ABSTRACT
1-Deoxy-D-xylulose-5-phosphate synthase (DXS), the first key enzyme in 2-methyl-D-erythritol-4-phosphate (MEP) pathway, catalyzes the condensation of glyceraldehyde-3-phosphate with pyruvate to 1-deoxy-xylose-5-phosphate (DXP). In this study, PgDXS1, PgDXS2, and PgDXS3 genes were cloned from the root of Platycodon grandiflorum (P. grandiflorum). The open reading frame (ORF) of PgDXS1, PgDXS2, and PgDXS3 were 2 160, 2 208, and 2 151 bp in full length, encoding 719, 735, and 716 amino acids, respectively. Homologous alignment results showed a high identity of PgDXSs with DXS in Hevea brasiliensis, Datura stramonium and Stevia rebaudiana. The recombinant expression plasmids of pET-28a-PgDXSs were constructed and transformed into Escherichia coli (E. coli) BL21 (DE3) cells, and the induced proteins were successfully expressed. Subcellular localization results showed that PgDXS1 and PgDXS2 were mainly located in chloroplasts, and PgDXS3 was located in chloroplasts, nucleus and cytoplasm. The expression of three DXS genes in different tissues of two producing areas of P. grandiflorum were assayed via real-time fluorescence quantitative PCR, and the results showed that all of them were highly expressed in leaves of P. grandiflorum from Taihe. Under methyl jasmonate (MeJA) treatment, the expression levels of three PgDXS genes showed a trend of first decreasing and then increasing at different time points (3 - 48 h), and the activity of DXS showed a trend of first increasing and then decreasing in three tissues of P. grandiflorum. This study provides a reference for further elucidating the biological function of PgDXS in terpenoid synthesis pathway in P. grandiflorum.
ABSTRACT
1-Deoxy-D-xylulose-5-phosphate synthase (DXS) is a rate-limiting enzyme involved in the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for terpenoid precursor biosynthesis. DXS plays an essential role in glycyrrhizic acid (GA) biosynthesis. Based on our previous transcriptome study, there was a negative correlation between DXS expression and GA content. Therefore, we explored the regulatory role of DXS in GA biosynthesis using both gene overexpression and gene knockout in a hairy root culture system. DXS was cloned from Glycyrrhiza glabra L. (GenBank Accession No. MN158121). A plant binary expression vector pCA-DXS was constructed by a gene fusion method. The sgRNA sequence was designed based on the first exon of DXS to construct the gene editing vector pHSE-DXS. Hairy roots overexpressing or knocking out DXS were generated through an Agrobacterium-mediated method with licorice hypocotyls as explants. Wild-type hairy roots and negative control hairy roots containing empty plasmids were also evaluated. UPLC was used to determine the GA content in each licorice hairy root line. Results showed that the content of GA in the hairy root group knocking out DXS was significantly higher than that in the wild-type and negative control groups, while in the hairy root group overexpressing DXS was significantly lower, suggesting that DXS plays a negative role in GA biosynthesis. This study provides a foundation for determining the function of DXS in terpenoid metabolism and for further establishment of a molecular regulatory network of GA biosynthesis.
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The objective of this research was to clone 1-deoxy-D-xylulose 5-phosphate reductoisomerase gene (MoDXR) and its promoter sequence from Morinda officinalis and carry out bioinformatic analysis, cis-acting elements analysis, and prokaryotic expression. On the basis of the MoDXR gene sequence obtained from the M. officinalis transcriptome and with NCBI-ORFfinder analysis, a pair of specific primers were designed, and used for RT-PCR amplification. The promoter region sequence at the 5′ end of MoDXR gene was isolated by the genome walking technique. Localization of MoDXR was carried out by subcellular analysis. The prokaryotic expression plasmid pET-28a-MoDXR was constructed and transfected into Escherichia coli BL21(DE3) chemically-competent cells; the recombiant plasmid expressed fusion protein after the induction by IPTG. The full-length cDNA of MoDXR was 2 015 bp,and open reading frame (ORF) size was 1 425 bp, and it encoded 474 amino acid residues and had a molecular mass of 51.27 kD. Sequence comparison with BlastP to the NCBI database revealed that MoDXR had high sequence similarity with many other DXRs, such as Coffea arabica DXR (CaDXR) and Rauvolfia verticillata DXR (RvDXR). A phylogenetic tree revealed that MoDXR had its closest relationship with DXR from Coffea arabica and Gardenia jasminoides. The subcellular localization revealed that MoDXR protein was located on the chloroplast. Plantcare analysis indicated that the promoter region sequence of MoDXR was 1 493 bp, covering multiple light, stress, and hormone-responsive cis-regulatory elements; protein electrophoresis showed that the expressed protein was the anticipated size. This research lays the foundation for further purification and structural and functional characterization of the MoDXR protein.
