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SmKSL overexpression combined with elicitor treatment enhances tanshinone production from Salvia miltiorrhiza hairy roots

https://doi.org/10.1016/j.bej.2020.107562Get rights and content

Highlights

  • The total tanshinone content increased by a maximum of 2.7-fold in Salvia miltiorrhiza hairy roots by overexpressing SmKSL.

  • Treatment with elicitors further increased the total tanshinone production in the SmKSL-overexpressing lines.

  • The tanshinone accumulation correlated positively with the transcriptional level of biosynthetic genes.

Abstract

Tanshinones are compounds of diterpenoid, which are used in the treatment of cardiovascular diseases; however there is an urgent need to improve their production using biotechnological methods. In the present study, Agrobacterium-mediated transformation was used to produce hairy roots of Salvia miltiorrhiza expressing kaurene synthase-like (SmKSL), a tanshinone biosynthesis gene. The results revealed that the yield of total tanshinone increased by a maximum of 2.7-fold in the transgenic Salvia miltiorrhiza hairy roots overexpressing SmKSL as compared with wild-type control lines. Treatment with elicitors further increased the total tanshinone production in the SmKSL-overexpressing lines. Yeast extract treatment of line p35S::SmKSL-15 produced the highest total tanshinone content (4.2 mg/g dry weight), representing an increase of 3.5-fold compared with that in the wild-type control lines. At the same time, qRT-PCR analysis indicated that tanshinone accumulation correlated positively with the transcriptional level of the two pathway genes.

Introduction

Salvia miltiorrhiza (also known as danshen) is a well-known herbal medicine, which has been listed in Chinese official Pharmacopoeia as being useful to treat cardiovascular and circulatory disease, menstrual disorders, and to prevent inflammation [[1], [2], [3]]. The two main active ingredients of S. miltiorrhiza are liposoluble tanshinones and hydrosoluble phenolic acid [4,5]. Tanshinones comprise abietane-type diterpenes, including cryptotanshinone, dihydrotanshinone I, tanshinone IIA and tanshinone I. The biological activities of tanshinones include, anti-oxidation, anti-ischemia, anti-tumor, and anti-inflammatory effects [6,7]. Tanshinone I, cryptotanshinone and tanshinone IIA, have antioxidant activities. Dihydrotanshinone I and cryptotanshinone have antimicrobial activities. Tanshinone IIA prevents DNA damage in hepatocytes by inhibiting the association of DNA with lipid peroxidation products [8]. However, traditional agricultural cultivation of tanshinones cannot meet the rapidly growing market demand because of their low content, the long growth cycle of S. miltiorrhiza, and high consumption. Thus the future of the tanshinone supply will depend on biotechnological means [9].

S. miltiorrhiza hairy root culture is a potential tanshinone production system [10,11]. Recent studies have shown that abiotic and biotic elicitors, e.g., yeast extract (YE), methyl jasmonate (MJ), Co2+, and Ag+, could improve tanshinone accumulation in S. miltiorrhiza hairy roots [12]. The contents of cryptotanshinone, dihydrotanshinone I, tanshinone IIA, and tanshinone I were 0.42 mg/g DW, 0.60 mg/g DW, 0.34 mg/g DW, and 0.82 mg/g DW after treatment with 15 μM Ag+ for 6 days, which were 4.40, 2.56, 1.42, and 1.46 fold higher compared with those in the control, respectively [13]. Furthermore, the cryptotanshinone, tanshinone I, and tanshinone IIA contents increased by 30.0, 0.87, and 3.90 fold, respectively, after 25 μM Ag+ treatment [14]. In addition, YE caused tanshinone accumulation in hairy roots of S. miltiorrhiza in a dose-dependent manner, resulting in the total tanshinone content increasing from 0.46 mg/g DW to 1.37 mg/g DW [15].

