Gene cloning and enzymatic activity analysis of phenylalanine ammonia-lyase from Sinopodophyllum hexandrum (Royle) Ying / 生物工程学报

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.

Molecular cloning and characterization of three phenylalanine ammonia-lyase genes from Schisandra chinensis / 中国天然药物

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.

Transcriptome analysis reveals key enzyme genes involved in lignin biosynthesis pathway in Arctium lappa / 中草药

Objective: To obtain the functional genes in Arctium lappa and analyze the key enzyme genes involved in biosynthesis pathway of lignin. Methods: The transcriptome dataset of roots of A. lappa was obtained by using the BGISEQ-500 sequencing platform. Unigenes were de novo assembled and annotated according to the existing nucleic acids and protein databases. The key enzyme genes involved in lignin biosynthesis pathway were analyzed and the three-dimensional model of phenylalanine ammonialyase (AlPAL) was generated by the SWISS-MODEL and PyMol. Results: A total of 54 215 Unigenes were obtained by de novo assembly, and 42 003 Unigenes were annotated in at least one public database. A total of 1 668 Unigenes were identified to be plant transcription factors (TFs), which belong to 54 TF families, and 423 Unigenes were found to be involved in the biosynthesis pathway of lignin. Structure model indicated that AlPAL was a homotypic tetramer, and each monomer was consisted of three domains, including 4-methyl-imidazole-5-ketone (MIO) domain, core domain and shield domain. The MIO domain contained three conserved amino acids (ASG), which formed the catalytic activity center of the enzyme. Conclusion: This study was the first de novo transcriptome assembly of A. lappa, which will lay the foundation for the identification of functional genes, secondary metabolic pathway and the study of regulation mechanism of biosynthesis pathway of lignin in A. lappa in the future.

Cloning and expression analysis of phenylalanine ammonia-lyase gene in Lonicera macranthoides / 中草药

Objective To clone the full length of LmPAL1 gene and analyze bioinformatics and expression patterns from Lonicera macranthoides. Methods The total RNA of L. macranthoides was extracted. The full-length cDNA sequence of LmPAL1 gene was cloned by RT-PCR and RACE technique; The genome sequence in bioinformatics was analyzed by using the relevant software; The relative expression of the gene in stem, leaf, and different flower period was determined by using real-time PCR. Results The cloned LmPAL1 gene open reading frame (ORF) was 2 145 bp, encoding 714 amino acids. It was predicted by bioinformatics analysis as hydrophilic protein, being located in the chloroplasts, containing PAL shielding structure domain (527-641 aa). This gene contained PAL/HAL active center sequence GTITASGDLVPLSYIAG (196-212 aa), which was highly similar to other phenylalanine ammonia-lyase. Real-time PCR results showed that the relative expression level of golden yellow flowering flower was higher in seven florescence periods. When comparing the stem, leaf, and white flower bud period, the relative expression of flower was the highest and the leaf was the lowest. Conclusion In this study, PAL1 gene of L. macranthoides was cloned successfully, laying a foundation for further study of the function of this gene and genetic improvement of L. macranthoides quality and providing the research basis for exploring the biosynthesis and regulation of chlorogenic acid in L. macranthoides.

Cloning, sequence characterization, expression profiling of hormones response of phenylalanine ammonia-lyase gene in Bletilla striata / 中草药

Objective: Phenylalanine ammonia-lyase gene (PAL) is one of the key enzymes associated with stress resistance in secondary metabolism pathway of plants. Exploring its sequence information and expression profiling information in stress response could comprehensively peep at the protein structure, functions and signal network of plant stress resistance. Methods: The full cDNA length of PAL from Bletilla striata was cloned by RT-PCR and RACE approaches. Physicochemical properties and conserved domain of BsPAL protein were determined by a series of bioinformatics tools as Protparam, SOPMA, SWISS-MODEL, etc. Multiple alignment and phylogenetic tree were achieved by DNAMAN and MEGA Software, respectively. The qPCR was employed to examine the expression profiles of BsPAL under exogenous hormone stress. Results: The full cDNA of BsPAL was 2 708 bp, encoding a 797 amino-acid protein with a molecular weight of 86 216.94 and an isoelectric point (pI) of 6.24. The BsPAL protein included the typical structural domain and active site of PALs in other plants, and without transmembrane region, which was more homologous with PALs of Dendrobium officinale and Phalaenopsis aphrodita. The qPCR Results: revealed the expression level of BsPAL in roots was much higher than that in leaves and stems. Under MeJA treatment, the expression trend of BsPAL was first gradually ascending and then descending, while SA treatment had the reverse effect. Conclusion: The BsPAL’s sequence characterizing, expression profiling and responding patterns against SA and MEJA provided a research basis for elucidating the metabolic pathways of phenylpropanoid and hormone signaling research in B. striata.

