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1.
Chinese Journal of Biotechnology ; (12): 2818-2838, 2023.
Article in Chinese | WPRIM | ID: wpr-981235

ABSTRACT

Phenylalanine ammonia-lyase (PAL) is the key entry enzyme of plant phenylpropanoid pathway. It plays an important role in the biosynthesis of podophyllotoxin, an anti-tumor lignan that is currently produced from its main natural source Sinopodophyllum hexandrum (Royle) Ying. In this study, we cloned the gene ShPAL encoding phenylalanine ammonia-lyase by RT-PCR from the root of S. hexandrum ecotype inhabited in the Aba' district, Sichuan, based on its public SRA transcriptome data-package. Bioinformatics analyses showed that the ShPAL-encoded protein is composed of 711 amino acids, contains the conserved domains of PAL, and has the signature motif within the active center of aromatic ammonia-lyases. Moreover, ShPAL protein was predicted to have a secondary structure mainly composed of α-helix and random coil, a typical 'seahorse' shape monomer tertiary structure, and a homologous tetramer three-dimensional structure by Swiss-Modelling. The phylogenetic lineage analysis indicated ShPAL was of the highest sequence identity and the shortest evolutionary distance with the PAL of Epimedium sagittatum from the same Berberidaceae family. Subcellular localization experiments showed that ShPAL protein was mainly distributed in the cytoplasm, despite of a minority on the endoplasmic reticulum membrane. Furthermore, ShPAL protein was recombinantly expressed in Escherichia coli and purified by histidine-tag affinity chromatography. Its enzymatic activity was determined up to 20.91 U/mg, with the optimum temperature of 41 ℃ and pH of 9.0. In contrast, the enzyme activity of its F130H mutant decreased by about 23.6%, yet with the same trends of change with temperature and pH, confirming that phenylalanine at this position does affect the substrate specificity of PAL. Both the wild type and the mutant have relatively poor thermostability, but good pH-stability. These results may help to further investigate the regulatory role of PAL in the process of podophyllotoxin biosynthesis and advance the heterologous synthesis of podophyllotoxin to protect the germplasm resource of S. hexandrum. They also demonstrate that ShPAL has a potential application in biochemical industry and biomedicine.


Subject(s)
Phenylalanine Ammonia-Lyase/metabolism , Podophyllotoxin , Phylogeny , Cloning, Molecular
2.
Chinese Journal of Natural Medicines (English Ed.) ; (6): 527-536, 2022.
Article in English | WPRIM | ID: wpr-939917

ABSTRACT

Phenylalanine ammonia-lyase (PAL), which catalyzes the conversion from L-phenylalanine to trans-cinnamic acid, is a well-known key enzyme and a connecting step between primary and secondary metabolisms in the phenylpropanoid biosynthetic pathway of plants and microbes. Schisandra chinensis, a woody vine plant belonging to the family of Magnoliaceae, is a rich source of dibenzocyclooctadiene lignans exhibiting potent activity. However, the functional role of PAL in the biosynthesis of lignan is relatively limited, compared with those in lignin and flavonoids biosynthesis. Therefore, it is essential to clone and characterize the PAL genes from this valuable medicinal plant. In this study, molecular cloning and characterization of three PAL genes (ScPAL1-3) from S. chinensis was carried out. ScPALs were cloned using RACE PCR. The sequence analysis of the three ScPALs was carried out to give basic characteristics followed by docking analysis. In order to determine their catalytic activity, recombinant protein was obtained by heterologous expression in pCold-TF vector in Escherichia coli (BL21-DE3), followed by Ni-affinity purification. The catalytic product of the purified recombinant proteins was verified using RP-HPLC through comparing with standard compounds. The optimal temperature, pH value and effects of different metal ions were determined. Vmax, Kcat and Km values were determined under the optimal conditions. The expression of three ScPALs in different tissues was also determined. Our work provided essential information for the function of ScPALs.


Subject(s)
Cloning, Molecular , Escherichia coli/metabolism , Phenylalanine/metabolism , Phenylalanine Ammonia-Lyase/chemistry , Recombinant Proteins , Schisandra/genetics
3.
China Journal of Chinese Materia Medica ; (24): 271-287, 2018.
Article in Chinese | WPRIM | ID: wpr-776390

ABSTRACT

The increasing demand of Chinese materia medica could not be supplied by wild resource, and the cultivated medicinal materials become popular, which led to decreased quality of many medicinal materials due to the difference of the circumstance between the wild and the cultivated. How to improve quality becomes key points of Chinese medicine resource. The leaves of Scutellaria baicalensis were sprayed with H₂O₂, the activities of superoxide dismutase (SOD) and catalase (CAT) changed little, but there had been a marked decrease of peroxidase (POD) and ascorbic oxidase (APX), which showed that the antioxidase system declined. Meanwhile, H₂O₂, as enhanced the expression of phenylalnine ammonialyase (PAL) and β-glucuronidase (GUS) as well as activity of PAL, promoted the biosynthesis and biotransformation of flavonoids. At the day 2 after treated, H₂O₂ of 0.004 μmol·L⁻¹ the contents of the baicalin and the wogonoside decreased slightly, but the contents of the baicalein and the wogonin increased significantly, the baicalein from 0.094% to 0.324%, the wogonin from 0.060% to 0.110%, i. e. increased 246% and 83.3%, respectively.


