|Year : 2018 | Volume
| Issue : 1 | Page : 15-20
Cloning and expression analysis of two dehydrodolichyl diphosphate synthase genes from Tripterygium wilfordii
Lin-Hui Gao1, Ping Su2, Yi-Feng Zhang1, Li-Chan Tu3, Yu-Jun Zhao2, Tian-Yuan Hu3, Jia-Wei Zhou3, Bao-Wei Ma3, Wei Gao4, Lu-Qi Huang2
1 School of Traditional Chinese Medicine, Capital Medical University; State Key Laboratory of Breeding Base Dao-di Herbs, National Resource Center for Chinese Materia Medical, China Academy of Chinese Medical Sciences, Beijing, PR China
2 State Key Laboratory of Breeding Base Dao-di Herbs, National Resource Center for Chinese Materia Medical, China Academy of Chinese Medical Sciences, Beijing, PR China
3 School of Traditional Chinese Medicine, Capital Medical University, Beijing, PR China
4 School of Traditional Chinese Medicine, Capital Medical University; Beijing Key Laboratory of Traditional Chinese Medicine Collateral Disease Theory Research, Capital Medical University, Beijing, PR China
|Date of Submission||04-Feb-2018|
|Date of Acceptance||12-Mar-2018|
|Date of Web Publication||10-Apr-2018|
State Key Laboratory Breeding Base of Dao-di Herbs, National Resource Center for Chinese Materia Medical, China Academy of Chinese Medical Sciences, Beijing 100700
School of Traditional Chinese Medicine, Capital Medical University, Beijing 100069
Source of Support: None, Conflict of Interest: None
Objective: To clone and investigate two dehydrodolichyl diphosphate synthase genes of Tripterygium wilfordii by bioinformatics and tissue expression analysis. Materials and Methods: According to the T. wifordii transcriptome database, specific primers were designed to clone the TwDHDDS1 and TwDHDDS2 genes via PCR. Based on the cloned sequences, protein structure prediction, multiple sequence alignment and phylogenetic tree construction were performed. The expression levels of the genes in different tissues of T. wilfordii were measured by real-time quantitative PCR. Results: The TwDHDDS1 gene encompassed a 873 bp open reading frame (ORF) and encoded a protein of 290 amino acids. The calculated molecular weight of the translated protein was about 33.46 kDa, and the theoretical isoelectric point (pI) was 8.67. The TwDHDDS2 encompassed a 768 bp ORF, encoding a protein of 255 amino acids with a calculated molecular weight of about 21.19 kDa, and a theoretical isoelectric point (pI) of 7.72. Plant tissue expression analysis indicated that TwDHDDS1 and TwDHDDS2 both have relatively ubiquitous expression in all sampled organ tissues, but showed the highest transcription levels in the stems. Conclusions: The results of this study provide a basis for further functional studies of TwDHDDS1 and TwDHDDS2. Most importantly, these genes are promising genetic targets for the regulation of the biosynthetic pathways of important bioactive terpenoids such as triptolide.
Keywords: Bioinformatic analysis, expression analysis, Tripterygium wilfordii, TwDHDDS gene
|How to cite this article:|
Gao LH, Su P, Zhang YF, Tu LC, Zhao YJ, Hu TY, Zhou JW, Ma BW, Gao W, Huang LQ. Cloning and expression analysis of two dehydrodolichyl diphosphate synthase genes from Tripterygium wilfordii. World J Tradit Chin Med 2018;4:15-20
|How to cite this URL:|
Gao LH, Su P, Zhang YF, Tu LC, Zhao YJ, Hu TY, Zhou JW, Ma BW, Gao W, Huang LQ. Cloning and expression analysis of two dehydrodolichyl diphosphate synthase genes from Tripterygium wilfordii. World J Tradit Chin Med [serial online] 2018 [cited 2018 Sep 25];4:15-20. Available from: http://www.wjtcm.net/text.asp?2018/4/1/15/229166
| Introduction|| |
Tripterygium wilfordii Hook F is a traditional Chinese medicinal herb that belongs to the family Celastraceae. The root is the main pharmacologically active part with a bitter taste and a cold essence according to the traditional Chinese medicine. It belongs to the four classics of the liver, heart, kidney, and stomach, and it is considered to have the functions of clearing the heat, detoxifying, removing wind and dehumidifying, relaxing tendons and activating blood circulation, eliminating swelling, relieving pain, and stopping itching. Modern pharmacological studies have shown that it has anticancer, anti-inflammatory, and immunoregulatory effects,,,, and it shows promise in the treatment of rheumatoid arthritis. The main bioactive components are triptolide, celastrol, and other terpenoids.,,, The current supply of these compounds is mainly dependent on extraction and isolation from the original plant. However, the content of active ingredients in the original plants is extremely low. The extraction and isolation from the original plant is, therefore, far too inefficient to meet the clinical needs. In recent years, the exploration of key biosynthesis genes and utilization of the principles of synthetic biology to design and modify microbial strains has become a very promising alternative approach to acquire natural products.,,