Cloning and expression of three PAL genes in Astragalus membranaceus / 中草药

Objective To disclose the molecular mechanism of calycosin-7-O-β-D-glucoside (CG) accumulation in Astragalus membranaceus, we cloned PAL genes and analyzed the expression patterns of them and changes of CG contents in different tissues of A. membranaceus. Methods PAL genes were cloned with the methods of homology cloning and RACE technique using the total RNA as template and the analysis of bioinformatics on the cloned genes was carried out, gene expressions in root, stem, and leaf were determined with real-time PCR method, and CG content in root, stem, and leaf were analyzed by HPLC methods. Results Three PAL genes were cloned from A. membranaceus. The genbank accession number was KY086279 (AmPAL1), KY086280 (AmPAL2), and KY086281 (AmPAL3), respectively; The full-length cDNA of them was 2 508 bp, 2 401 bp, and 2 498 bp, respectively; And they all consisted of 2 157 bp open reading frame encoding 718 amino acids. Deduced AmPAL proteins had typical active sequences of PAL proteins, they were homology with other PAL proteins, and they shared the highest identities with PAL proteins of leguminous plants. Phylogenetic tree analysis showed AmPAL1 belonged to the different sub-class with the sub-class of AmPAL2 and AmPAL3. Real-time PCR analysis indicated that expression levels of AmPALs were different from each other, the expression level of AmPAL1 was the highest, the expression level of AmPAL2 was the next, and that of AmPAL3 was lowest in all detected tissues, and only the expression levels of AmPAL2 was similar to the changes of CG contents in different tissues (root > stem > leaf). Conclusion The cloned AmPAL1, AmPAL2, and AmPAL3 from A. membranaceus were typical genes of PAL, each might have different function in developing of different tissues, and AmPAL2 might involve in CG accumulation in different tissues.

Directing construction of CRISPR/Cas9 vector of SmPAL1 in Salvia miltiorrhiza by target efficiency detection in vitro / 中国中药杂志

To construct CRISPR/Cas9 vectors for the editing of SmPAL1 in the phenylpropane metabolic pathway of Salvia miltiorrhiza, CIRSPR/Cas9 target sites of SmPAL1 were designed by online software. Its target efficiencies were detected in vitro by enzyme digestion and sequences with highly efficiency were constructed into CRISPR/Cas9 vectors. Three possible CRISPR target sequences (SmPAL1-g1, SmPAL1-g2, SmPAL1-g3) were designed and the enzyme digestion efficiencies were 53.3%, 76.6% and 10.0%. SmPAL1-g1 and SmPAL1-g2 were constructed into vector VK005-03 named as VK005-03-g1 and VK005-03-g2. The results of sequencing showed that the two CRISPR/Cas target sequences were all constructed into VK005-03. Here we first laid the foundation for the study of SmPAL1 and provided an effective strategy for the screening of sgRNA.

Induction of defense-related enzymes in patchouli inoculated with virulent Ralstonia solanacearum

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.

The phenylalanine ammonia-lyase gene family in Isatis indigotica Fort.: molecular cloning, characterization, and expression analysis / 中国天然药物

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.

cDNA cloning, Phylogenic Analysis and Gene Expression Pattern of Phenylalanine ammonia-lyase in Sugarcane (Saccharum officinarum L.)

The aim of the present study was to clone and characterize a full length cDNA of sugarcane (Saccharum officinarum) phenylalanine ammonia-lyase (SoPAL). Differential tissue expression pattern of the SoPAL transcript and its enzyme activity was also analyzed during the tillering stage of growth. The full-length of SoPAL cDNA was 2118 bp long and contained a protein with 706 amino acids, determined by encoding technique. The amino acid sequence and phylogenic analysis of the cloned SoPAL showed high similarity to PAL from other monocotyledonous such as sorghum (96%), maize (93%) and Bamboos (87.12%). The highest levels of SoPAL transcript were observed in the root and stem, while its minimal gene expression levels were in the leaves and sheath, respectively. The highest level of SoPAL enzyme activity was in the leaves. These results helped to understanding the characteristics of PAL biosynthesis and its regulation at the molecular level in sugarcane. This information could be critical for the manipulation of phenylpropanoid biosynthesis in the plant using biotechnological processes.