Subject(s)
Ascorbate Oxidase , Metabolism , Catalase , Metabolism , Drugs, Chinese Herbal , Chemistry , Flavanones , Flavonoids , Glucosides , Glucuronidase , Metabolism , Hydrogen Peroxide , Peroxidase , Metabolism , Phenylalanine Ammonia-Lyase , Metabolism , Scutellaria baicalensis , Metabolism , Secondary Metabolism , Superoxide Dismutase , Metabolism
4.
Electron. j. biotechnol ; 27: 63-69, May. 2017. graf
Article in English | LILACS | ID: biblio-1010394

ABSTRACT

Background: Defense-related anti-oxidative response is a vital defense mechanism of plants against pathogen invasion. Ralstonia solanacearum is an important phytopathogen. Bacterial wilt caused by R. solanacearum is the most destructive disease and causes severe losses in patchouli, an important aromatic and medicinal plant in Southeast Asia. The present study evaluated the defense response of patchouli inoculated with virulent R. solanacearum. Results: Results showed that the basic enzymatic activities differed not only between the leaves and stems but also between the upper and lower parts of the same organ of patchouli. POD, SOD, PPO, and PAL enzymatic activities were significantly elevated in leaves and stems from patchouli inoculated with R. solanacearum compared to those in control. The variation magnitude and rate of POD, PPO, and PAL activities were more obvious than those of SOD in patchouli inoculated with R. solanacearum. PAGE isoenzymatic analysis showed that there were one new POD band and two new SOD bands elicited, and at least two isoformic POD bands and two SOD bands were observably intensified compared to the corresponding control. Conclusion: Our results suggest that not only defense-related enzymatic activities were elevated but also the new isoenzymatic isoforms were induced in patchouli inoculated with R. solanacearum.


Subject(s)
Ralstonia solanacearum/pathogenicity , Pogostemon/enzymology , Pogostemon/microbiology , Phenylalanine Ammonia-Lyase/metabolism , Superoxide Dismutase/metabolism , Virulence , Catechol Oxidase/metabolism , Peroxidase/metabolism , Ralstonia solanacearum/physiology , Electrophoresis, Polyacrylamide Gel , Enzymes/immunology , Enzymes/metabolism , Native Polyacrylamide Gel Electrophoresis , Pogostemon/immunology , Antioxidants
5.
Chinese Journal of Natural Medicines (English Ed.) ; (6): 801-812, 2016.
Article in English | WPRIM | ID: wpr-812572

ABSTRACT

Phenolic compounds, metabolites of the phenylpropanoid pathway, play an important role in the growth and environmental adaptation of many plants. Phenylalanine ammonia-lyase (PAL) is the first key enzyme of the phenylpropanoid pathway. The present study was designed to investigate whether there is a multi-gene family in I. Indigotic and, if so, to characterize their properties. We conducted a comprehensive survey on the transcription profiling database by using tBLASTn analysis. Several bioinformatics methods were employed to perform the prediction of composition and physicochemical characters. The expression levels of IiPAL genes in various tissues of I. indigotica with stress treatment were examined by quantitative real-time PCR. Protoplast transient transformation was used to observe the locations of IiPALs. IiPALs were functionally characterized by expression with pET-32a vector in Escherichia colis strain BL21 (DE3). Integration of transcripts and metabolite accumulations was used to reveal the relation between IiPALs and target compounds. An new gene (IiPAL2) was identified and both IiPALs had the conserved enzymatic active site Ala-Ser-Gly and were classified as members of dicotyledon. IiPAL1 and IiPAL2 were expressed in roots, stems, leaves, and flowers, with the highest expression levels of IiPAL1 and IiPAL2 being observed in stems and roots, respectively. The two genes responded to the exogenous elicitor in different manners. Subcellular localization experiment showed that both IiPALs were localized in the cytosol. The recombinant proteins were shown to catalyze the conversion of L-Phe to trans-cinnamic acid. Correlation analysis indicated that IiPAL1 was more close to the biosynthesis of secondary metabolites than IiPAL2. In conclusion, the present study provides a basis for the elucidation of the role of IiPALs genes in the biosynthesis of phenolic compounds, which will help further metabolic engineering to improve the accumulation of bioactive components in I. indigotica.


