Mol Biol Rep (2012) 39:8803–8812 DOI 10.1007/s11033-012-1743-4
Functional characterization and differential expression studies of squalene synthase from Withania somnifera Neha Gupta • Poonam Sharma • R. J. Santosh Kumar Rishi K. Vishwakarma • B. M. Khan
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Received: 20 December 2011 / Accepted: 7 June 2012 / Published online: 21 June 2012 Ó Springer Science+Business Media B.V. 2012
Abstract Squalene synthase (SQS: EC 2.5.1.21) is a potential branch point regulatory enzyme and represents the first committed step to diverge the carbon flux from the main isoprenoid pathway towards sterol biosynthesis. In the present study, cloning and characterization of Withania somnifera squalene synthase (WsSQS) cDNA was investigated subsequently followed by its heterologous expression and preliminary enzyme activity. Two different types of WsSQS cDNA clones (WsSQS1and WsSQS2) were identified that contained an open reading frames of 1,236 and 1,242 bp encoding polypeptides of 412 and 414 amino acids respectively. Both WsSQS isoforms share 99 % similarity and identity with each other. WsSQS deduced amino acids sequences, when compared with SQS of other plant species, showed maximum similarity and identity with Capsicum annuum followed by Solanum tuberosum and Nicotiana tabacum. To obtain soluble recombinant enzymes, 24 hydrophobic amino acids were deleted from the carboxy terminus and expressed as 6X His–Tag fusion protein in Escherichia coli. Approximately 43 kDa recombinant protein was purified using Ni–NTA affinity chromatography and checked on SDS-PAGE. Preliminary activity of the purified enzymes was determined and the products were analyzed by gas chromatograph–mass spectrometer (GC–MS). Quantitative real-time PCR (qRTPCR) analysis showed that WsSQS expresses more in young leaves than mature leaves, stem and root.
Neha Gupta and Poonam Sharma contributed equally to this study. N. Gupta P. Sharma R. J. Santosh Kumar R. K. Vishwakarma B. M. Khan (&) Plant Tissue Culture Division, National Chemical Laboratory, Dr. Homi Bhabha Road, Pune 411008, Maharashtra, India e-mail:
[email protected]
Keywords Withania somnifera Squalene synthase Gas chromatograph–Mass Spectrometer (GC–MS) qRT-PCR Abbreviations IPTG Isopropyl b-D-1-thiogalactopyranoside SQS Squalene synthase SQS Squalene synthase gene TBME Tert-butyl methyl ether qRT-PCR Quantitative real-time PCR
Introduction Withania somnifera (L.) Dunal of solanaceae is an erect evergreen shrub distributed throughout the drier parts of India. It, commonly called as Ashwagandha, is well known since thousands of years for its pharmacological and medicinal properties which are attributed to the characteristic secondary metabolites of the plant, called withanolides [1–3]. These bioactive compounds are structurally diverse steroidal compounds with an ergosterol skeleton. The different withanolides are classified according to their structural skeleton [4] and their structural variation is responsible for the wide array of pharmacological activities. Withanolides have been widely studied for their antioxidant [5, 6], antiinflammatory [7], antitumor [8, 9], immunomodulating [10], antistress [11], hemopoetic effect [12], rejuvenating effect [13] and for the protection against CCl4 induced hepatotoxicity [4]. Several withanolides and sitoindosides (a withanolide containing a glucose molecule at carbon 27) have been isolated and reported from aerial parts, roots and berries of Withania species. They are found to be localized mainly in leaves and their concentration usually ranges from 0.001 to 0.5 % dry weight [14].
