elevated. In summary, this functional identification of SlTRI has provided for a better understanding of tropane alkaloid biosynthesis. It also provides a candidate gene for enhancing tropane alkaloid biosynthesis in S. lurida via metabolic engineering.

Promoting scopolamine biosynthesis in transgenic Atropa belladonna plants with pmt and h6h overexpression under field conditions

Atropa belladonna is one of the most important plant sources for producing pharmaceutical tropane alkaloids (TAs). T1 progeny of transgenic A. belladonna, in which putrescine N-methyltransferase (EC. 2.1.1.53) from Nicotiana tabacum (NtPMT) and hyoscyamine 6β-hydroxylase (EC. 1.14.11.14) from Hyoscyamus niger (HnH6H) were overexpressed, were established to investigate TA biosynthesis and distribution in ripe fruits, leaves, stems, primary roots and secondary roots under field conditions. Both NtPMT and HnH6H were detected at the transcriptional level in transgenic plants, whereas they were not detected in wild-type plants. The transgenes did not influence the root-specific expression patterns of endogenous TA biosynthetic genes in A. belladonna. All four endogenous TA biosynthetic genes (AbPMT, AbTRI, AbCYP80F1 and AbH6H) had the highest/exclusive expression levels in secondary roots, suggesting that TAs were mainly synthesized in secondary roots. T1 progeny of transgenic A. belladonna showed an impressive scopolamine-rich chemotype that greatly improved the pharmaceutical value of A. belladonna. The higher efficiency of hyoscyamine conversion was found in aerial than in underground parts. In aerial parts of transgenic plants, hyoscyamine was totally converted to downstream alkaloids, especially scopolamine. Hyoscyamine, anisodamine and scopolamine were detected in underground parts, but scopolamine and anisodamine were more abundant than hyoscyamine. The exclusively higher levels of anisodamine in roots suggested that it might be difficult for its translocation from root to aerial organs. T1 progeny of transgenic A. belladonna, which produces scopolamine at very high levels (2.94–5.13 mg g−1) in field conditions, can provide more valuable plant materials for scopolamine production.

Activating antioxidant enzymes, hyoscyamine and scopolamine biosynthesis of Hyoscyamus niger L. plants with nano-sized titanium dioxide and bulk application

Application of nanotechnology is now widely distributed overall the life, especially in agricultural systems. This study intended to indicate the impacts of nano-sized titanium dioxide particles (NT) and bulk (BT) on antioxidant enzymes activities including superoxide dismutase (SOD), peroxidase (POX) and catalase (CAT), and variations of two major tropane alkaloids such as hyoscyamine (HYO) and scopolamine (SCO) in Hyoscyamus niger L. Plants were treated with different concentrations of NT and BT (0, 20, 40 and 80 mg l-1). Alkaloids extracted were identified by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) analysis. Results showed that SOD activity increased with increasing titanium dioxide concentration in both nano-particles and bulk treated plants. However, the highest and the lowest POX activity were observed in plants exposed to NT at 40 mg l-1 and control, respectively. Generally, all tested enzymes activities were higher in NT treated plants that those of BT except CAT activity at 80 mg l-1. The highest alkaloids content values, HYO: 0.286 g kg-1 and SCO: 0.126 g kg-1, were achieved in plants treated with NT at 80 and 20 mg l-1, respectively. The maximum and minimum plant biomass and subsequently total alkaloids yield were obtained in plants exposed to NT at 40 mg l-1 and controls, respectively. Our results suggest that NT in appropriate level (40 mg l-1) may act as an elicitor for biochemical responses and tropane alkaloids biosynthesis in H. niger plants.

Digital gene expression analysis of Microsporum canis exposed to berberine chloride.

Berberine, a natural isoquinoline alkaloid of many medicinal herbs, has an active function against a variety of microbial infections including Microsporum canis (M. canis). However, the underlying mechanisms are poorly understood. To study the effect of berberine chloride on M. canis infection, a Digital Gene Expression (DGE) tag profiling was constructed and a transcriptome analysis of the M. canis cellular responses upon berberine treatment was performed. Illimina/Hisseq sequencing technique was used to generate the data of gene expression profile, and the following enrichment analysis of Gene Ontology (GO) and Pathway function were conducted based on the data of transcriptome. The results of DGE showed that there were 8476945, 14256722, 7708575, 5669955, 6565513 and 9303468 tags respectively, which was obtained from M. canis incubated with berberine or control DMSO. 8,783 genes were totally mapped, and 1,890 genes have shown significant changes between the two groups. 1,030 genes were up-regulated and 860 genes were down-regulated (P<0.05) in berberine treated group compared to the control group. Besides, twenty-three GO terms were identified by Gene Ontology functional enrichment analysis, such as calcium-transporting ATPase activity, 2-oxoglutarate metabolic process, valine catabolic process, peroxisome and unfolded protein binding. Pathway significant enrichment analysis indicated 6 signaling pathways that are significant, including steroid biosynthesis, steroid hormone biosynthesis, Parkinson’s disease, 2,4-Dichlorobenzoate degradation, and tropane, piperidine and Isoquinoline alkaloid biosynthesis. Among these, eleven selected genes were further verified by qRT-PCR. Our findings provide a comprehensive view on the gene expression profile of M. canis upon berberine treatment, and shed light on its complicated effects on M. canis.

Expression Pattern of pmt, erf1 and jap1 Genes in Nicotiana benthamiana and Atropa belladonna Plants under UV Radiation, Wounding and Methyl Jasmonate Treatments

The Solanaceae plants produce a variety of interesting biologically active products including the steroid alkaloids solanidine, nicotine and tropane alkaloids. Putrescine N-methyltransferase (PMT) is an enzyme that catalyses s-adenosylmethionine-dependent methylation of putrescine in one of the primary steps of nicotine and tropane alkaloids biosynthesis pathway. Two tobacco members of the AP2/ERF-domain transcription factors family called NtORC1 and NtJAP1 were shown to up regulate the activity of the NtPMT promoter in Nicotiana tabacum L. under environmental stresses. In this study, semiquantitave RT-PCR analysis showed that expression patterns of PMT, ERF1 and JAP1 genes in shoots and roots of Nicotiana benthamiana Domin and Atropa belladonna L. were examined under methyl jasmonate, UV radiation and wounding treatments. Plants were harvested half an hour after each treatment. The expression pattern of examined genes showed differences between plant tissues under different treatments.