key: cord-0826518-3avp66bg
authors: Ma, Chunying; Liu, Chunsheng; Wang, Wenquan
title: Molecular cloning and characterization of GuHMGR, an HMG-CoA reductase gene from liquorice (Glycyrrhiza uralensis)
date: 2011-09-09
journal: Front Agric China
DOI: 10.1007/s11703-011-1121-3
sha: 40576184de4756734299f52f0293e672e3ff7aa7
doc_id: 826518
cord_uid: 3avp66bg

A full length cDNA encoding HMGR (designated as GuHMGR) was isolated from liquorice (Glycyrrhiza uralensis) based on degenerated PCR and genome walking. The full length cDNA of GuHMGR was 2330 bp with a 1518-bp open reading frame (ORF) encoding a 505-aa polypeptide. Bioinformatics analysis indicated that there were two trans-membrane domains in GuHMGR. A molecular model of tertiary structure showed that GuHMGR is a novel HMGR with a similar spatial structure to other plant HMGRs. The deduced polypeptide of GuHMGR has an isoelectric point (pI) of 6.41 and a calculated molecular weight of about 54.7 kDa. Sequence comparison and phylogenetic tree analysis showed that GuHMGR had the highest homology with HMGRs from Pisum sativum and Medicago truncatula, indicating that GuHMGR belongs to the plant HMGR group. Expression analysis showed the similar amount of transcript level of GuHMGR in roots and leaves, suggesting that this gene was expressed constitutively in plants. Therefore, this novel HMGR gene would possibly provide a new strategy for studying the glycyrrhizin metabolism at the molecular level in the future.

Liquorice (Glycyrrhiza uralensis Fisch.), also known as GANCAO or TIAN CAO in Chinese, is one of the many valuable medicinal plants for its great therapeutic effects. Usually, the roots and rhizomes of liquorice are used as sources of medicinal materials. Its major bioactive component is glycyrrhizin, which has the medicinal effects of antiinflammation (Matsui et al., 2004) and hepatoprotection (Van Rossum et al., 1998; Shibata, 2000) , etc. Glycyrrhizin also has antivirus effects, such as being anti-HIV (Ito et al., 1987) and against severe acute respiratory syndrome (SARS) associated coronavirus (Cinatl et al., 2003) . In the meantime, glycyrrhizin is widely used in large quantities as a wellknown natural sweetener and flavoring additive because of its sweet taste. This many a day, glycyrrhiza plants used as medicinal materials were collected from wild resources. The everincreasing demand of glycyrrhiza plants has resulted in a dramatic decline in the wild resource reserves and the meadow desertification by collecting the wild glycyrrhiza plants. In the past several decades, the glycyrrhiza plants have been cultivated as additional resources in order to increase the glycyrrhiza resources. However, the glycyrrhizin content is far lower from cultivated glycyrrhiza plants than from wild ones.

Glycyrrhizin belongs to triterpenoid saponin with a similar structure to terpenoid. All terpenoids are produced through a common biosynthetic pathway. Based on the previous studies (Hayashi et al., 2001; Jiang et al., 2006) , the glycyrrhizin synthesis pathway of triterpenoid saponins is listed in Fig. 1 . The series of enzyme catalyzed reactions suggested the glycyrrhizin biosynthesis pathway is a complex course. 3hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), catalyzing the conversion of HMG-CoA to MVA, is considered as the first step in the MVA pathway in plants (Chappell, 1995) . It is also considered a major rate-limiting step in the isoprenoid biosynthetic pathway (Bach, 1995) .

To date, a large number of plant genes for HMG-CoA reductase have been isolated and sequenced. The gene encoding HMGR has been cloned from some plants such as Arabidopsis thaliana (Caelles et al., 1989; Learned and Fink, 1989; Enjuto et al., 1994) , potato (Park et al., 1992) , tomato (Choi et al., 1992) , Catharanthus roseus (Maldonado-Mendoza et al., 1992) , Camptotheca acuminate (Maldonado-Mendoza et al., 1997) , Taxus media (Liao et al., 2004) , Eucommia ulmoides oliver (Jiang et al., 2006) and Ginkgo biloba (Shen et al., 2006) . The HMGR gene cloning might provide the basis for increasing terpenoid production by genetic engineering. However, there is no report yet on the cloning of HMGR from liquorice species. In the present study, we isolated and characterized a novel gene encoding HMGR from G. uralensis, aiming at further exploration in the biochemical regulation mechanism of glycyrrhizin biosynthesis and improving the glycyrrhizin concentration in cultivated plants based on bioengineering approach in the future.