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The biosynthetic pathways of withanolides are still unclear and they are supposed to be derived from cholesterol (Fig. 1). Squalene synthase (SQS: EC 2.5.1.21) is an important regulatory enzyme of cholesterol biosynthetic pathway. It is a bifunctional enzyme which catalyses the condensation of two molecules of farnesyl pyrophosphate (FPP) in a head-to-head manner to form presqualene diphosphate (PSPP) and then converts the PSPP to squalene in the presence of NADPH and Mg2? (Fig. 1). As studied in engineered yeasts [15, 16], down regulation of the squalene synthase in the sterol biosynthetic pathway leads to the accumulation of FPP, which is redirected away from this pathway and toward the synthesis of other commercially important isoprenoids. As a key enzyme in the regulation of isoprenoid biosynthesis, SQS encoding genes have been cloned and characterized from several organisms including bacteria [17], yeasts [18, 19], Ganoderma lucidum [20], protozoa
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and animals [21–23] and human beings [24, 25]. Plant squalene synthase genes have also been characterized in Nicotiana tabacum, Panax ginseng, Glycyrrhiza glabra, Euphorbia tirucalli and other plants [26–31]. SQS amino acid sequence analysis provided insights for engineering more soluble variants. Jennings et al. [18] cloned the yeast squalene synthase gene and suggested that the enzyme consisted of a large cytosolic domain anchored to the endoplasmic reticulum by a single C-terminal transmembrane helix. Subsequently, a soluble and fully active version of recombinant yeast squalene synthase was constructed by deletion of a C-terminal hydrophobic region from the enzyme [19, 32]. Similar C-terminal hydrophobic domains were also found in the Schizosaccharomyces pombe [24] and Homo sapiens proteins [33]. Membrane bound SQS enzyme has been purified to homogeneity from microsomal membranes of Saccharomyces cerevisiae [34] and in a truncated soluble form from rat liver [22]. In plants, the enzyme has been solubilized and partially purified from daffodil microsomal membranes [35] and from tobacco cell-suspension cultures [36]. C-terminal truncation was also carried out in Capsicum annuum by removing last 24 amino acids and fully active SQS protein was purified from recombinant Escherichia coli [37]. The ayurvedic properties of the plant W. somnifera are credited into the account of withanolides. Squalene synthase has proved to play an imperative role in steroids biosynthesis in plants. But the function of this gene is still unexplored in this plant. Hence, the present study is aimed at the molecular studies on squalene synthase from W. somnifera with regard to cDNA cloning and functional characterization of in vitro enzyme reaction to provide insights into the regulation of withanolide biosynthetic pathway. Here, we also report the quantitative real-time PCR analysis of WsSQS providing information about its abundance in different organs of the plant at the transcript level.
Materials and methods Plant material
Fig. 1 Schematic representation of important steps involved in withanolides biosynthesis pathway. HMGS hydroxymethylglutarylCoA synthase, HMGR hydroxymethylglutaryl-CoA reductase, MVA mevalonic acid, IPP isopentenyl diphosphate, DMAPP dimethylallyl diphosphate, GPP geranyl diphosphate, FPPS farnesyl diphosphate synthase, SQS squalene synthase and SQE squalene epoxidase. The reaction catalyzed by SQS is shown in inset where presqualene pyrophosphate (PSPP) is the intermediate. Multiple step reactions are depicted by dashed arrows
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Withania somnifera plants were obtained from the green house of National Chemical Laboratory. Withania cultures were also maintained by sub culturing of meristems after every 6 weeks. Total RNA extraction and cDNA synthesis Total RNA was extracted from 100 mg W. somnifera leaves using Trizol (Sigma) reagent. The purity and concentration of RNA was checked on Nanovue (GE Healthcare). Ideally