The genes encoding key enzymes in the biosynthetic pathway of terpenoids in T. wilfordii have already been cloned and identified.,,,,,,, The biosynthetic pathway consists of an upstream isoprenoid pathway, in which two common C5 building blocks, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate, are formed through either the cytoplasmic mevalonate or the plastidic 2-C-methyl-D-erythritol-4-phosphate pathway, then prenyltransferase-catalyzed condensations of these two C5 units, result in the formation of linear elongated prenyldiphosphates including the C10 monoterpene precursor geranyl diphosphate, the C30 triterpene (e.g., celastrol) precursor farnesyl diphosphate (FPP), and the C20 diterpene (e.g., triptolide) precursor geranylgeranyl diphosphate. However, little is known about the terpenoid bypass metabolic pathway. Based on this knowledge, we identified two dehydrodolichyl diphosphate synthase (DHDDS) genes from T. wilfordii by analyzing transcriptome data. DHDDS participates in the terpenoid bypass metabolic pathway, where it continuously condensates FPP with multiple IPP units to generate dehydrodolichyl diphosphate (dedol-PP) which is the precursor of dolichol [Figure 1]. Dolichol has a long chain composed of isopentenyl group and plays an important role in the process of glycosylation as a glycosyl carrier.
|Figure 1: The biosynthesis pathway of dehydrodolichyl diphosphate in Tripterygium wilfordii. MVA: Mevalonate, MEP: 2-C-methyl-D-erythritol-4-phosphate, OPP: Octaprenyl diphosphate, IPP: Isopentenyl diphosphate, DMAPP: Dimethylallyl diphosphate, GPP: Geranyl diphosphate, GGPP: Geranylgeranyl diphosphate, FPP: Farnesyl diphosphate, DHDDS: Dehydrodolichyl diphosphate synthase|
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In this study, two TwDHDDS genes were cloned and investigated by bioinformatics and tissue expression analysis. The data enrich our knowledge of the terpenoid bypass metabolic pathway of T. wilfordii and provide genetic targets for the subsequent use of CRISPR-Cas9 gene editing technology to eliminate bypass genes or inhibit terpenoid bypass metabolic pathways to increase the metabolic flux toward the synthesis of valuable bioactive terpenoids such as triptolide.
| Materials and Methods|| |
Whole plants of T. wilfordii Hook F were obtained from the experimental fields of Fujian Agriculture and Forestry University (Fujian Province, China). Fresh leaves, stems, roots, and flowers were cleaned and stored at −80°C.
Isolation and purification of total RNA from different tissues of Tripterygium wilfordii
The total RNA of different tissues of T. wilfordii was extracted using the cetyltrimethylammonium bromide method  and purified by RNA Purification Kit (TransGen Biotech, Beijing, China). The concentration of RNA was measured using a NanaDrop 2000 nucleic acid/protein quantifier (Thermo Scientific, USA).
Cloning and sequencing of TwDHDDS from Tripterygium wilfordii
The extracted RNA was converted to first-strand cDNA templates using the FastKing RT Kit (Tiangen Biotech, Beijing, China). Specific primers [Table 1] were designed based on sequences in the T. wilfordii transcriptome database. The 50 μL reaction mixtures comprised 25 μL Phusion HF MM (New England Biolabs, USA), 2.5 μL (10 μM) each of the forward and reverse primers, 1 μL cDNA, and 19 μL ddH2O. The primers and polymerase chain reaction (PCR) conditions are listed in [Table 1]. The PCR product was purified and cloned into the pEASY-T3 vector (TransGen Biotech) and transformed into Escherichia coli Trans 5α competent cells (TransGen Biotech). Positive clones were selected and sequenced to obtain the TwDHDDS gene sequence. Primer synthesis and sequencing was conducted by Shanghai Majorbio Bio-pharm Technology Co., ltd., PR China.