Subject(s)
Amino Acid Sequence , Base Sequence , Cloning, Molecular , Gene Expression Regulation, Plant , Isatis , Genetics , Molecular Sequence Data , Multigene Family , Phenylalanine Ammonia-Lyase , Chemistry , Genetics , Metabolism , Phylogeny , Plant Proteins , Chemistry , Genetics , Metabolism , Sequence Alignment
6.
Chinese Journal of Biotechnology ; (12): 222-230, 2016.
Article in Chinese | WPRIM | ID: wpr-242299

ABSTRACT

To investigate roles of nitric oxide (NO) signal in accumulations of phenolic acids in abscisic.acid (ABA)-induced Salvia miltiorrhiza hairy roots, S. miltiorrhiza hairy roots were treated with different concentrations of sodium nitroprusside (SNP)-an exogenous NO donor, for 6 days, and contents of phenolic acids in the hairy roots are determined. Then with treatment of ABA and NO scavenger (2-(4-carboxy-2-phenyl)-4,4,5,5-tetramethylimidazoline-1- oxyl-3-oxide, c-PTIO) or NO synthase inhibitor (NG-nitro-L-arginine methyl ester, L-NAME), contents of phenolic acids and expression levels of three key genes involved in phenolic acids biosynthesis were detected. Phenolic acids production in S. miltiorrhiza hairy roots was most significantly improved by 100 µmoL/L SNP. Contents of RA and salvianolic acid B increased by 3 and 4 folds. ABA significantly improved transcript levels of PAL (phenylalanine ammonia lyase), TAT (tyrosine aminotransferase) and RAS (rosmarinic acid synthase), and increased phenolic acids accumulations. However, with treatments of ABA+c-PTIO or ABA+L-NAME, accumulations of phenolic acids and expression levels of the three key genes were significantly inhibited. Both NO and ABA can increase accumulations of phenolic acids in S. miltiorrhiza hairy roots. NO signal probably mediates the ABA-induced phenolic acids production.


Subject(s)
Abscisic Acid , Pharmacology , Benzofurans , Metabolism , Free Radical Scavengers , Pharmacology , Hydroxybenzoates , Metabolism , Nitric Oxide , Metabolism , Phenylalanine Ammonia-Lyase , Metabolism , Plant Roots , Metabolism , Salvia miltiorrhiza , Metabolism , Tyrosine Transaminase , Metabolism
7.
Braz. j. microbiol ; 46(3): 701-706, July-Sept. 2015. tab
Article in English | LILACS | ID: lil-755833

ABSTRACT

The bacterial spot of tomato, caused by Xanthomonas spp., is a very important disease, especially in the hot and humid periods of the year. The chemical control of the disease has not been very effective for a number of reasons. This study aimed to evaluate, under greenhouse conditions, the efficacy of leaf-spraying chemicals (acibenzolar-S-methyl (ASM) (0.025 g.L−1), fluazinam (0.25 g.L−1), pyraclostrobin (0.08 g.L−1), pyraclostrobin + methiran (0.02 g.L−1 + 2.2 g.L−1), copper oxychloride (1.50 g.L−1), mancozeb + copper oxychloride (0.88 g.L−1 + 0.60 g.L−1), and oxytetracycline (0.40 g.L−1)) on control of bacterial spot. Tomatoes Santa Clara and Gisele cultivars were pulverized 3 days before inoculation with Xanthomonas perforans. The production of enzymes associated with resistance induction (peroxidase, polyphenol oxidase, phenylalanine ammonia-lyase, β-1,3-glucanase, and protease) was quantified from leaf samples collected 24 hours before and 24 hours after chemical spraying and at 1, 2, 4, 6, and 8 days after bacterial inoculation. All products tested controlled bacterial spot, but only ASM, pyraclostrobin, and pyraclostrobin + metiram increased the production of peroxidase in the leaves of the two tomato cultivars, and increased the production of polyphenol oxidase and β-1,3-glucanase in the Santa Clara cultivar.

.


Subject(s)
Disease Resistance/drug effects , Fungicides, Industrial/pharmacology , Solanum lycopersicum/microbiology , Plant Diseases/microbiology , Xanthomonas/growth & development , Catechol Oxidase/metabolism , /metabolism , Solanum lycopersicum/enzymology , Solanum lycopersicum/immunology , Peptide Hydrolases/metabolism , Peroxidase/metabolism , Phenylalanine Ammonia-Lyase/metabolism , Plant Diseases/immunology , Xanthomonas/drug effects
8.
Rev. bras. plantas med ; 17(1): 143-149, Jan-Mar/2015. tab, graf
Article in Portuguese | LILACS | ID: lil-742930