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2 lg of this extracted RNA was directly reverse transcribed with dNTPs using AMV reverse transcriptase (Promega, USA) and an oligo (dT)15 primer in a total volume of 20 ll for 1 h at 42 °C. The resulting cDNA mixture was directly used as a template for PCR amplification. Cloning, characterization and phylogenetic analysis of squalene synthase Based on highly conserved amino acid sequences from reported squalene synthase of several plants, primers were synthesized for the PCR amplification of the corresponding W. somnifera cDNA. The forward (SQSF1) and reverse (SQSR2) primers were 50 -GAA AGC GGA (G/A)CA GAT CCC-30 and 50 -AAA TCT CAC AAA ACA T-30 respectively. PCR was carried out under the following conditions: 35 cycles of 95 °C for 1 min, 55 °C for 30 s and 72 °C for 1.30 min using above cDNA as a template. An amplicon of expected size was recovered, cloned into pGEM-T Easy vector (Promega, USA) and transformed into E. coli XL10 competent cells. Plasmid DNA were isolated and subjected to nucleotide sequencing. The obtained nucleotide sequence for partial SQS showed maximum similarity with Solanum tuberosum. So for full length gene, forward primer SQSF0 (50 -ATG GGA ACA TTG AGG GCG ATT-30 ) and reverse primer SQSR0 (50 -CTA GAA TCG GTT GCC AGG AAG TTG T-30 ) were directly synthesized from the S. tuberosum SQS sequence. Full length SQS was then amplified by using above primers (SQSF0–SQSR0) at the same PCR conditions. The fragment was eluted from the gel using gel elution kit (Axygen), ligated in pGEM-T Easy vector and transformed in E. coli competent cells. The clone was further confirmed by sequencing. To find out the 50 and 30 UTRs and to confirm the full length SQS sequence, 50 RACE and 30 RACE PCR (BD Clonetech, USA) were performed with gene specific primers designed from the already obtained SQS. The primer sequence for 50 RACE were RACESQSR 50 -GGC CAG AAC ATA CGG CAC TTG GGC ACC T-30 and RACESQSNR 50 -GAG AAT CTG GAG CCA GAT CTT CCT TCC CA-30 ; and for 30 RACE were RACESQSF 50 CTT GGG AAC CAT TAT CAG CAG GC-30 and RACESQSNF 50 -GAC GAA TAT TGT CAC TAT GTA GCT G-30 . Two step PCR was carried out for 35 cycles with a program of 94 °C, 1 min; 73 °C, 30 s for 50 RACE and 60 °C, 30 s for 30 RACE; 72 °C, 1 min and a final extension at 72 °C, 10 min. The 50 RACE and 30 RACE PCR products were cloned and subjected to sequencing. The sequence analyses of WsSQSs were carried out at the NCBI server (http://www.ncbi.nlm.nih.gov). The physicochemical properties of the polypeptide like pI, molecular weight, etc. were calculated using expasy tools
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(http://expasy.org/tools). The nucleotide and deduced amino acid sequences were analyzed and sequence comparison was conducted through database search using BLAST tool (http://www.ncbi.nlm.nih.gov). Multiple sequence alignment was done by CLUSTAL W. The conserved domains in WsSQSs were determined by Conserved Domain Database software (http://www.ncbi.nlm. nih.gov/Structure/cdd/cdd.shtml). The known SQS amino acid sequences from wide range of different organisms including plants, animals, fungi and bacteria were downloaded from NCBI database and were aligned using CLUSTAL X2. A phylogenetic tree was created based on 500 bootstrap replications with the MEGA4 program [38] by neighbor-joining method [39]. Bacterial expression and purification of WsSQS SQS is a membrane bound enzyme and is anchored to the endoplasmic reticulum through its hydrophobic amino acids at the carboxy terminal [32, 40]. To make the recombinant enzymes soluble and to avoid problems regarding their expression, C-terminal truncated WsSQSs were generated using the forward primer that harbored the NdeI restriction site (50 -CAT ATG GGA ACA TTG AGG GCG-30 ) and a reverse primer (50 -CTC GAG GTA ATT TGG CTC ACT C-30 ) that contained a XhoI restriction site (restriction sites underlined). The reverse primer was located 24 amino acids upstream from the native stop codon in the full length WsSQS. The resulting amplified product with appropriate sites was first cloned in pGEM-T Easy vector. Clones were digested from pGEM-T Easy vector with the same restriction enzymes and the resultant digested products were then directionally cloned in pET30b (?) expression vector (Novagen) already flanked with same restriction enzymes. The constructs pET30b (?)