Plant Molecular Biology Reporter

, Volume 26, Issue 1, pp 1–11

Ri-mediated Transformation of Glycyrrhiza uralensis with a Squalene Synthase Gene (GuSQS1) for Production of Glycyrrhizin

Authors

  • Hong-Yu Lu
    • Department of Breeding and GeneticsChina Pharmaceutical University
  • Jing-Mei Liu
    • Chinese Academy of Agricultural Sciences
  • Hai-Chao Zhang
    • Department of Breeding and GeneticsChina Pharmaceutical University
  • Tao Yin
    • College of Agriculture and BiotechnologyZhejiang University
    • Department of Breeding and GeneticsChina Pharmaceutical University
Article

DOI: 10.1007/s11105-008-0018-7

Cite this article as:
Lu, H., Liu, J., Zhang, H. et al. Plant Mol Biol Rep (2008) 26: 1. doi:10.1007/s11105-008-0018-7

Abstract

Root of Glycyrrhiza uralensis, one of the most important medicinal plants, containing bioactive triterpene saponins (glycyrrhizin). Squalene synthase (SQS) plays a regulatory role in the biosynthesis of triterpene saponins. In the present investigation, SQS coding sequence from G. uralensis was cloned by polymerase chain reaction (PCR) and a transgenic system was developed for G. uralensis through Agrobacterium rhizogenes-mediated transformation. The SQS gene placed under a CaMV 35S promoter was transferred into G. uralensis using A. rhizogenes strain ACCC10060. The transformed hairy roots were selected on Murashige and Skoog (1962)-containing phosphinothricin (PPT) and root lines were established. The integration of SQS gene was confirmed by PCR and Southern blot. Three transgenic root lines UP1, UP24, UP31 were obtained and their growth rates were detected. The result showed that transgenic root lines but UP1 line grew faster than control hairy roots; high-performance liquid chromatography (HPLC) analysis demonstrated the highest glycyrrhizin content of transgenic roots was 2.5 mg/g dry weight and was about 2.6 times higher than control hairy roots.

Keywords

GlycyrrhizinSqualene synthase (SQS)Glycyrrhiza uralensisHairy roots

Abbreviations

SQS

squalene synthase

 

β-amyrin synthase

OSC

oxido- squalene cyclase

GuSQS

Glycyrrhiza uralensis squalene synthase

PPT

phosphinothricin

GgSQS

Glycyrrhiza glabra squalene synthase

AS

acetosyringone

Kana

kanamycin sulphate

cef

cefotaxim sodium

cb

carbenicillin

Introduction

Licorice (Glycyrrhizae Radix, Gancao in Chinese and Kanzo in Japanese), one of the most popular medicinal plants in the world, is widely used in many fields such as flavoring agent, medicament, commodity, tobacco. The root and stolons of this plant accumulate a large amount of glycyrrhizin, an oleanane-type triterpene saponins, which is a well-known natural sweetener and is 50 times sweeter than sugar (Hanrahan 2001). Glycyrrhizin and its hydrolysis product, glycyrrhetinic acid, possess many important pharmacological activities, including antiinflammatory (Gibson 1978; Capasso et al. 1983; Shibata 2000), antiulcer (Duke 1985; Dehpour et al. 1994), antitumor (Tokuda et al. 1986; Wang et al. 1991; Abe et al. 1987), antibacterial (Hyung et al. 2002), and antiallergic (Murray 1995) activities. Some derivatives from glycyrrhizin have been used for treatment of chronic hepatitis (Van Rossum and Vulto et al. 1999) and gastric ulcer in clinics.