ABSTRACT

Foram desenvolvidos dois experimentos com objetivo de avaliar o potencial de preparados de cavalinha (Equisetum sp.) na síntese de metabólitos de defesa em cotilédones de soja (Glycinemax L.) e o efeito sobre o crescimento de Rhizoctonia solani, in vitro. O delineamento experimental utilizado para os experimentos foi inteiramente casualizado em esquema fatorial 3x5 (formas de extração x concentrações), com quatro repetições. As formas de extração foram extrato alcoólico, infusão e maceração, nas concentrações de zero; 1; 10, 20 e 40%. No primeiro experimento foi avaliada a indução de compostos de defesa vegetal em cotilédones de soja em resposta aos derivados a base de cavalinha, sendo quantificada a atividade da enzima fenilalanina amônia-liase (FAL), via espectofotometria, a fitoalexina gliceolina, e o teor de fenóis totais. No segundo experimento, in vitro, a unidade experimental foi uma placa de Petri, sendo os preparados de cavalinha incorporados ao meio BDA (Batata-dextrose e Agar) e avaliado o crescimento micelial de R. Solani. Os preparados de extrato alcoólico, infusão e maceração de cavalinha apresentaram capacidade de indução das fitoalexinas gliceolinas em cotilédones de soja, bem como, ativaram o metabolismo de compostos fenólicos. Entre os preparados, o extrato alcoólico e a maceração, se sobressaem sobre a infusão. Os preparados de extrato alcoólico, infusão e maceração de cavalinha em todas as suas concentrações inibem o crescimento do fungo R. solani, in vitro. .


Two experiments were carried out in the Federal Technological University of Paraná - Dois Vizinhos Campus - with the aim to evaluate the potential of horsetail (Equisetum sp.) derivatives for the synthesis of defense metabolites in soybean (Glycine max L.) cotyledons and their effect on the in vitro growth of Rhizoctonia solani. The experimental design was completely randomized in a 3 x 5 factorial design (extraction form x concentration), with four replications. The extraction forms were alcoholic extract, infusion and maceration and the concentrations tested were zero, 1, 10, 20 and 40%. In the first experiment, we evaluated the induction of plant defense in soybean cotyledons as a response to horsetail derivatives through spectrophotometry according to phytoalexin glyceollin, phenylalanine ammonia lyase enzyme activity (PAL) and total phenols. In the second experiment, in vitro, the experimental unit was a Petri dish, and the horsetail derivatives were incorporated into medium culture (potato dextrose agar), and we evaluated the mycelial growth of R. solani. The alcoholic extract, infusion and maceration of horsetail derivatives presented phytoalexin glyceolin induction in soybean cotyledons, in addition to activating the metabolism of phenolic compounds. Among the derivatives, the alcoholic extract and the maceration form of extraction were superior in relation to the infusion. The alcoholic extract, infusion and maceration of horsetail derivatives inhibited the in vitro growth of R. solani in all concentrations.


Subject(s)
Rhizoctonia/classification , Glycine max/classification , Cotyledon/classification , Equisetum/physiology , Metabolism , Phenylalanine Ammonia-Lyase/chemical synthesis
9.
Mycobiology ; : 327-332, 2015.
Article in English | WPRIM | ID: wpr-729629

ABSTRACT

Phenylalanine ammonia-lyase (PAL) gene is known to be expressed in plants, and is involved in the differentiation, growth and synthesis of secondary metabolites. However, its expression in fungi remains to be explored. To understand its expression in mushroom fungi, the PAL gene of the edible mushroom Flammulina velutipes (Fvpal) was cloned and characterized. The cloned Fvpal consists of 2,175 bp, coding for a polypeptide containing 724 amino acids and having 11 introns. The translated amino acid sequence of Fvpal shares a high identity (66%) with that of ectomycorrhizal fungus Tricholoma matsutake. Distinctively, the Fvpal expression in the mycelium was higher in minimal medium supplemented with L-tyrosine than with other aromatic amino acids. During cultivation of the mushroom on sawdust medium, Fvpal expression in the fruit body correspondingly increased as the mushroom grew. In the fruiting body, Fvpal was expressed more in the stipe than in the pileus. These results suggest that F. velutipes PAL activity differs in the different organs of the mushroom. Overall, this is first report to show that the PAL gene expression is associated with mushroom growth in fungi.


Subject(s)
Agaricales , Amino Acid Sequence , Amino Acids , Amino Acids, Aromatic , Clinical Coding , Clone Cells , Cloning, Organism , Flammulina , Fruit , Fungi , Gene Expression , Introns , Mycelium , Phenylalanine Ammonia-Lyase , Phenylalanine , Tricholoma , Tyrosine
10.
Chinese Journal of Biotechnology ; (12): 1219-1229, 2015.
Article in Chinese | WPRIM | ID: wpr-240562

ABSTRACT

We analyzed the best light intensity for callus induction and maintenance in Vitis vinifera and explored the mechanism of grape callus browning. Tender stem segments of grape cultivar "gold finger" were used to study the effects of different light intensities (0, 500, 1 000, 1 500, 2 000, 2 500, 3 000 and 4 000 Lx) on the induction rate, browning rate and associated enzyme activity and gene expression during Vitis vinifera callus formation. The callus induction rate under 0, 500, 1 000 and 1 500 Lx was more than 92%, significantly higher than in other treatments (P < 0.05). A lower browning rate and better callus growth were also observed during subculture under 1 000 and 1 500 Lx treatments. We found that chlorogenic acid, caffeic acid, p-hydroxybenzoic acid and coumaric acid contents were correlated with the browning rate of callus, among which chlorogenic acid content was positively correlated with the browning rate (P < 0.05). Peroxidase (POD) and polyphenol oxidase (PPO) activities were negatively correlated with the browning rate of callus (P < 0.01). The POD, PPO and phenylalanine ammonialyase (PAL) expression levels were positively correlated with the browning rate at P < 0.05 or P < 0.01. An appropriate light intensity for the tissue culture of Vitis vinifera was 1 000-1 500 Lx, higher or lower light intensities significantly impaired normal callus growth.