–WsSQS1 and pET30b (?)– WsSQS2, were transformed into the E. coli strain BL21 (DE3). For WsSQS expression, the recombinant cells were grown till the OD600 reaches 0.5–0.6. Then this exponentially growing culture was induced by adding IPTG to a final concentration of 1 mM and kept at 30 °C for 3 h. The recombinant cells were harvested by centrifuging them at 8,000 g for 15 min at 4 °C. The pellet was resuspended in lysis buffer (50 mM Tris–Cl, pH 8.0, 300 mM NaCl, 0.5 % Triton X-100, 10 % glycerol) and then sonicated at 4 °C for 3–4 min (30 s pulse of on/off) at 75 % amplitude. Lysozyme was added to a final concentration 0.1 mg/ml and kept on ice for 20 min. The sonicated sample was then centrifuged at 10,000 g for 20 min at 4 °C and supernatant was used as soluble lysate. The pellet containing inclusion bodies was dissolved in denaturing urea buffer (8 M Urea, 150 mM NaCl). Protein concentration of both fractions
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was estimated by Bradford method [41] (Biorad, USA) using BSA standard graph and analyzed on SDS-PAGE by Coomassie Blue staining. The protein from lysate was purified using Ni–NTA affinity column chromatography. The column was first washed with sterile distilled water and equilibrated with binding buffer (pH 8.0 Tris–Cl 50 mM, NaCl 300 mM, imidazole 20 mM). The lysate was then loaded onto the column and kept for binding for 2 h. The column was then washed with wash buffer (pH 8.0 Tris–Cl 50 mM, NaCl 300 mM, imidazole 30 mM) until OD280 becomes zero and protein was eluted using elution buffer (pH 8.0 Tris–Cl 50 mM, NaCl 300 mM, imidazole 250 mM). The purified fractions of proteins were collected and analyzed on SDSPAGE by silver staining. Enzyme assay and product analysis Assay for SQS activity is based on the conversion of FPP to squalene. The reaction mixture of the SQS assay was as follows: 200 lg purified protein, 200 lM FPP, 1 mM NADPH and the total reaction volume was made to 400 ll with buffer (1 mg/ml BSA, 1 % Tween, 1 % b-mercaptoethanol, 10 mM MgCl2 and 20 mM HEPES buffer). The reaction mixture was incubated at 30 °C for 2 h and then extracted using NaCl and TBME solvent. This was then concentrated using N2 bubbles and then loaded into GC column. The product formation after the reaction was analyzed by a gas chromatography mass spectrometer (Agilent 5975C mass selective detector interfaced with an Agilent 7890A gas chromatograph) fitted with a b-cyclodextrin column (30 m 9 0.32 mm 9 0.25 lm, J & W Scientific). The sample size was 1 ll with a splitless injection. The injector temperature was 200 °C. Expression analysis by qRT-PCR In order to investigate the expression profiles of WsSQS in different organs, total RNA was extracted from young leaf, mature leaf, stem and root of same age (3 months old) in vitro grown plant and 2 lg of this RNA was reverse transcribed into cDNA as mentioned above. This cDNA was then used as a template in qRT-PCR with primers SQS-F (50 -TTT ATG ATC GTG AAT GGC ACT TTT C-30 ) and SQS-R (50 -AGC GGT TGA AAC ATG ATG GAA C-30 ) specific to the common coding sequence of WsSQS. The concentration of primers was optimized to overcome the problem of dimer formation and 120 nM concentration was used finally. Amplification was performed under the following conditions: 95 °C for 10 min, 40 cycles of amplification (95 °C for 30 s, 55 °C for 45 s, 72 °C for 30 s), 95 °C for 1 min, 55 °C for 30 s and 95 °C for 30 s. A primer set of 18S-F (50 -GCA CGC GCG CTA CAA TGA AAG-30 )