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1-deoxy-D-xylulose-5-phosphate synthase2(DXS2) is the first key enzyme of the MEP pathway,which plays an important role in terpene biosynthesis of plants. According to the data of Swertia mussotii transcriptome, DXS2 gene(Gen Bank number MH535905) was cloned and named as Sm DXS2. The bioinformatics results showed that Sm DXS2 has no intron,with a 2 145 bp open reading frame encoding a polypeptide of 714 amino acids. They are belonging to 20 kinds of amino acids,and the most abundant amino acids include Ala,Gly and Trp. The predicted protein molecular weight was 76. 91 k Da and its theoretical isoelectric point(p I) was6. 5,which belonging to a hydrophilic protein. α-Helix and loop were the major motifs of predicted secondary structure of DXS2. The three function domains are TPP＿superfamily,Transket＿pyr＿ superfamily and Transketolase＿C superfamily,respectively. The Sm DXS2 protein shared high identity with other DXS2 proteins of plants. Phylogenetic analysis showed that Sm DXS2 protein is grouped with the gentian DXS2 protein. The recombinant protein of Sm DXS2 gene in Escherichia coli was approximately 92. 00 k Da(containing sumo-His tag protein 13 k Da),which was consistent with the anticipated size.This work will provide a foundation for further functional research of Sm DXS2 protein and increasing the product of iridoid compound by genetic engineering in S. mussotii.
Subject(s)
Amino Acid Sequence , Cloning, Molecular , DNA, Complementary , Genetics , Genes, Plant , Iridoids , Phylogeny , Plant Proteins , Genetics , Swertia , Genetics , Transcriptome , Transferases , GeneticsABSTRACT
is famous for its important therapeutic effects. Saponins are bioactive compounds found in different parts and developmental stages of plants. Thus, it is urgently to study saponins distribution in different parts and growth ages of plants. In this study, potential biomarkers were found, and their chemical characteristic differences were revealed through metabolomic analysis. High-performance liquid chromatography data indicated the higher content of saponins (, Rg1, Re, Rd, and Rb1) in the underground parts than that in the aerial parts. 20()-Protopanaxadiol saponins were mainly distributed in the aerial parts. Additionally, the total saponin content in the 3-year-old plant (188.0 mg/g) was 1.4-fold higher than that in 2-year-old plant (130.5 mg/g). The transcriptomic analysis indicated the tissue-specific transcription expression of genes, namely, , , , , and , which encoded critical synthases in saponin biosyntheses. These genes showed similar expression patterns among the parts of plants. The expression levels of these genes in the flowers and leaves were 5.2fold higher than that in the roots and fibrils. These results suggested that saponins might be actively synthesized in the aerial parts and transformed to the underground parts. This study provides insights into the chemical and genetic characteristics of to facilitate the synthesis of its secondary metabolites and a scientific basis for appropriate collection and rational use of this plant.
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ABSTRACT The apicomplexan parasite Theileria parva, the causative agent of ECF, is an important pathogen affecting both domestic and wild animals, causing major economic losses in the world. Problems such as high cost of drugs, development of resistance, and absence of effective vaccines prevent effective combating of the pathogen. Thus, it is necessary to explore new targets for affordable and higher therapeutic value drugs. 1-Deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) in the non-mevalonate isoprene biosynthesis pathway is vital to the organism and therefore has been selected as a target for developing antitheilerial drugs. In this study, the 3D structure of TpDXR was identified by template-based in silico homology modelling method, the constructed model was validated and structurally analysed, and possible ligand binding pockets were identified for the first time in the literature. A reliable 3D model for TpLDH was modelled by using 3AU9 chain 'A' Plasmodium falciparum as a template. The obtained result showed that the model has a good resolution structure with 86.768 overall quality factor and a -9.15 z-score for TpDXR. The present study promises the possibility of exploiting new and safe inhibitors using the structure-based drug design that is effective against ECF through docking studies.