Bioinformatics analysis of TwDHDDS
The open reading frames (ORFs) were identified using ORF Finder (https://www.ncbi.nlm.nih.gov/orffinder) to obtain the protein sequences, which were compared with those from other species using NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi), and the protein sequences of other species were downloaded. Multiple sequence alignments were performed using DNAMAN software is DNAMAN V6 software (Lynnon Biosoft, Quebec, QC, Canada) and the phylogenetic tree was constructed using molecular evolutionary genetics analysis (MEGA 6.0) software  based on the results of the homology comparison. Interpro (http://www.ebi.ac.uk/interpro) was used to identify functional domains. The theoretical isoelectric point (pI) and molecular weight (Mw) were determined using the Compute pI/Mw tool (https://web.expasy.org/compute_pi/). The TargetP 1.1 server (http://www.cbs.dtu.dk/services/TargetP/) was used to analyze signal peptides. Subcellular localization was analyzed using Psort (http://psort1.hgc.jp/form.html). Transmembrane domains were analyzed using TMHMM server v2.0 (http://www.cbs.dtu. dk/services/TMHMM/). PredictProtein (http://www.predictprotein.org) was used to predict the secondary structure of the proteins. The 3D structure homology models were built using SWISS-MODEL (https://www.swissmodel.expasy.org/interactive).
Tissue expression analysis of TwDHDDS
The eukaryotic translation elongation factor 1α (Ef1α) gene was used as an endogenous control to normalize the expression level. The specific primers for quantitative real-time (qRT) PCR were designed according to the TwDHDDS sequence [Table 1]. The 10 μL reaction mixes comprised 5 μL qPCR SuperMix (TransGen Biotech), 0.2 μL (10 μM) each of the forward and reverse primers, 1 μL cDNA, and 3.6 μL ddH2O, and the reactions were performed on a Roche LightCycler 480 Real-Time PCR System (Roche, Switzerland). PCR conditions are listed in [Table 1]. The expression of TwDHDDS was evaluated using the 2−ΔΔCt method  with triplicate measurements based on three biological replicates. The error was estimated by evaluating the 2−ΔΔCt term using ΔΔCt + standard deviation and ΔΔCt − standard deviation.
| Results|| |
Cloning and sequence homology analysis of TwDHDDS1 and TwDHDDS2
The full-length cDNA sequences of TwDHDDS1 and TwDHDDS 2 were obtained through RT-PCR with specific primers [Figure 2]. TwDHDDS1 (GenBank accession number: MG787474) encodes a protein of 290 amino acids with high sequence identity (78%) with known DHDDS from Corchorus olitorius and Gossypium raimondii. TwDHDDS2 (GenBank accession number: MG7874745) encodes a protein of 255 amino acids, which is 66% identical to the DHDDS from Malus domestica and Pyrusx bretschneideri [Figure 3] and [Table 2]. The domain analysis conducted using InterPro showed that TwDHDDS1 and TwDHDDS2 share a conserved domain, in which TwDHDDS1 has a conserved site of polyprenyltransferase activity.
|Figure 2: The polymerase chain reaction amplification results of TwDHDDS1 and TwDHDDS2 using specific primers. M: Trans 2K DNA marker|
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|Figure 3: Sequence alignment of TwDHDDS1 (a) and TwDHDDS2 (b) with homologous proteins|
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|Table 2: The Basic Local Alignment Search Tool analysis of TwDHDDS1 and TwDHDDS2 protein sequences|
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Phylogenetic tree analysis of TwDHDDS1 and TwDHDDS2
Sequence divergences were calculated using the Kimura two-parameter (K2P) distance model. A neighbor-joining tree was created using the MEGA software to evaluate the homology of the DHDDS sequences [Figure 4]. Bootstrapping was performed with 1000 replications and was reported as percentages. The results showed that TwDHDDS1 shared more identity with the DHDDS from C. olitorius, Durio zibethinus, and G. raimondii, which clustered into one clade. By contrast, TwDHDDS2 clustered close to the dehydrodolichyl diphosphate synthase complex subunit NUS1 sequences from Carica papaya, Vitis vinifera, and Populus euphratica, indicating that TwDHDDS2 and dehydrodolichyl diphosphate synthase complex subunit NUS1 have a closer genetic relationship.