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Mol Biol Rep (2012) 39:8803–8812 Fig. 2 a Multiple alignment of WsSQS1 and WsSQS2 with the c following other plant SQSs: S. tuberosum SQS (BAA82093.1), S. lycopersicum SQS (ACY25092.1), C. annuum SQS (AAD20626.1), D. inoxia SQS (AAY22201.1), N. tabacum SQS (AAB08578.1) and D. kaki SQS (ACN69082.1). Six highly conserved domains are: substrate binding pocket (yellow highlighted), substrate–Mg2? binding site (blue highlighted), active site lid residues (green highlighted), catalytic residues (residues in red), aspartate-rich region 1 and aspartate-rich region 2 embedded in substrate–Mg2? binding site (DTVED and DYLED respectively). b Putative conserved domains of squalene synthase of W. somnifera. (Color figure online)
and 18S-R (50 -TCT GTA CAA AGG GCA GGG ACG-30 ) were also used at the same time to amplify the 18S rRNA gene (highly conserved house-keeping gene) as an internal control to estimate if equal amounts of RNA was used among the samples. Brilliant SYBR Green QPCR master mix (29 with low ROX, Stratagene, USA) was used for the reaction. The amplified products were analysed with MxPro software attached to qRT-PCR machine (Stratagene Mx3000P, USA). Data was analyzed by comparative Ct method [42, 43].
Results cDNA cloning and phylogenetic analysis of WsSQS Oligonucleotide PCR primers designed from the already known consensus sequences successfully amplified *600 bp fragment of a putative SQS cDNA which showed maximum similarity with S. tuberosum. For full length WsSQS, PCR was carried out with different set of gene specific primers designed from S. tuberosum SQS. Approximately 1.2 kb amplicon was cloned and plasmid was isolated from putative positive clones and confirmed by sequencing. The sequence analysis of about 10–15 colonies resulted in the identification of two different isoforms of squalene synthase from W. somnifera. Further RACE PCR was done to confirm the primer region of the full length sequence. Preliminary confirmation of two isoforms was done by PCR based approach, by designing a new reverse primer (50 -C CTT CGT GCC ACC TAA CA-30 ) from the region different in both the clones with the previous forward primer common to both the isoforms. This gave an amplification of expected size with only one isoform. The overall sequence information revealed that ORF of WsSQS1 was 1,236 bp (GenBank Accession No. GU 181386) and WsSQS2 was 1,242 bp (GenBank Accession No. GU732820), flanked by 85 bp of a 50 UTR, 257 bp of a 30 UTR followed by polyA tail. The molecular mass of WsSQS proteins was predicted to be about 45 kDa from the deduced amino acid sequence. WsSQS1 and WsSQS2 amino acid sequences share 99 % similarity and identity. They are also similar to the squalene
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Fig. 3 Phylogenetic tree analysis of SQSs from different organisms constructed by the neighbor-joining method. Accession numbers: N. tabacum, AAB08578.1; D. inoxia, AAY22201.1, C. annuum, AAD20626.1; S. tuberosum, BAA82093.1; S. lycopersicum, ACY25092.1; W. somnifera, ADC95435.1; W. somnifera clone 1, GU181386; W. somnifera clone 2, GU732820; E. tirucalli, BAH23428.1; G. max, BAA22559.1; L. japonicus, BAC56854.1; G. uralensis, ACS66750.1; D. kaki, ACN69082.1; A. elata, ADC32654.1; P. ginseng, ACV88718.1; A. annua, AAR20329.1; Z. mays, NP_001104839.1; A. thaliana, AAB61927.1; H. sapiens, NP_004453.3; S. scrofa, NP_001161120.1; M. musculus, NP_034321.2; C. glabrata, BAB12207.1; S. cerevisiae, ACD03847.1; T. reesei, EGR47283.1; A. niger, XP_001395275.1; S. aureus, YP_001317747.1. Distances between each clone and group are calculated with Mega 4 program