Recent environmentally irresponsible overcollection of wild Glycyrrhiza plants has resulted in the decrease and extinction of wild Glycyrrhiza resources and in desertification. In 2000, the Chinese government imposed restrictions on the collection of wild glycyrrhiza plants. As a result, to meet the increasing demand for glycyrrhizin, attempts at attaining a mass of glycyrrhizin in licorice cells by means of biotechnology have been made (Henry and Chantalat-Dublanche 1984; Hayashi et al. 1988, 1990, 2003; Ayabe et al. 1990). However, some of their results showed that cell suspension cultures of licorice did not produce glycyrrhizin. It would therefore be interesting and important to increase accumulation of glycyrrhizin in licorice plants and/or organs by genetic engineering. One effective approach toward achieving this goal has been to modify the triterpene biosynthesis pathway leading to the formation of glycyrrhizin.

In licorice, triterpene saponins are synthesized from β-amyrin, which is catalyzed by β-amyrin synthase (bAS), an oxidosqualene cyclase (OSC). OSCs catalyze the cyclization of 2,3-oxidosqualene, a common intermediate of both triterpene and phytosterol biosyntheses (Abe et al. 1993; Haralampidis et al. 2002; Hayashi et al. 2003). 2,3-oxidosqualene comes from squalene, which is catalyzed by squalene synthase (SQS). Squalene synthase catalyzes the first enzymatic step in sterol and triterpenoid biosynthesis (Abe et al. 1993). Since SQS is situated at a putative key branch point in the isoprenoid biosynthetic pathway, it may play a regulatory role in this pathway. At present, many reports show the important roles of the SQS gene in the regulation of triterpene and phytosterols biosynthesis in plants (Devarenne et al. 1998, 2002; Suzuki et al. 2002; Wentzinger et al. 2002; Lee et al. 2004; Seo et al. 2005). Overexpressing SQS gene may possibly affect the accumulation of glycyrrhizin and related triterpene saponins. The reports on transformation of SQS gene in licorice have not been found as yet. In this paper, we describe the effect on accumulation of glycyrrhizin by introducing a GuSQS gene (Glycyrrhiza uralensis SQS1 gene, Acession No. AM182329) to hairy roots of G. uralensis with an Agrobacterium-Ri binary vector.

Materials and Methods

Isolation and Cloning of Glycyrrhiza uralensis Squalene Synthase(GuSQS)

Total RNA was isolated by a total RNA isolation system (Promega) from the leaf of G. uralensis and was used as template to synthesize cDNA coding squalene synthase (SQS).Two oligonucleotide primers were synthesized based on the reported two SQS cDNA sequences available at National Centre for Biotechnology Information (NCBI) database (GenBank Accession no. D86409 and D86410) as follows: forward primer: 5′-ATGGGGAGTTTGGGAGCGAT-3′; reverse primer: 5′-CTA(A/g)TTAT(C/t)(t/A)TGG(C/t)G(T/g)TT(A/g)GCAG -3′. To achieve T-clone, A ‘T’ nucleotide was added to 5′end and 3′end of the respective primers for GuSQS coding sequence. A 20-μl polymerase chain reaction (PCR) reaction mix contained 2.0 μl 10 × High Fidelity Taq buffer (Promega), 0.5 μl of 10 μM each of forward and reverse primers,0.5 μl of 10 mM deoxyribonucleotide triphosphates (dNTPs),100 ng of template cDNA, 2.0 units of High Fidelity Taq DNA polymerase (Promega). Thirty-five cycles of amplification were carried out at 94°C for 3.5 min, 55°C for 30 s, 72°C for 1 min with a 10 min final extension at 72°C. The PCR-amplified product was cloned into pMD 18-T vector (Takara) to generate pMDGuSQS and the nucleotide sequences were determined and verified with the reported sequence.