The perennial rhizomes of G. uralensis were collected from Inner Mongolia Autonomous Region, China and transplanted in a greenhouse located in Agricultural University of Hebei, China. Routine cultivation was conducted for normal growth of G. uralensis plants. One month later, the fresh tissues of G. uralensis were sampled for genomic DNA extraction, gene cloning and gene expression analysis.

Total DNA and RNA isolation All tissue materials including leaves and roots were obtained from G. uralensis plants and then triturated by mortar and pestle in liquid nitrogen. Total RNA was isolated with cetyltrimethylammonium bromide (CTAB)-containing extraction buffer, essentially as described previously (Chang et al., 1993) . Genomic DNA was extracted from leaves of G. uralensis using CTAB method with different ingredient concentrations. The RNA and DNA quality were determined by agarose gel electrophoresis and spectrophotometer analysis before use.

Cloning partial genomic sequence of GuHMGR According to the conserved sequence of HMGR gene shared by other plant species, a pair of primers GuHMGRF1 (5′-TTCTTCTCCGTCGCGTACTTTCT-3′) and GuHMGRR1 (5′-GCTAGATCTGTTGTATTTCATGTG-3′) were designed and synthesized. The predicted PCR product contained the terminal codon for the reverse primer in the position of 3′ nontranslated region. PCR was carried out in a total volume of 20 µL reaction which contained 2 µL 10 Â buffer (plus Mg 2+ ), 1.6 µL 10 mM each of dNTPs, 0.4 µL 10 µM GuHMGRF1, 0.4 µL 10µM GuHMGRR1, 1µL genomic DNA and 0.1 µL Ex Taq DNA polymerase (TaKaRa, China). The thermal reactions were as follows: denaturation at 94ºC for 3 min, followed by 40 cycles for 30 s at 94ºC, 30 s at 53ºC, and 2 min at 72ºC, with a final extension at 72ºC for 10 min. The amplified product was purified, ligated into pUCm-T vector and cloned into E. coli strain DH5а followed by sequencing.

Cloning of the full-length DNA sequence of GuHMGR Based on the above partial DNA fragment of GuHMGR, genome walking was carried out to obtain the full gene sequence of GuHMGR. For that, in total, three 5′ primers with reverse orientation were designed including GuHMGRSP1 (5′-CTTTTGGTGGGAGCTTCGGGGAG-3′), SP2 (5′-GAT-GAGGGAGACGATGGCGGC-3′) and SP3 (5′-GAGGA-GAAAGTACGCGACGGAGAA-3′). The genome walking procedure was performed as the manufacturer's recommendation by using the GenomeWalker TM Universal Kit (TaKaRa, China). The amplified products were purified, ligated into pUCm-T vector and cloned into E. coli strain DH5α followed by sequencing. The full-length DNA of GuHMGR was obtained by assembling these two sequences derived from PCR and genome walking.

Cloning of partial cDNA sequence of GuHMGR According to the full length DNA sequence of GuHMGR, a pair of primers GuHMGRF1 (5′-TTCTTCTCCGTCGCGTA -CTTTCT-3′) and GuHMGRR3 (5′-TTTTGATACCATGTT-CATCCCCAT-3′) were designed and synthesized. RT-PCR was carried out to amplify the conserved fragment of HMGR gene from G. uralensis. The PCR reaction solution and the PCR course were similar to that used for cloning of the partial genomic sequence of GuHMGR, but the template was the transcribed cDNA from roots instead of the genomic DNA. The amplified product was purified, ligated into pUCm-T Figure 1 The glycyrrhizin biosynthesis pathway. HMG-CoA stands for 3-hydroxy-3-methylglutaryl coenzyme A, MVA for mevalonic acid, IPP for isopentenyl pyrophosphate, DMAPP for dimethylallyl pyrophosphate, GPP for geranyl pyrophosphate, and FPP for farnesyl pyrophosphate, respectively. HMGR, the enzyme that catalyze the second biochemical reaction from HMG-CoA to MVA, represents 3-hydroxy-3-methylglutaryl-CoA reductase.

vector and cloned into E. coli strain DH5α followed by sequencing from the forward strands.