Subject(s)
Caffeic Acids , Chemistry , Catechol Oxidase , Chemistry , Culture Media , Chemistry , Gene Expression Regulation, Plant , Light , Peroxidase , Metabolism , Phenylalanine Ammonia-Lyase , Metabolism , Plant Stems , Radiation Effects , Tissue Culture Techniques , Vitis , Radiation Effects
11.
Electron. j. biotechnol ; 17(4): 156-161, July 2014. graf, tab
Article in English | LILACS | ID: lil-719106

ABSTRACT

Background Three oligosaccharides (EOS, WOS and SOS) were respectively prepared from the corresponding polysaccharides, namely exopolysaccharide (EPS), water-extracted mycelial polysaccharide (WPS) and sodium hydroxide-extracted mycelial polysaccharides (SPS) from the endophytic fungus Fusarium oxysporum Dzf17. In this study, the effects of EOS, WOS and SOS on the activities of the defense-related enzymes, namely phenylalanine ammonia lyase (PAL), polyphenoloxidase (PPO) and peroxidase (POD) in its host plant Dioscorea zingiberensis cultures were investigated. Results For the suspension cell cultures of D. zingiberensis, the highest PAL activity was induced by 0.5 mg/mL of WOS at 48 h after treatment, which was 4.55-fold as that of control. Both PPO and POD activities were increased to the maximum values by 0.25 mg/mL of WOS at 48 h after treatment, which were respectively 3.74 and 3.45-fold as those of control. For the seedling cultures, the highest PAL activity was elicited by 2.5 mg/mL of EOS at 48 h after treatment, which was 3.62-fold as that of control. Both PPO and POD reached their maximum values treated with 2.5 mg/mL of WOS at 48 h after treatment, which were 4.61 and 4.19-fold as those of control, separately. Conclusions Both EOS and WOS significantly increased the activities of PAL, PPO and POD in the suspension cell and seedling cultures of D. zingiberensis. The results suggested that the oligosaccharides from the endophytic fungus F. oxysporum Dzf17 may be related to the activation and enhancement of the defensive mechanisms of D. zingiberensis suspension cell and seedling cultures.


Subject(s)
Oligosaccharides/metabolism , Phenylalanine Ammonia-Lyase/metabolism , Catechol Oxidase/metabolism , Peroxidase/metabolism , Endophytes , Fusarium , Polysaccharides , Suspensions , Cell Culture Techniques , Dioscorea , Plant Cells , Disease Resistance
12.
Indian J Exp Biol ; 2014 Mar; 52(3): 252-260
Article in English | IMSEAR | ID: sea-150355

ABSTRACT

Activity differences of the first (phenylalanine ammonia lyase, PAL) and the last (cinnamyl alcohol dehydrogenase, CAD) enzymes of phenylpropanoid pathway in the roots of resistant (Yangambi Km5 and Anaikomban) and susceptible (Nendran and Robusta) banana cultivars caused by root lesion nematode, Pratylenchus coffeae, were investigated. Also, the accumulation of phenolics and deposition of lignin polymers in cell walls in relation to resistance of the banana cultivars to the nematode were analyzed. Compared to the susceptible cultivars, the resistant cultivars had constitutively significantly higher PAL activity and total soluble and cell wall-bound phenolics than in susceptible cultivars. The resistant cultivars responded strongly to the infection of the nematode by induction of several-time higher PAL and CAD enzymes activities, soluble and wall-bound phenolics and enrichment of lignin polymers in cell wall and these biochemical parameters reached maximum at 7th day postinoculation. In addition, profiles of phenolic acid metabolites in roots of Yangambi Km5 and Nendran were analyzed by HPLC to ascertain the underlying biochemical mechanism of bananas resistance to the nematode. Identification and quantification of soluble and cell wall-bound phenolic acids showed six metabolites and only quantitative, no qualitative, differences occurred between the resistant and susceptible cvs. and between constitutive and induced contents. A very prominent increase of p-coumaric, ferulic and sinapic acids, which are precursors of monolignols of lignin, in resistant cv. was found. These constitutive and induced biochemical alterations are definitely the chemical defenses of resistant cvs. to the nematode infection.