Construction of Plant Expression Cassette for Agrobacterium-mediated Plant Transformation

The plasmid pMDGuSQS1 was digested completely with XbaI and then digested incompletely with HincIIto obtain the 1.2-kb fragment containing GuSQS1 coding sequence. The gel-purified fragment was directionally cloned into a 130/pLRP1-HB-based plant expression double-T vector (Conservation in our department), which contains bar gene as a selection marker (under the control of CaMV35S promoter and nopaline synthase terminator) in one T-DNA section, regulatory elements consisting of CaMV35S promoter and nopaline synthase terminator for constitutive expression of GuSQS gene in another T-DNA section. The GuSQS1 gene from pMDGuSQS1 clone was inserted in between XbaI and SmaI site located between CaMV35S promoter and nopaline synthase terminator of above-mentioned 130/pLRP1-HB (Fig. 1), forming the vector p130/35S-GuSQS1 that was then used for G. uralensis transformation.
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Fig. 1

Construction of plant expression vector p130/35S-GuSQS1 containing GuSQS1 gene

Transformation and Culture of A. rhizogenes

After amplification in E. coli strain DH5α, plasmid of 130/p35S-GuSQS1 was used to transform Agrobacterium rhizogenes strain ACCC 10060, which was then selected on YEB medium (yeast extract 1.0 g, beef extract 5.0 g, peptone 5.0 g, sucrose 5.0 g, MgSO4•7H2O 0.49 g, pH 7.0) supplemented with 50 mg/l kanamycin sulfate and validated strictly by PCR. The wild type of this strain was cultured in YEB medium without Kan. Growth condition was set at 28°C with constant shaking (180 rpm). When optical density (O.D.) of the liquid containing A. rhizogenes cells was up to 0.7 at 600 nm, the cells were collected by centrifugation and suspended in same cubage of liquid Murashige and Skoog (MS) medium containing 200 μM acetosyringone (AS) as before centrifugation and then used for plant transformation.

Transformation, Induction and Selection of G. uralensis Hairy Root

Induction and selection of G. uralensis hairy root were performed as follows. The seeds of G. uralensis from the northern parts of Mongolia, were surface- sterilized and germinated on Murashige and Skoog (1962) medium containing 2% sucrose and 0.8% agar in dark for 1 day and then transferred to light (16 h light and 8 h dark) for 3 days. Then the hypocotyls and cotyledonary nodes from 4-day-old seedlings were collected and floated in above-mentioned MS liquid medium containing A. rhizogenes cells and incubated for 20 min at 28°C with gentle shaking. After infection, the explants were blotted with sterile filters and transferred to MS solid medium in dark at 25°C for 2 days. Then the explants were washed with sterile water and transferred onto fresh solid MS medium supplemented with 500 mg/l cefotaxim sodium (cef) to eliminate A. rhizogenes. The same medium supplemented with 0, 0.5, 0.8, 1.2, 1.5, and 2.0 mg/l phosphinothricin (PPT) were then used to select the transformations. The explants were subcultured every 5 days in fresh medium containing 500 mg/l cef until adventitious roots were 2 cm in length. Those hairy roots were excised from explants and transferred to fresh medium with 250 mg/l cef and subcultured weekly until the residual bacteria were completely killed. The PPT-resistant hairy roots were further examined by PCR and southern blot for confirmation of the genetic transformation. Liquid cultures were established with 20-mm-long tips from 15-day-old bacteria-free hairy roots. The roots were grown in liquid MS medium at 25°C on a rotary shaker (100 rpm) in the dark.

DNA Isolation, PCR and Southern Blotting Analysis

DNA was isolated from hairy roots using a CTAB method. PCRs were carried out by 30 cycles of 94°C for 3 min, 94°C for 40 s, 56°C for 30 s, and 72°C for 30 s, followed by a final extension step of 72°C, 10 min. DNA (50 ng) isolated from hairy roots was used as templates. Two pairs of primers Bar-F (BF) (5-TGCACCATCGTCAACCACTACATC-3), Bar-R (BR) (5-GCTGCCAGAAACCCACGTCAT-3) and GuSS1-F (5′-AAAGAACTATCAAGCAGCAA-3′), GuSS1-R (5′-AAAGTCAAAGAAAGCACCAT-3′) were synthesized according to bar gene and GuSQS1 cDNA sequences and simultaneously used for PCR amplification. PCR products were electrophoresed on a 1% agarose gel and visualized by staining with ethidium bromide.