The full length cDNA sequence of GuHMGR was deduced based on the comparison between the partial cDNA GuHMGR, full length genomic DNA of GuHMGR, and the full-length cDNA of Pisum sativum L. (GenBank accession No. AF303583).

The homologous genes of GuHMGR were identified by BLAST search in which the full length cDNA sequence was the query. The obtained sequences were then analyzed using bioinformatics tool at websites (http://www.ncbi.nlm.nih. gov/, http://www.expasy.org/). The deduced GuHMGR and other known plant HMGRs retrieved from GenBank were aligned with DNAStar software.

To investigate the GuHMGR expression pattern in different parts of G. uralensis, total RNA was extracted from taproots, lateral roots, old leaves and new leaves, respectively. Semiquantitative one-step RT-PCR was carried out according to the manufacturer's instructions (TaKaRa, China). An aliquot of 20 µL reaction solution containing 2 µL 10 Â buffer (without Mg 2+ ), 1.2 µL 25 mM each of MgCl 2 , 1.6 µL 10 mM each of dNTPs, 0.4 µL 10 µM GuHMGRF1, 0.4 µL 10 µM GuHMGRR3, 1 µL cDNA and 0.15 µL rTaq DNA polymerase (TaKaRa, China). The template was denatured at 94ºC for 3 min, followed by 40 cycles of amplification (30 s at 94ºC, 30 s at 53ºC, 2 min at 72ºC) and a final extension at 72ºC for 10 min. The PCR results were determined by agarose gel electrophoresis.

Using primers GuHMGRF1 and GuHMGRR1, a band of 2633 bp was specially amplified (Fig. 2A) . Sequence analysis showed that it was highly homologous to HMGRs in plant species. To obtain the full length of GuHMGR genomic sequence, genome walking was conducted in which three primers including SP1, SP2 and SP3 were then designed for the PCR amplification of the unknown 5′ region. A product of 437 bp was specially amplified (Fig. 2B) . The genomic DNA of GuHMGR was then obtained by assembling the sequences of the 2633 bp and 437 bp.

Comparison between the GuHMGR genomic sequence and the cDNA of Pisum sativum showed that the genomic DNA contained two introns. The lengths of three exons were 1206 bp, 181 bp and 943 bp, respectively. The lengths of the two introns were 87 bp and 245 bp, respectively.

The full length cDNA of GuHMGR was obtained based on the comparison between the partial cDNA GuHMGR, full length genomic DNA of GuHMGR, and the full-length cDNA of Pisum sativum L. (GenBank accession No. AF303583). Sequencing results showed that the GuHMGR was 2330 bp in length and contained a 1518 bp ORF, encoding a protein of 505 amino acids (Fig. 3) , with an isoelectric point of 6.41. The calculated molecular weight of GuHMGR was about 54.7 kDa, which is similar to previously reported plant HMGRs. The GuHMGR showed a homology with Pisum sativum HMGR (67.3), Medicago truncatula HMGR3 (65.3) Figure 2 The PCR amplified pattern of the GuHMGR gene from Glycyrrhiza uralensis Fisch. A is 2633 bp conservative sequence of the GuHMGR gene using the primer pair (GuHMGRF1, GuHMGRR1); B is Genome walking products of the GuHMGR gene. M is Molecular weight marker DL2000; 1-4 represent 2633 bp conservative sequence of the GuHMGR gene, the first round PCR product of genome walking, the second round PCR product of genome walking, and the third round PCR product of genome walking respectively. and HMGR2 (64.1) and HMGR1 (63.3), and Litchi chinensis HMGR (64.1) (Fig. 4) .