synthase protein reported from other plants. WsSQS1 and WsSQS2 show 97.6 and 96.6 % similarity and 94.6 and 93.7 % identity with C. annuum; 98.1 and 97.1 % similarity and 94.4 and 93.5 % identity with S. tuberosum; 97.3 and 96.4 % similarity and 93.2 and 92.3 % identity with N. tabacum; 91.5 and 90.6 % similarity and 88.3 and 87.4 % identity with Datura inoxia; 84.8 and 83.9 % similarity and 70.1 and 69.3 % identity with Arabidopsis thaliana respectively. WsSQS show specific hits with trans-isoprenyl diphosphate synthases and belongs to superfamily of isoprenoid biosynthesis class 1 enzyme. According to conserved domain database six highly conserved peptide domains have been identified in the known squalene synthases (Fig. 2b). The amino acid residues in these domains are also conserved in the WsSQS polypeptide. These domains are situated between 40 and 340 amino acid residues. Amino acids at the carboxy terminal region of the protein
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exhibit least sequence identity because carboxy terminal is very hydrophobic and it is considered to anchor the protein in the ER membrane. Domain I is substrate binding pocket or chemical binding site which consists of about 18 amino acids distributed throughout the polypeptide. Domain II is substrate–Mg2? binding site which consists of 10 amino acids divided in two patches of 5 each in which domain V and VI (aspartate rich region 1 and 2 respectively) are embedded. Domain III is active site which is composed of about 9 amino acid residues. Domain IV is catalytic domain of about 14 amino acids distributed throughout the polypeptide. The catalytic site is composed of the large central cavity formed by antiparallel alpha helices with two aspartate rich regions (DXXXD) on opposite walls [44–47] (Fig. 2a). These residues are considered to play role in binding of prenyl phosphates by binding Mg2? ions. A phylogenetic tree (Fig. 3) was constructed by using known SQS sequences from various different type of
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organisms suggesting that the different form of SQS were evolved from a single ancestral gene. Phylogenetic tree was divided in four clusters. Cluster I and II were composed of dicots. All mammalian enzymes were grouped in cluster III and cluster IV consisted of all fungal SQS enzymes. WsSQS1 and WsSQS2 fall in cluster I and were found closely related to Solanum species followed by C. annuum. Expression and purification of WsSQS in E. coli
Fig. 4 SDS-PAGE analysis of WsSQSs expressed in E. coli. Lane M protein molecular size marker, lane 1 inclusion bodies of WsSQS1, lane 2 lysate of WsSQS1; lane 3, second eluted fraction from lysate of WsSQS1, lane 4 third eluted fraction from lysate of WsSQS1, lane 5 inclusion bodies of WsSQS2, lane 6 lysate of WsSQS2, lane 7 second eluted fraction from lysate of WsSQS2, lane 8 third eluted fraction from lysate of WsSQS2. Position of purified eluents is about 43 kDa
Fig. 5 GC–MS detection of WsSQS catalysed product squalene. a Gas chromatogram of authentic squalene, its mass spectrum is shown in (d); b gas chromatogram of an in vitro reaction mixture of
The truncated WsSQS (that contained a deletion of the last 24 amino acids at carboxy terminus), were inserted into vector pET30b (?) and transformed into E. coli BL21 (DE3). The lysate and inclusion bodies of both the construct were checked on SDS-PAGE and predicted molecular mass of approximately 43 kDa was detected. The same was done with full length WsSQS but it was found that the expression was prominent in case of truncated WsSQS (data not shown). The truncated protein was further purified using
purified WsSQS, its mass spectrum is shown in (e); c gas chromatogram of an in vitro reaction mixture in absence of NADPH
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Fig. 6 Transcript expression analysis of WsSQS (qRT-PCR). Fold expression of WsSQS versus four different parts of the plant. Maximum expression was observed in young leaves and least in roots. Data is expressed as mean ± standard deviation of triplicates