The hairy root DNA was digested with EcoR I at 37°C overnight and fractionated on a 1.0% agarose gel. The DNA was then transferred onto a NC membrane. The DNA fragment, obtained by PCR amplification using a pair of primers GuSS1-Fand GuSS1-R, was labeled with digoxigenin (DIG)-dUTP using the PCR DIG Probe Synthesis kit (Roche Molecular Biochemicals). The blot was hybridized with the probe and the signal was detected according to the manufacturer protocol (Roche Molecular Biochemicals).

Analysis of Growth Yield and Glycyrrhizin Content of Hairy Root

Thirty days after they were subcultured, the hairy roots were harvested, dried to be constant weight. The dry weight of hairy roots was used as the growth yield. Glycyrrhizin (100 mg dry weight) were extracted with 4 ml 50% ethanol twice using ultrasonic assisted method (Ultrasonic power 1,000 W), each for 30 min at room temperature. The extract was filtered through a 0.45-mm membrane, and total volumes of the solution were pinpointed 10 ml. An Agilent HPLC 1100 system (America) consisted of a G1311A pump, a multisolvent delivery system, and a 6000LP ultraviolet (UV) detector. The column was a Phenomenex Luna C 18(2), 3.0 × 150 mm. The mobile phase was composed of 0.05 M AcONa/60% acetonitrile featuring gradient elution steps as follows: 0 min, 65:35; 24 min, 40:60; 32 min, 0:100; 50 min, 65:35. The flow rate of mobile phase was 1 ml/min with UV absorbance detection at 254 nm at room temperature. Glycyrrhizic acid ammonium salt (Sigma) was used as a standard. At this wavelength, standard curve was obtained by repeating the same procedure for different concentrations of the standard.

Results and Discussion

Cloning of GuSQS Gene from G. uralensis and Construction of the Plant Expression Vector

The GuSQS coding sequence synthase was amplified by PCR as described in the “Materials and Methods” section. Two sequence of 98.79% and 99.11% respective similarity were obtained from the PCR-amplified product while aligned with the reported gene sequences of GgSQS1 D86409 and GgSQS2 D86410 (Hayashi et al. 1996, 1999) through pairwise online BLAST analysis, and amino acid sequences similarity 98.29% and 98.54%, respectively.

Hayashi et al. (1996) first cloned two SQS cDNAs from cultured cells of G. glabra and characterized their function through prokaryotic expression and designated as GgSQS1 and GgSQS2, respectively. They deemed that the two SQS maybe differentially regulated in the cultured licorice cells (Hayashi et al. 1996, 1999). Their result from prokaryotic expression of the two squalene synthases activity by the recombinant plasmids showed that activity of GgSQS1 was stronger than GgSQS2. However, it is unknown how the differential expression of squalene synthase genes was performed in cultured cells and intact plants of licorice. Here, we also attained two isoforms of squalene synthase from the young leaves of G. uralensis and named GuSQS1 and GuSQS2, respectively. In this study, GuSQS1 gene first was used to construct GuSQS1 plant expression cassette and inserted between the control of CaMV- 35S promoter and the nos terminator element in 130/pLRP1-HB. The recombinant plasmid was designated 130/p35S-GuSQS1 (Fig. 1). A. rhizogenes strain ACCC10060 was used to transfer the T-DNA containing this construct to hairy roots of G. uralensis.