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1-Deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) is the second rate-limiting enzyme of terpenoid biosynthesis in the methylerythritol-4-phosphate pathway. According to the transcriptome database of Cinnamomum camphora, the DXR cDNA was cloned by rapid amplification of cDNA ends (RACE) from C. camphora, and was named CcDXR1 (GenBank number:KU886266). The ORF of CcDXR1 is composed of 1 413 bp, and it encodes 470 amino acids. The bioinformatics analysis suggests that the molecular weight of the encoded protein is 51.1 kD and the theoretically isoelectric point is 6.62, and there is no signal peptide and transmembrane structure in putative protein. The analysis of sequence alignment and phylogenetic tree showed that the CcDXR1 belonged to the DXR family. The results of the realtime PCR indicated that expression level of CcDXR1 in mature leaves was higher than tender leaves, which in roots was similar to leaves and the lowest in branches. The camphor is divided into five chemotypes, according to the main chemical compounds in C. camphora. It also showed that the expression level of CcDXR1 in borneol C. camphora was highest than that in cineol, iso-nerolidol, camphor and linalool. Our results revealed that the expression level of CcDXR1 exhibits diversity among plant tissues, growth periods and five chemical types, and the research provides foundation for further study of the terpenoids biosynthetic pathway in C. camphora.
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Objective: Based on the data of transcriptome sequencing of Magnolia officinalis, MoDXS1 and MoDXS2 genes were completed in detail by using bioinformatics methods. Methods: MoDXS1 and MoDXS2 genes were analyzed and predicted by the tools of bioinformatics in the following aspects: physical and chemical characteristics of amino acid sequences, function domain, hyophobicity or hydrophilicity, secondary structure and tertiary structure of protein, molecular phylogenetic evolution, and so on; The expression levels of MoDXS1 and MoDXS2 were identified by real-time PCR. Results: ORF Finder indicated that MoDXS1 and MoDXS2 genes were full-length, and they all were unstable hydrophobic proteins; Structural domain of MoDXS1 and MoDXS2 showed high homology with other plants; The secondary structures all were hybrid architecture, and alpha helixes were the major motifs, tertiary structure of protein was predicted by Homology modeling; Sequence alignment that MoDXS family had relative close relationship to the DXS of Nicotiana tabacum, Salvia miltiorrhiza, and Arabidopsis thaliana; The results of evolutionary relationship analysis showed that MoDXS1 had relative close relationship to angiosperm, but MoDXS2 was clustered in a clade solely; The expression levels of DXS1 in M. officinalis and M. officinalis var. biloba were not significantly different, but the expression level of DXS2 in M. officinalis var. biloba was higher than that in M. officinalis. By applying the technique of GC-MS, the contents of major volatile components β-caryophyllene, β-caryophyllene oxide, and β-eudesmol in M. officinalis var. biloba are higher than those in M. officinalis. Conclusion: The results provide theoretical reference for studies on secondary metabolic regulation in terpenoid of M. officinalis.
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We cloned and analyzed the two genes of the 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase (HDR) gene family from Huperzia serrate. The two transcripts coding HDR, named HsHDR1 and HsHDR2, were discovered in the transcriptome dataset of H. serrate and were cloned by reverse transcription-polymerase chain reaction (RT-PCR). The physicochemical properties, protein domains, protein secondary structure, and 3D structure of the putative HsHDR1 and HsHDR2 proteins were analyzed. The full-length cDNA of the HsHDR1 gene contained 1431 bp encoding a putative protein with 476 amino acids, whereas the HsHDR2 gene contained 1428 bp encoding a putative protein of 475 amino acids. These two proteins contained the conserved domain of 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase (PF02401), but without the transmembrane region and signal peptide. The most abundant expression of HsHDR1 and HsHDR2 was detected in H. serrate roots, followed by the stems and leaves. Our results provide a foundation for exploring the function of HsHDR1 and HsHDR2 in terpenoid and sterol biosynthesis in Huperziaceae plants.
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Objective: To clone the 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) gene from Houttuynia cordata and analyze the differential expression. Methods: The cDNA sequence of DXR was cloned from H. cordata by using RT-PCR strategy. The physical and chemical properties, secondary structure, and three-dimensional structure of the DXR protein were forecasted and analyzed, and its function was predicted. The differential expression of DXR gene in rhizome, stems, leaves, and flowers was analyzed by fluorescent quantitative PCR. Results: The cDNA contained a 1 416 bp open reading frame and encoded a predicted protein of 471 amino acids. Bioinformatics predicted that no transmembrane region and signal peptide were present in DXR. Relative real-time PCR analysis indicated that DXR showed the highest transcript abundance in leaves, moderate level in rhizomes, lower level in stems, and the lowest level in flowers. Conclusion: This study clones DXR gene from H. cordata for the first time, and provides a foundation for exploring the mechanism of this gene for the terpenoid biosynthesis in H. cordata.