|Figure 4: Phylogenetic tree of dehydrodolichyl diphosphate synthase sequences from different species|
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Physicochemical properties and three-dimensional structure prediction of TwDHDDS1 and TwDHDDS2
The calculated Mw of TwDHDDS1 was 33.46 kDa, and the predicted pI was 8.67. TwDHDDS1 is a secretory protein with a signal peptide, predicted to be localized to microbody. The results of transmembrane domain analysis showed that the amino acids 18–44 and 61–83 were located in the transmembrane region. The results of the secondary structure prediction showed a predominantly helix and loop structure, with 54.5% α-helices and 35.2% loops and 10.3% β-strands. Using the structure 2 vg3.1.B as a template for homology modeling, a 3D structure was generated [Figure 5]a, with 27.87% identity. The calculated Mw of TwDHDDS2 was 29.19 kDa, and the predicted pI was 7.72. TwDHDDS2 is also a secretory protein with a signal peptide and was also predicted to be localized to microbody. However, transmembrane domain analysis showed that the TwDHDDS2 protein does not have a transmembrane region. The results of secondary structure prediction showed a predominantly α-helical structure, with 71% α-helices, 20.4% loops, and 8.6% β-strands. Using 3wyi. 1.A as a template for homology modeling, 3D structure was generated [Figure 5]b, with 18.97% identity.
|Figure 5: Homology models of the three-dimensional structures of the two TwDHDDS proteins from this study. (a) TwDHDDS1 (b) TwDHDDS2|
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Tissue expression analysis of TwDHDDS1 and TwDHDDS2
To investigate the physiological roles of TwDHDDS1 and TwDHDDS2 in T. wilfordii, we performed qRT-PCR to evaluate the corresponding mRNA levels in various organs including root, stem, leaf, and flower. The results showed that TwDHDDS1 and TwDHDDS2 were expressed practically ubiquitously in all sampled organ tissues, with the highest transcription levels in stems [Figure 6].
|Figure 6: The transcript levels of TwDHDDS1 and TwDHDDS2 in different tissues of Tripterygium wilfordii|
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| Discussion|| |
DHDDS are conserved and widespread in a variety of organisms including yeasts, bacteria, animals, and plants.,,, They belong to the isopentenyl transferase family and can be subdivided into different classes according to the structural features of their respective substrates and products such as cis or trans structure and the number of carbon atoms. The tissue expression analysis revealed that both TwDHDDS1 and TwDHDDS2 were expressed practically ubiquitously in all sampled organ tissues, and showed the highest transcription levels in stems which was different from the expression pattern of Arabidopsis thaliana DHDDS gene. These results suggested that stem tissues need an more abundant supply of dedol-PP to sustain an active synthesis of dolichol. The results of BLAST and phylogenetic tree analysis showed that TwDHDDS1 had a close genetic relationship with dehydrodolichyl diphosphate synthases from other species, while TwDHDDS2 and the dehydrodolichyl diphosphate synthase NUS1 subunit had a closer genetic relationship. Hence, this study provides the basis for further functional investigations of TwDHDDS1 and TwDHDDS2.
FPP and IPP are important precursors of monoterpenes, sesquiterpenes, diterpenes (e.g., triptolide), and triterpenes (e.g., celastrol)., DHDDS can continuously condense FPP and multiple IPP units to produce dehydrodolichyl diphosphate and the biosynthesis of dolichol will competitively consume these precursors. The genes encoding key enzymes in the upstream biosynthetic pathways of triptolide and celastrol have already been cloned and identified and the accumulation of triptolide and celastrol can be directly increased by overexpressing the key enzymes involved in the upstream pathway through genetic manipulation. Here, two TwDHDDS genes involved in the dolichol synthetic pathway were cloned and studied, enriching our knowledge of the T. wilfordii terpenoid bypass pathway. The blockage of this synthetic pathway by CRISPR-Cas9-based genome editing can provide a new strategy to improve the production of important bioactive terpenoids.
Financial support and sponsorship
This work was supported by the National Natural Science Foundation of China (81773830 to W.G.), High-level Teachers in Beijing Municipal Universities in the Period of 13th Five-year Plan (CIT&TCD20170324 to W.G.) and Key project at central government level: The ability establishment of sustainable use for valuable Chinese medicine resources (2060302 to L.H.).
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
[Table 1], [Table 2]