Induction and Selection of Transformed G. uralensis Roots

The hypocotyls and cotyledonary nodes from 4-day-old seedlings of G. uralensis were infected with either A. rhizogenes strain ACCC10060 harboring the binary vector 130/p35S-GuSQS1, or the wild type of this strain. The hairy root-inducing rates were 96% for cotyledonary nodes and 88.5% for the hypocotyls, respectively. The hairy roots from different explants, which grew normally and rapidly, were selected and hairy root lines were established. Different concentrations of PPT, ranging from 0, 0.5, 0.8, 1.2, 1.5, and 2.0 mg/l, were applied to the medium to select for PPT resistant hairy root. All lines of hairy roots stopped growing, following browning and dying after two weeks of culture in medium containing 1.2 mg/l, 1.5 and 2.0 mg/l PPT. On the medium containing 0.5–0.8 mg/l PPT, 19 hairy root lines could grow rapidly as well as on the medium free of PPT. Therefore, a concentration of 0.8 mg/l PPT seemed to be optimal for selection of transgenic hairy roots of G. uralensis containing the bar gene and 19 hairy root lines were established.

The PPT resistant hairy roots were examined by PCRs using primers BF and BR specific to the bar gene and primers GuSS1-F and GuSS1-R specific to the GuSQS1. Specific products (431 and 616 bp, respectively) were amplified from some of PPT resistant lines (Fig. 2). Three lines, UP1, UP24, and UP31, were further analyzed by Southern blot. The result showed that the foreign GuSQS1 and bar gene were integrated into the genome of G. uralensis hairy root lines UP1, UP24, and UP31(Fig. 3). Since double-T vector was used in gene transformation, the hairy roots selected in medium containing 0.8 mg/l PPT should be examined by PCRs using two different primers to ensure two sections T to be transformed simultaneously. Although GuSQS1 gene was derived from G. uralensis, PCRs showed that no specific products were amplified from DNA template of wild hairy roots, which might attribute to many introns in GuSQS1 gene and, consequently, the anticipative products was too long to obtain. Plentiful studies have shown that introns are ubiquitous and take up a large proportion of whole genes in eukaryotes, sometimes up to 90% (Doolitile 1987). Therefore, PCR analysis could not attain products in DNA of the wild hairy roots. The same possibility was shown in the southern blot analysis (Fig. 3).
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Fig. 2

PCR analysis of the transgenic hairy root. 1 Marker (5 kb); 25 are transgenic hairy root lines UP1, UP30, UP31, and UP24, respectively; 6 positive control plasmid of 130/35S- GuSQS1; 7 control

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Fig. 3

Southern blotting analysis of the transformed hairy root with GuSQS1 gene. 1, 3, and 4 are transgenic hairy roots lines UP1, UP24, and UP31, respectively; 2 control hairy root

Successful attempts of transforming G. uralensis by A. rhizogenes and subsequent culture of the hairy roots have been made in some studies (Ko et al. 1987; Saito et al. 1990, 1991). Thereunto, Saito et al. (1990) achieved successfully the transformation with a binary vector system of an Ri plasmid, pRi15834, and a mini Ti vector, pGSGluc1, containing chimeric neo and gus genes. Their results also showed the frequency of double transformation with Ri and a mini Ti was rather high, up to 80%. Successful transformations of binary vector by A. rhizogenes have also been realized in other plant species (Stougaard et al. 1987; Shahin et al. 1986; Simpson et al. 1986; Hamil et al. 1987; Chen et al. 1999).

Growth Characteristics of Transgenic Hairy Roots

Transgenic G. uralensis hairy roots expressing the GuSQS1 gene showed some different characters from control hairy roots. The notable tendencies were more frequent formation of callus and vitrification from most of transgenic hairy roots in the course of subculture on solid MS medium than control. When the roots were cultured on MS solid medium or MS solid medium containing 2.0 mg/l GA3, the degree of vitrification and of formation of callus were modified. However, when transgenic roots with a few calli were cultured in MS liquid medium, normal roots reproduced from most of these roots and grew rapidly. Rate of growth of UP24 and UP31 was 1.3–1.6 times than control root line in a subculture cycle (30 days). Instead, UP1 root lines grew more slowly than control (Fig. 4).
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Fig. 4

Growth yield of G. uralensis hairy root lines. Lane 1: control hairy roots; lane 2: hairy roots of UP1; lane 3: hairy roots of UP24; lane 4: hairy roots of UP31