Cloning and sequence analysis of phenylalanine ammonia-lyase gene from Curcuma longa / 中草药

Objective: This study aimed at cloning the phenylalanine ammonia-lyase (PAL) gene from Curcuma longa (CurPAL) and analyzing the bioinformatics. Methods: PAL gene was cloned by RT-PCR and RACE strategy with the template of RNA extracted from C. longa leaves. The bioinformatic analysis of this gene and its corresponding protein were performed. Results: One unique sequence of PAL, named as CurPAL (GenBank NO. KJ780359), was cloned from C. longa. The full-length of CurPAL cDNA was 1 293 bp, including 243 bp of 5'-UTR, 123 bp of 3'-UTR, and 927 bp of ORF encoding 308 amino acids. The molecular weight and theroretical isoelectric point (pI) of the deduced CurPAL protein were 33 000 and 5.76, respectively. The protein of CurPAL was stable and soluble. The domination sites and catalytic active sites in PAL protein of Nerium oleander were also found in CurPAL. It was found that the amino acid sequence of CurPAL had more than 75% homology with PAL of Prunus salicina, Camellia chekiangoleosa, Capsicum chinense, and Musa acuminate via multiple alignments. It revealed that CurPAL had closer relationship with PALs from Zingiberales plants than from other plants by phylogenetic tree analysis. Secondary and tertiary structures indicate that CurPAL is a full α protein contained by homotetramer. Conclusion: The cDNA encoding PAL from C. longa is cloned and reported for the first time. This work provides a scientific basis for exploring the biosynthetic pathway of the medicinal ingredient and improving its quality in C. longa.

Activity of enzymes associated with the enzymatic browning of minimally processed potatoes

The purpose of the present study was to evaluate the effect of different potato cultivars and storage temperatures on the specific activity of phenylalanine ammonia-lyase (PAL), polyphenol oxidase (PPO) and peroxidase (POD) in minimally processed potatoes. Potato cultivars Agata, Asterix and Monalisa were selected, washed, peeled, diced, sanitized, centrifuged, vacuum- packed and stored at 5 and 15°C for 9 and 5 days, respectively. There was an increase in the enzymatic activity in all the cultivars stored at 15°C. The cultivars 'Agata' and 'Asterix' stored at 5ºC did not differ significantly between them for the PAL, PPO and POD activities. The PAL, PPO and POD activities were also influenced by the storage temperature. The cultivars Agata and Asterix were more suitable in minimal processing than 'Monalisa', which was more susceptible to oxidative browning.

Fungal and Plant Phenylalanine Ammonia-lyase

L-Phenylalanine is one of the essential amino acids that cannot be synthesized in mammals in adequate amounts to meet the requirements for protein synthesis. Fungi and plants are able to synthesize phenylalanine via the shikimic acid pathway. L-Phenylalanine, derived from the shikimic acid pathway, is used directly for protein synthesis in plants or metabolized through the phenylpropanoid pathway. This phenylpropanoid metabolism leads to the biosynthesis of a wide array of phenylpropanoid secondary products. The first step in this metabolic sequence involves the action of phenylalanine ammonia-lyase (PAL). The discovery of PAL enzyme in fungi and the detection of 14CO2 production from 14C-ring-labeled phenylalanine and cinnamic acid demonstrated that certain fungi can degrade phenylalanine by a pathway involving an initial deamination to cinnamic acid, as happens in plants. In this review, we provide background information on PAL and a recent update on the presence of PAL genes in fungi.