Subject(s)
Alcohol Oxidoreductases/genetics , Alcohol Oxidoreductases/metabolism , Animals , Disease Resistance/genetics , Metabolic Networks and Pathways , Musa/enzymology , Musa/genetics , Musa/growth & development , Musa/parasitology , Nematoda/genetics , Nematoda/pathogenicity , Phenols/chemistry , Phenols/metabolism , Phenylalanine Ammonia-Lyase/genetics , Phenylalanine Ammonia-Lyase/metabolism , Plant Diseases/genetics , Plant Diseases/parasitology , Plant Roots/enzymology , Plant Roots/metabolism , Polymers/chemistry , Propanols/chemistry , Propanols/metabolism
13.
Acta Pharmaceutica Sinica ; (12): 1498-1502, 2013.
Article in Chinese | WPRIM | ID: wpr-298052

ABSTRACT

The expression of phenylalnine ammonia lyase (LJPAL1) is closely related to the content of active compounds in Lonicera japonica. In this paper, a prokaryotic expression vector is constructed and LJPAL1 protein is expressed in E. coli. Three antigen sites were synthesized to peptide antigen and prepared polyclonal antibody of Anti-LJT-1, Anti-LJT-2 and Anti-LJT-3, separately. Antibody Anti-LJT-2 was screened using Western blotting. And indirect ELISA was built using Anti-LJT-2. The results of this study will be a base for honeysuckle chemical quality and evaluation kits.


Subject(s)
Antibodies , Allergy and Immunology , Metabolism , Antibody Specificity , Blotting, Western , Enzyme-Linked Immunosorbent Assay , Escherichia coli , Genetics , Metabolism , Flowers , Chemistry , Gene Expression Regulation, Plant , Genetic Vectors , Lonicera , Chemistry , Phenylalanine Ammonia-Lyase , Chemistry , Allergy and Immunology , Plants, Medicinal , Chemistry , Plasmids , Protein Structure, Secondary , Protein Structure, Tertiary
14.
IJB-Iranian Journal of Biotechnology. 2012; 10 (1): 32-39
in English | IMEMR | ID: emr-122604

ABSTRACT

Phenylalanine amonia-lyase [PAL] is one of the most important enzymes that plays a key role in regulation of phenylpropanoid production in plants. It catalyzes the first step of the phenylpropanoid pathway in which L-phenylalanine is deaminated to trans-cinnamic acid. This step is significant for metabolic engineering and hyper-expression of the major phenylpropanoid, methyl chavicol. We followed gene expression and activity of PAL in Ocimum basilicum L. at different stages of growth including seedling, beginning and middle of growth phase, budding stage and flowering, and their correlation with final concentration of phenylpropanoid compounds. The level of gene expression was monitored by semi quantitative RT-PCR and phenylpropanoid compounds were identified by gas chromatography/mass spectrometry [GC/MS]. PAL activity was assayed using spectrophotometer. The results indicated that the level of gene expression and activity of PAL enzyme are altered during the plant development, where the highest expression and activity [0.851 [micromol cinnamic acid/mg/min] was achieved at budding stage. In this experiment, changes of methylchavicol content were correlated to the transcription and activity of PAL enzyme


Subject(s)
Gene Expression , Phenylalanine Ammonia-Lyase , Oils, Volatile , Anisoles , Seedlings , Reverse Transcriptase Polymerase Chain Reaction , Gas Chromatography-Mass Spectrometry , Spectrophotometry
15.
Chinese Journal of Biotechnology ; (12): 1359-1369, 2012.
Article in Chinese | WPRIM | ID: wpr-342390

ABSTRACT

We studied the influence of the concentration of Ca2+ (0-50 mmol/L) in culture medium on the synthesis of rosmarinic acid (RA) and related enzymes in Salvia miltiorrhiza suspension cultures. Using verpamil (VP, a calcium channel antagonist) and ionophore A23187, we studied the mechanism of secondary metabolites of Salvia miltiorrhiza suspension cultures influenced by the concentration of Ca2+ in the culture medium. The synthesis of intracellular RA in 6-day incubation was significantly dependent on the medium Ca2+ concentration. At the optimal Ca2+ concentration of 10 mmol/L, a maximal RA content of 20.149 mg/g biomass dry weight was reached, which was about 37.3% and 20.4% higher than that at Ca2+ concentrations of 1 and 3 mmol/L, respectively. The variation of the activity of PAL and TAT, two key enzymes of the two branches of RA, could be affected by the concentration of Ca2+ in culture medium. The change of their activity occurred prior to the accumulation of RA, which suggested both of the key enzymes be involved in the synthesis of RA. Meanwhile, the enzymatic action of PAL was more distinct than TAT. The treatment of VP and A23187, respectively, indicated that the influence of RA affected by the concentration of Ca2+ in the culture medium was accomplished by the intracellular Ca2+, and the flow of Ca2+ from the extracellular to the intracellular environment could also participate in this process.