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Objective: To clone the 1-deoxy-D-xylulose-5-phosphate synthase 1 (DXS1) gene from Houttuynia cordata and to analyze the expression difference. Methods: The cloning primers were designed based on the transcriptome dataset of H. cordata, one unique sequence encoding DXS1 was discovered. The sequence of DXS1 was cloned from H. cordata by RT-PCR. The physicochemical properties, secondary structure, and three-dimensional structure of the DXS1 protein were forecasted and analyzed, and its structure and function were predicted. And the different expression levels of DXS1 gene in rhizome, stems, leaves, and flowers were analyzed by fluorescent quantitative PCR. Results: The cDNA (named as DXS1) contains a 2 172 bp open reading frame and encodes a predicted protein of 723 amino acids. No transmembrane region and signal peptide were present in DXS1. The conserved domain of DXS was present in DXS1. Relative real-time PCR analysis indicated that DXS1 showed the highest transcript abundance in the flowers, moderate level in the leaves, lower level in the rhizomes, and the lowest level in the stems. Conclusion: This study cloned the DXS1 gene from H. cordata for the first time. The results will lay a foundation for exploring the mechanism of terpenoid biosynthesis in H. cordata plants.
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Objective: To clone the gene sequence of the key enzyme in synthesis pathway of terpenoids, 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) from Gentiana macrophylla (GmDXR) and to analyze its characteristic and expression. Methods: GmDXR gene was cloned and the sequence characterization was analyzed with bioinformatics methods, then the expression patterns of GmDXR were studied by real-time PCR. Results: GmDXR gene contained a completed open reading frame of 1 428 bp encoding 475 amino acids, GmDXR has high homology (≥85%) with DXR proteins from Rauvolfia verticillata, Lycopersicon esculentum, and other plants. Further analysis showed that GmDXR had non-transmembrane domain structure and signal peptide, which mainly located in the chloroplast. Quantitative PCR results showed that GmDXR mainly expressed in the leaves of G. macrophylla and could be induced by plant hormone such as methyl jasmonate (MeJA). Conclusion: GmDXR contains conserved structures of DXR protein. GmDXR expresses variously in different organs of G. macrophylla and could be induced by MeJA. It is very helpful for the future research on biosynthetic mechanism of iridoid compounds in G. macrophylla.
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Objective: To clone the conserved cDNA fragment of 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) gene from Angelica sinensis and to analyze its tissue-specific expression in roots, stems, and leaves. Methods: A pair of degenerate primers were designed according to the conservative sequences of the cloned DXR from other plant species. The total RNA from the leaves of A. sinensis was as template, DXR fragment was obtained by reverse transcription polymerase chain reaction (RT-PCR), connected to pGEM-T Easy vector then transformed into Escherichia coli DH5α. The positive clone identified by PCR was sequenced. Taking Actin of A. sinensis as a reference gene, SYBR Green quantitative RT-PCR was used to detect the relative expression levels of DXR different tissues of A. sinensis. Results: The DXR fragment of A. sinensis containing 564 bp encoding 187 amino acids was registered in Genbank (No. KJ000259). The sequence identity analysis suggested that both the obtained nucleotide sequence and its corresponding amino acid sequence shared more than 80% of homology with GenBank DXRs from Gossypium barbadense and other five higher plant species. DXR was expressed at the highest level in the leaves, and the relative expression levels in the leaves were 2.5 and 3.2 times relative to the stems and roots, respectively (P<0.01). Conclusion: A novel DXR fragment is cloned from A. sinensis and its tissue-specific expression in A. sinensis is investigated. This work might establish an experimental basis for the effective application of DXR in the future.
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The transcript encoding 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) was discovered from the transcriptome data of Huperzia serrata. The transcript contained an open reading frame with length of 1,440 bp and coded 479 amino acids. The full length of HsDXR1 had been cloned using RT-PCR method. Ac-cording the bioinformatic analysis, the molecular weight of HsDXR1 protein was 51.4961 kDa and the pI was 6.44. No signal peptide and transmembrane site was discovered in HsDXR1, and the protein was most likely to be located in chloroplast. HsDXR1 had the same domain similar to the DXR protein of Arabidopsis and Oryza sativa. The expression level of HsDXR1 was most abundantly in H. serrata stem, followed by root and leaf. This study cloned and analyzed HsDXR1 gene from H. serrata for the first time. The result will provide a foundation for exploring the mechanism of terpene biosynthesis in H. serrata plants.