Naringenin inhibits the growth and stimulates the lignification of soybean root

The flavanone naringenin, an intermediate in flavonoid biosynthesis, was tested for its effect on root growth, phenylalanine ammonia-lyase (PAL) and peroxidase (POD) activities, as well as phenolic compounds and lignin contents in soybean (Glycine max L. Merrill) seedlings. Three-day-old seedlings were cultivated in half-strength Hoagland nutrient solution (pH 6.0), with or without 0.1 to 0.4 mM naringenin in a growth chamber (25°C, 12-h photoperiod, irradiance of 280 µmol m-2 s-1) for 24 h. Inhibitory effects on root growth (length, weight, cell viability), PAL and soluble POD activities were detected after naringenin treatments. These effects were associated with stimulatory activity of the cell wall-bound POD followed by an increase in the lignin contents, suggesting that naringenin-induced inhibition in soybean roots could be due to the lignification process.

Os efeitos de naringenina, um intermediário da biossíntese de flavonóides, foram avaliados sobre o crescimento das raízes, as atividades da fenilalanina amônia liase (PAL) e peroxidases, bem como sobre os teores de compostos fenólicos e de lignina em plântulas de soja (Glycine max L. Merrill). Plântulas de três dias foram cultivadas em solução nutritiva de Hoagland, meia-força (pH 6,0), contendo ou não, naringenina 0,1 a 0,4 mM, em uma câmara de germinação (25°C, fotoperíodo de 12 h, 280 µmol m-2 s-1) durante 24 h. Efeitos inibitórios no crescimento das raízes (comprimento, massa e viabilidade celular) e nas atividades da PAL e POD solúvel foram constatados após os tratamentos com naringenina. Estes efeitos foram associados com atividade estimulatória da POD ligada à parede celular, seguido por aumento nos teores de lignina, sugerindo que a inibição do crescimento das raízes pode ser devido ao processo de lignificação.

Cloning and sequence analysis of a novel phenylalanine ammonia-lyase gene from Astragalus membranaceus / 中草药

Objective To clone and sequence the cDNA encoding phenylalanine ammonia-lyase(PAL)gene from Astragalus membranaceus.Methods RT-PCR and RACE Techniques were used to clone a phenylalanine ammonia-lyase gene from A.membranaceus roots with the total RNA as the template.Results The cloned gene named as AmPAL and the Genbank registry number is EF567076.Squence analysis showed that the full-length of AmPAL cDNA was 2 650 bp,including a 2 154 bp open reading frame(ORF).AmPAL was a new number of PAL family that consisted of 718 amino acids with prediated mole-cular weight of 7.805?104 and isoelectric point(PI)of 5.96.At the same time,AmPAL had the homo-logy with PAL of known leguminous plants and shared above 80% identity of amino acid sequences.Conclusion It is the first report that a novel PAL gene is cloned from A.membranaceus.This work lays a foundation for regulating phenylpropanoid pathway of medical plant with AmPAL.

Effect of pH on callus growth, PAL activity and paclitaxel content of plants of Taxus L / 中草药

Object To study the effect of pH on callus tissue growth and paclitaxel content of Taxus cuspidata Sieb. et Zucc. and Taxus chinensis var. mairei (Lemee et Le ′vl.) Cheng et L. K. Fu Methods The callus tissues were cultured on B5 medium with defferent pH values, and the growth rate, PAL activity and paclitaxel content were determined. Results Different callus tissues of taxus need different optimum pH values. The pH values, which could promote callus growth significantly, were all inhabitory to PAL activity and accumulation of paxlitaxel. Conclusion pH had great influences on the growth of the callus and the level of secondary metabolism, thereby the synthesis and accumulation of paclitaxel were affected.

Expression of phenylalanine ammonia-lyase cDNA from rice in E. coli BL21DE3 / 中国病理生理杂志

AIM: To study the expression and its kinetics of rice phenylalanine ammonia-lyase gene encoding into E. coli as the basis of treatment for phenylketouria. METHODS: The phenylalanine ammonia-lyase-1-cDNA(rPAL-1-cDNA) from rice was recombined into E. coli high expression vector pET-28c and transformed into E. coli host strain BL21DE3. Engineering bacteria was then inducted by isopropyl-?-D-thiogalactoside (IPTG) for 1, 3, 5, 7 hours, in order to obtain high level expression. RESULTS: After induction, the expression level of fusion protein was 21.40%, 30.60%, 35.40%, 35.43% respectively. The fusion protein exhibited a band of 78 6 kD on SDS-PAGE analysis,but was not found in controls.The target protein was mainly existed in the form of inclusion body. CONCLUSION:Rice PAL gene expressing E. coli was established by gentic engineering technique.