Subject(s)
Calcium , Pharmacology , Cinnamates , Metabolism , Culture Media , Culture Techniques , Methods , Depsides , Metabolism , Phenylalanine Ammonia-Lyase , Metabolism , Salvia miltiorrhiza , Chemistry , Tyrosine Transaminase , Metabolism
16.
China Journal of Chinese Materia Medica ; (24): 3793-3798, 2012.
Article in Chinese | WPRIM | ID: wpr-346836

ABSTRACT

<p><b>OBJECTIVE</b>To study the developmental phase on the growth and active compounds in Scutellaria baicalensis.</p><p><b>METHOD</b>Seeds of wild plants were collected from Laiwu and sowed in Fangshan (Beijing) and Laiwu (Shandong). Samples of aerial and underground parts were collected in five growth periods of sprouts, seedlings, flowering, seed drop and withered periods respectively. The length of taproot, fresh weight of root, diameter of taproot and the length of stem were determined. The content of active compounds and total flavonoids were determined by HPLC and ultraviolet spectrophotometry respectively. The transcripted level of PAL1, PAL2, PAL3, C4H, 4CL, CHS, GUS and UBGAT were analyzed with RT-PCR.</p><p><b>RESULT</b>The results showed that the aerial part of S. baicalensis grew quickly before flowering stage, and the underground part grew mostly between the periods of flowering and withered. In the whole growing developmental periods, the content of total flavonoids was not changed significantly, the content of baicalin was increased gradually and the content of baicalein was decreased gradually. Expression level of PAL and 4CL was the highest in withered period, CHS was increased between flowering and seed drop and decreased in withered period.</p><p><b>CONCLUSION</b>Seedlings and withered periods may be the key phase affecting the growth and active compounds in S. baicalensis.</p>


Subject(s)
Acyltransferases , Genetics , Metabolism , Chromatography, High Pressure Liquid , Coenzyme A Ligases , Genetics , Metabolism , Flavonoids , Metabolism , Flowers , Genetics , Metabolism , Gene Expression Regulation, Developmental , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Plant , Glucuronidase , Genetics , Metabolism , Glucuronosyltransferase , Genetics , Metabolism , Phenylalanine Ammonia-Lyase , Genetics , Metabolism , Plant Proteins , Genetics , Metabolism , Plant Roots , Genetics , Metabolism , Plant Stems , Genetics , Metabolism , Reverse Transcriptase Polymerase Chain Reaction , Scutellaria baicalensis , Genetics , Metabolism , Seedlings , Genetics , Metabolism , Spectrophotometry, Ultraviolet , Time Factors , Trans-Cinnamate 4-Monooxygenase , Genetics , Metabolism
17.
China Journal of Chinese Materia Medica ; (24): 754-759, 2012.
Article in Chinese | WPRIM | ID: wpr-288712

ABSTRACT

<p><b>OBJECTIVE</b>To study the function of ABA and fluridone on the contents of penolic acids and two key synthetases (PAL and TAT).</p><p><b>METHOD</b>Conducted 4 different concentrations in the hairy root of Salvia miltiorrhiza after culturing 18 days and treated with fluridone. One day later, harvested the hairy root and measured the activity of PAL and TAT; Treatment for 6 days, gathered and determined the contents of phenolic acids.</p><p><b>RESULT</b>In certain concentration of ABA, lower ABA could induced the production of growth and higher ABA inhibitor the growth in hairy roots of S. miltiorrhiza; ABA induced the accumulation of caffeic acid considerably, and the effect on the contents of coffee acid show positive correlation; As for the RA and LAB, the low dosage of ABA simulated the production and higher ABA inhibited the production of them; the ABA biosynthetic inhibitor fluridone can decreases ABA's the effect; The different of ABA activated the activity of PAL and TAT, but the impact were discriminating, when treatment with ABA and fluridone, the inducing were declined.</p><p><b>CONCLUSION</b>ABA induced the accumulation of.</p>


Subject(s)
Abscisic Acid , Metabolism , Pharmacology , Antioxidants , Metabolism , Biomass , Caffeic Acids , Metabolism , Herbicides , Pharmacology , Hydroxybenzoates , Metabolism , Medicine, Chinese Traditional , Phenylalanine Ammonia-Lyase , Metabolism , Plant Roots , Pyridones , Pharmacology , Salvia miltiorrhiza , Time Factors , Tyrosine Transaminase , Metabolism
18.
Chinese Journal of Biotechnology ; (12): 320-328, 2012.
Article in Chinese | WPRIM | ID: wpr-304490

ABSTRACT

Rosmarinic acid (RA), a phenolic acid, is one of the important secondary metabolites produced in Salvia miltiorrhiza. To observe the influence of salicylic acid (SA), an elicitor, on the synthesis of RA and related enzymes, we treated the cell suspension cultures of S. miltiorrhiza with SA and L-a-aminooxy-beta-phenylpropionic acid (AOPP), a competitive inhibitor of tyrosine aminotransferase (TAT). Under this condition, the activities of related enzymes, such as phenylalanine ammonia-lyase and TAT were traced and assayed; the accumulative amount of RA was measured. The results showed that the PAL activity reached the peak at 4 h, 124% higher than that of the control, and the content of RA reached its maximum ((5.914 +/- 0.296) mg/g dry weight) at 8 h, after treated by 6.25 mg/L SA on day 6 of the suspension culture. The results of treatment with 0.1 micromol/L AOPP showed that AOPP affected little on the TAT activity, while the PAL activity was significantly influenced, with 44% lower than that of the control at 6 h. Meanwhile, the reduced accumulation of RA ((4.709 +/- 0.204) mg/g dry weight) paralleled with the decrease in PAL activity. The co-treatment by 0.1 micromol/L AOPP and 6.25 mg/L SA relieved the restriction imposed by AOPP on PAL, and made the cell cultures accumulate more RA than sole treatment with AOPP, indicated that SA induced the accumulation of RA in suspension cell culture of S. miltiorrhiza, and the rate-limiting effect of PAL was stronger than TAT.