Ni–NTA affinity chromatography and purified fractions of both the isoforms were checked by SDS-PAGE (Fig. 4). To confirm WsSQS as a functional gene and whether the purified enzymes are active or not, preliminary enzyme assays were conducted using GC–MS (Fig. 5). A comparison of the retention time and mass fragmentation pattern of the samples with standard squalene (Sigma) confirms that the recombinant WsSQS enzymes are active and catalyzed two-step cyclization reaction of FPP to squalene via PSPP. In contrast, no such peak and mass fragmentation pattern were detected in control where enzyme was not added in the reaction mixture. The assay was also done in absence of NADPH where squalene peak was not obtained which clearly indicates that NADPH is indispensable component for the reaction. Expression profile of WsSQS in different organs To analyze WsSQS transcript accumulation in different parts of the plant, qRT-PCR was performed. The data revealed that the gene has highest expression in young leaves while the lower amount was detected in roots. Young leaves accumulate 13-folds more SQS transcript as compared to roots and 1.5-folds more than mature leaves. Around 5-folds expression of SQS was observed in stem as compared to roots (Fig. 6).
Discussion Though the plant, W. somnifera has been well characterized in terms of pharmaceutical activities as well as
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phytochemical profiles, not much is known about the genes responsible for biosynthesis of these compounds. To gain new insights into the role of SQS in the isoprenoid biosynthetic pathway in higher plants, we isolated cDNA coding Withania SQS. This is the first attempt of cloning and characterization of this gene from W. somnifera. We isolated two full length SQS isoforms which were confirmed preliminarily by PCR based approach. The two SQS genes share 99 % identity and show a high level of sequence similarity in contrast to the SQS isoforms reported from A. thaliana which share 79 % identity and 88 % similarity [30]. Both WsSQS genes have been deposited in GenBank and characterized bioinformatically. Phylogenetic analysis showed WsSQSs are clustered in dicots group and are closely related to Solanum spp. and C. annuum. CDD search revealed 6 conserved peptide domains in WsSQS which is consistent with the SQS from S. pombe [24], Taxus cuspidata [48], G. lucidum [20] and C. annuum [37] although there are differences in some amino acid residues of these domains. The truncated version of the WsSQS was obtained to get more protein in soluble fraction and purified recombinant protein was obtained after Ni–NTA affinity chromatography. GC–MS analysis confirmed that the recombinant SQS proteins could catalyze the formation of squalene from FPP and NADPH is essential requirement of the reaction. Somewhat similar product estimation of SQS was also reported in E. tirucalli [29]. Results of qRT-PCR analyses of WsSQS expression demonstrated that WsSQS has tissue specific expression with highest expression in leaves and lowest in roots. The similar results were found with the expression of some other genes of the same pathway [49, 50] while some different observation were seen in T. cuspidata where the gene expresses constitutively in all tissues and highest in roots [48] and tobacco where SQS was found to be localized predominantly in shoot apical meristem [26]. The rationale of the present study is to regulate the isoprenoid biosynthesis through the over-expression of SQS gene so as to enhance the production of withanolides in W. somnifera. Further work of development of transgenic Withania with altered withanolides content is underway which will provide clear insights about the role of SQS in sterol biosynthesis. Such studies will not only be beneficial for better understanding of this pathway but will also provide molecular wealth for biotechnological improvement of this medicinal plant. Acknowledgments The authors thank Dr. H. V. Thulasiram, Organic chemistry, National Chemical Laboratory (Pune, India) for providing GC–MS facility; Council of Scientific and Industrial Research (CSIR), New Delhi, India for financial support and University Grants Commission (UGC), New Delhi, India for providing fellowship.
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