Sequence comparisons by performing Blast Search in GenBank database (http:// www.ncbi.nih.gov) and multiple alignment analysis revealed that GuHMGR had a high homology with many other plant HMGRs, such as Pisum sativum HMGR (PsHMGR), Hevea brasiliensis HMGR (HbHMGR), Medicao truntula HMGR1-4 (MtHMGR), etc. The results suggested that GuHMGR belongs to the plant HMGR superfamily. Using TMHMM 2.0 (http//www.cbs. dtu.dt/services/TMHMM-2.0), two transmembrane regions were predicted with one located between Pro 20 and Leu 42 and the other located between Ile 63 and Ile 85 along the polypeptide chain. A likely signal peptide cleavage site between Ala 28 and Phe 29 was found using SignalP3.0 (http://www.cbs.dtu.dk/services/signalP/). To better characterize the GuHMGR protein, comparative 3-D modeling was performed by SWISS-MODEL (Schwede et al., 2003) with the HMGR from human as the template, the crystal structure of which has been elucidated (Fig. 5) .

To investigate GuHMGR expression pattern in different tissues of G. uralensis, the one-step RT-PCR analysis was performed using same amount of total RNA isolated from different tissues including old leaves, new leaves, the taproots and lateral roots as the templates and the HMGRF1 and HMGRR3 as the primers. The result showed that GuHMGR is expressed in all four tested tissues at similar transcriped levels, suggesting that this gene is expressed in a constitutive pattern in plants (Fig. 6) .

The full-length cDNA sequence and deduced amino acid sequence of G. uralensis 3-hydroxy-3-methylglutary1-CoA reductase (GuHMGR). The start codon (ATG) and the stop codon (TAA) are boxed.

HMGR was studied initially in mammals because of its significant role in regulating the cholesterol biosynthesis (Goldstein and Brown, 1990 ). Subsequently, the HMGRs have been widely studied in plant species. In tomato, a HMG1::GUS reporter gene fusion was used to analyze the regulation of HMG1 gene expression which suggested that the role of HMG1 was to supply the MVA demand associated with cell division and growth (Jelesko et al., 1999) .

Till now, only few glycyrrhizin biosynthetic genes have been successfully isolated from glycyrrhiza plants. An oxidosqualene cyclase cDNA encoding β-amyrin synthase, was isolated from cultured cells of Glycyrrhiza glabra (Hayashi et al., 2001) . Two cDNAs of squalene synthase were isolated from G. glabra (Hayashi et al., 1999) . Molecular cloning of 3-hydroxy-3-methylglutaryl coenzyme A reductase in G. uralensis provides a useful tool to elucidate the glycyrrhizin biosyntheses in glycyrrhiza plants. Further experiments are required to study the combined function of the glycyrrhiza gene in plant for glycyrrhizin biosyntheses.

In this paper, the full length genomic DNA sequence and cDNA of GuHMGR gene were cloned from G. uralensis. The deduced polypeptide of GuHMGRs share high identities with other plant HMGRs, such as HMGR from Pisum sativum and Medicago truncatula. This suggested that GuHMGR, the HMGR gene derived from glycyrrhiza plants possibly plays an important role in the biosynthesis in glycyrrhizin.

Usually root and rhizome are used as the sources of medicinal materials. But some reports suggest that other organs of the glycyrrhiza plants also contain the medicinal content such as glycyrrhizin. Hence, it is of great value to determine whether the GuHMGR expression is correlated with the glycyrrhizin content in different tissues of G. uralensis. In this study, the one-step RT-PCR analysis was performed using total RNA from different tissues, including old leaves, new leaves, the taproots and lateral roots with the primers HMGRF1 and HMGRR3. The results showed that GuHMGR was expressed in all four tissues, which suggested that the GuHMGR may be a constitutively expressed gene. However, the expression in roots was higher than that in leaves, corresponding with the fact that the roots are usually used as medicinal material and more glycyrrhizin is accumulated in roots than leaves. Further study will be undertaken to identify the functions of GuHMGR in improving glycyrrhizin biosynthesis based on bioengineering approach in the future.

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Molecular cloning of a HMG-CoA reductase gene from Eucommia ulmoides Oliver

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Structure and nucleotide sequence of tomato HMG2 encoding 3-hydroxy-3-methyl-glutaryl coenzyme A reductase

SWISS-MODEL: An automated protein homology-modeling server

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glycyrrhizin as a potential treatment for chronic hepatitis C

This research was supported by the State Natural Sciences Foundation Projects (No. 30572328 and No. 30672615), and the Ministry of Science and Technology of the People's Republic of China. It was also supported by the Province Natural Sciences Foundation Projects of Hebei (No. C2009000583).