Subject(s)
Cell Culture Techniques , Methods , Cinnamates , Metabolism , Depsides , Metabolism , Phenylalanine Ammonia-Lyase , Metabolism , Plant Cells , Metabolism , Salicylic Acid , Pharmacology , Salvia miltiorrhiza , Cell Biology , Metabolism , Suspensions , Tyrosine Transaminase , Metabolism
19.
China Journal of Chinese Materia Medica ; (24): 3238-3243, 2011.
Article in Chinese | WPRIM | ID: wpr-251158

ABSTRACT

<p><b>OBJECTIVE</b>To clone and characterize the DNA and cDNA sequences of phenylalanine ammonia-lyase gene (PAL) from Fagopyrum dibotrys, and investigate the biological activity of the obtained PAL.</p><p><b>METHOD</b>Using homology cloning and RT-PCR techniques, the DNA and full-length cDNA sequences of PAL gene were amplified from F. dibotrys. The obtained sequences were analyzed by bioinformatics software. The ORF of PAL gene was cloned into expression vector pET-30b(+) and transformed into Escherichia coli BL21 (DE3) for expression the recombined protein. The catalytic activity of the recombined protein was determined by Spectrophotometer and thin layer chromatography (TLC) methods.</p><p><b>RESULT</b>The DNA sequence of PAL gene (designated as FdPAL, GenBank accession number: HM628904) was 2 583 bp in size, of which consisted two extrons and a single intron, and the full-length cDNA of FdPAL was 2 169 bp in size, which contained an ORF. The deduced protein of FdPAL contained 722 amino acids with calculated molecular weight (MW) of 78.31 kDa and an isoelectric point (pI) of 5.94. The SDS-PAGE results showed that the molecular weight of recombinant FdPAL protein was 75.37 kDa, which is consistent with the predictions. After 4 hours of induction, the enzymatic specific activity of FdPAL reached the summit, up to 4 386 nmol x g(-1) x min(-1). The reaction products were also identified by TLC, using L-Phe and trans-cinnamic acid as the internal standard.</p><p><b>CONCLUSION</b>The PAL gene (both DNA sequence and full-length cDNA sequence) was cloned from F. dibotrys, and it has the same classic characters as other PALs in plants. The recombinant FdPAL was efficiently expressed in E. coli and had the activity for catalyzing the conversion from L-phenylalanine to cinnamic acid.</p>


Subject(s)
Amino Acid Sequence , Base Sequence , Cloning, Molecular , Escherichia coli , Genetics , Metabolism , Fagopyrum , Classification , Genetics , Molecular Sequence Data , Phenylalanine Ammonia-Lyase , Chemistry , Genetics , Metabolism , Phylogeny , Protein Conformation , Recombinant Proteins , Chemistry , Genetics , Metabolism
20.
China Journal of Chinese Materia Medica ; (24): 1707-1710, 2011.
Article in Chinese | WPRIM | ID: wpr-354138

ABSTRACT

<p><b>OBJECTIVE</b>Study on correlation between H2O2 scavenging system and flavonoids accumulation of Scutellaria baicalensis.</p><p><b>METHOD</b>The content of baicalin and baicalein in suspension cell of S. baicalensis was determined by HPLC. The content of total flavonoids and H2O2, the activity of POD and PAL was detected by UV spectrophotometry.</p><p><b>RESULT</b>The content of total flavonoid and the activity of PAL increased significantly in 12 days after 40 degrees C, dark and PEG stress. Around 12 days after NPA, NPA +40 degrees C, 40 degrees C, NPA + dark, dark and PEG stress, the content of baicalin declined and the content of baicalein rise, the activity of POD showed an increasing trend, and level of H2O2 remain stable.</p><p><b>CONCLUSION</b>Moderate environmental stress could promote the accumulation of total flavonoids in S. baicalensis, baicalin convert to baicalein by POD, and maintaining the stability of H2O2 content to avoid oxidative damage.</p>


Subject(s)
Anti-Infective Agents , Metabolism , Antioxidants , Metabolism , Cells, Cultured , Darkness , Flavanones , Metabolism , Flavonoids , Metabolism , Hot Temperature , Hydrogen Peroxide , Metabolism , Oxidants , Metabolism , Oxidative Stress , Peroxidase , Metabolism , Phenylalanine Ammonia-Lyase , Metabolism , Plant Extracts , Chemistry , Plant Roots , Chemistry , Metabolism , Plants, Medicinal , Polyethylene Glycols , Pharmacology , Scutellaria baicalensis , Chemistry , Metabolism , Stress, Physiological
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