Chen et al. (1999) reported that formation of callus was discovered in transgenic hairy roots expressing the foreign farnesyl diphosphate synthase (FDS) gene. They presume overexpressing FDS may cause changes of carbon flow in the isoprenoid pathway, from which many phytohormones are derived, and subsequently affect related endogenous hormone levels, giving rise to changes of morphogenesis or characters of transgenic tissues. Squalene synthase plays an important regulation role in biosynthesis of triterpenes and phytosterols, and squalene is a common intermediate of triterpenes and phytosterols. Modification of squalene synthase may have an effect on the synthesis of phytohormones. However, we now have no direct evidence to approve it. More investigations are needed.

Production of Glycyrrhizin in Hairy Roots

The result of HPLC analysis showed different glycyrrhizin production in three transgenic hairy roots expressing foreign G. uralensis squalene synthaseI gene (Fig. 5). For UP31 and UP24 root line, the highest yield of 2.5 mg/g dry weight of glycyrrhizin content was detected, which is about 2.6 and 1.4 times higher than control, respectively (Fig. 6). However, the yield of UP1 of glycyrrhizin content was lower 0.8 times than control (Fig. 6).
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Fig. 5

Spectrometric HPLC of glycyrrhizin in hairy root. (a) Transgenic root line UP31; (b) control hairy roots

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Fig. 6

Contents of glycyrrhizin in hairy roots detected by HPLC. Bar 1: control hairy roots; bar 2: hairy roots of UP1; bar 3: hairy roots of UP24; bar 4: hairy roots of UP31

So far, there have been reports about glycyrrhizin in plant cell, tissue, and organ cultures of licorice (Ko et al. 1987; Saito et al. 1990), which showed different results. Ko et al. detected the glycyrrhizin in hairy roots of G. uralensis. Their results showed 2.2–4.7% glycyrrhizin content in hairy roots of G. uralensis. However, the results from Kazuki showed no detectable glycyrrhizin in the hairy roots of G. uralensis. In our study, low glycyrrhizin could be detected in the wild hairy roots of G. uralensis cultured for 30 days. Maybe un-uniform conditions of hairy roots culture and glycyrrhizin detection lead to different results.

In the isoprenoid biosynthetic pathway, the carbon flow starting from the farnesyl diphosphate may follow in two different pathways containing synthesis of sesquiterpenes catalyzed by sesquiterpene synthase and synthesis of sterols and saponins catalyzed by squalene synthase. Here, glycyrrhizin yields of transgenic root lines UP24 and UP31 by overexpressing squalene synthase were increased, while glycyrrhizin yield of UP1, one of transgenic root lines, was declined. This might be explained by cosuppression following foreign gene transformation in UP1 root line. We will demonstrate the result through further investigations.

In licorice, soyasaponin as well as glycyrrhizin, which is a type of tritrepene saponin, is derived from a common intermediate, β-amyrin. Hayashi et al. (1988, 2003) reported that cell suspension cultures of licorice did not produce glycyrrhizin, but produce soyasaponin. Moreover, upregulation of soyasaponin biosynthesis as a result from the upregulation of related enzyme activities was discovered in cultured cells of G. glabra treated by methyl jasmonate. The related enzymes include SQS and BAS. Subsequently, the content of soyasaponin in transgenic hairy roots producing higher glycyrrhizin has a possible change. The results from Lee et al. (2004) showed overexpression of squalene synthase followed by the upregulation of all the downstream synthase genes expression. Therefore, it is necessary to us to investigate downstream synthase genes expression and production of other related metabolites in transgenic hairy roots.

Acknowledgments

This work was supported by grants from Nation Crop Molecular Design Center and from Nation ‘863’ Program in Peking University, China. We thank Prof. Xi-Ping Wang and Mrs Xie Yin for their help and advices.

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© Springer-Verlag 2008