The biosynthesis of diterpenoids such as tanshinone comprises three stages. First, two different isoprene biosynthesis pathways: the cytosolic MVA (mevalonate) pathway and/or the plastid-based MEP (2-C-methyl-D-erythritol 4-phosphate) pathway, synthesize isoprene precursor IPP (isopentenyl diphosphate) and its isomer dimethylallyl diphosphate [[16], [17], [18]]. Second, the precursors of geranylgeranyl diphosphate are synthesized by GGPPS (geranylgeranyl diphosphate synthase). In the final stage, a variety of terpenoids are synthesized by terpenoid synthase/cyclase catalysis, including CPS (copalyl diphosphate synthase), KSL (kaurene synthase-like), CYP76AH1 (cytochrome P450 76ah1), and other enzymes [12,19,20]. The first steps toward genetic engineering to enhance tanshinone content in S. miltiorrhiza comprised the successful isolation of S. miltiorrhiza terpenoid biosynthetic genes, including SmHMGR (hydroxymethylglutaryl-CoA reductase), SmDXR (1-deoxy-D-xylulose 5-phosphate reductoisomerase), SmDXS 1-deoxy-D-xylulose 5-phosphate synthase), and SmGGPPS [21]. KSL catalyses diphosphate ester ionization and its subsequent rearrangement into miltiradiene, a key intermediate of tanshinones [22]. However, to the best of our knowledge, enhancing the tanshinone production by overexpression of the SmKSL gene has not been attempted in S. miltiorrhiza.

In the present study, we overexpressed the SmKSL gene in hairy roots of S. miltiorrhiza to increase the tanshinone content. Moreover, the transgenic hairy root lines were treated by different elicitors with the aim of further increasing tanshinone production. We also assessed the expression of genes in both the MEP and MVA pathways for their correlation with tanshinone accumulation.

Section snippets

Construction of the plant expression vector

The SmKSL gene (EF635966.1) was PCR-amplified from a cDNA library of S. miltiorrhiza seedlings using primers containing Bam HI and Xma I restriction enzyme digestion sites at their 5′ ends (Supplementary Table S1). The construction of SmKSL overexpression vector was performed as described previously [23]. The disarmed A. tumefaciens strain C58C1, which contained the A. rhizogenes Ri plasmid and the recombinant SmKSL binary vector plasmid, was utilized to genetically transform S. miltiorrhiza.

Transformation of S. miltiorrhiza

S.

Production of transgenic hairy root lines

The modified Agrobacterium strain C58C1, which contained the plant expression vectors, mediated the transformation of S. miltiorrhiza leaves, resulting in transgenic hairy roots. Hairy roots with normal phenotype, kanamycin resistance and normal growth were selected for further analysis. S. miltiorrhiza hairy root genomic DNA was isolated, and the N-terminal region of the SmKSL gene and the CaMV35S promoter were amplified by PCR using specifically designed primers. The primer pair rol A-F and

Discussion

Extensive studies have been made of the activities and complex structures of secondary metabolites identified in plant tissues and cell cultures [28,29]. As a system for secondary metabolites production, hairy roots have certain advantages, such as the ease of establishing a transformation procedure using Agrobacterium rhizogenes Ri plasmid infection of wounded plant tissues, and high genetic stability [[30], [31], [32]]. In addition, hairy roots and the normal roots show no significant

Conclusions

The results of the present study revealed that a combination of SmKSL overexpression and elicitor treatments, such as YE or MJ, could enhance the tanshinone content effectively in the hairy roots of S. miltiorrhiza. Moreover, tanshinone accumulation correlated positively with key gene expression, including, SmDXR, SmHDS, SmHMGR, SmPMK, SmGPPS, SmCPS, SmKSL, and SmCPR in transgenic hairy roots. Our study demonstrated that it was a more effective strategy to enhance tanshinone level in S.

Authors contribution

Tao Wei, Yong Zhang, Chunguo Wang and Chengbin Chen designed the experiments, analyzed the data and wrote the manuscript. Tao Wei and Kejun Deng performed the main experiments in this study. Yonghong Gao contributed reagents, materials, and helped in the tanshinone detection experiments. Li Chen and Wenqin Song contributed to data analyses and discussion.

Declaration of Competing Interest

All authors read and approved the final manuscript and declared no conflict of interest.

Acknowledgments

This work was supported by the National Basic Research Program of China (2017YFD01005050102), the National GMO New Variety Breeding Major Project (2018ZX08020003-001-003), the Fundamental Research Funds for the Central Universities (2672018ZYGX2018J078), the National Natural Science Foundation of China (81872957). We would like to thank the native English speaking scientists of Elixigen Company (Huntington Beach, California) for editing our manuscript.

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