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Molecular cloning and characterization of two 1-deoxy-d-xylulose-5-phosphate synthase genes involved in tanshinone biosynthesis in Salvia miltiorrhiza

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Abstract

The synthesis of diterpenoid tanshinones, which are some of the main bioactive ingredients of Danshen (the roots of Salvia miltiorrhiza), begins with the plastidial methylerythritol 4-phosphate (MEP) pathway. 1-Deoxy-d-xylulose-5-phosphate synthase (DXS) catalyzes the first step of the MEP pathway and is considered to be a key rate-limiting enzyme. To confirm the function of DXS genes, in this study two isoforms of DXS (SmDXS1 and SmDXS2) were isolated by rapid amplification of DNA ends from leaves of S. miltiorrhiza. Bioinformatic analyzes indicated that two SmDXSs had high homology with other plant DXSs. Tissue expression pattern analysis revealed that SmDXS1 and SmDXS2 exhibited distinct expression patterns in S. miltiorrhiza. SmDXS1 was expressed in all analyzed tissues; the highest expression was observed in leaves, followed by stems, with weak expression in roots. In contrast, SmDXS2 transcripts were predominantly detected in roots, but very weakly. Subcellular localization analysis revealed that SmDXS1 and SmDXS2 were both localized in the chloroplast. Agrobacterium-mediated transformation was used for induction of transgenic hairy root lines and tanshinone yield was assessed by high-performance liquid chromatography. Compared with the control, overexpression of SmDXS1 and SmDXS2 significantly enhanced tanshinone accumulation in transgenic roots, whereas only down-regulation of SmDXS2 led to a significant decrease. Our results indicated that SmDXS2 was an important rate-limiting gene in the MEP pathway involved in tanshinone biosynthesis and could be an effective regulatory target for controlling tanshinone accumulation in S. miltiorrhiza hairy root cultures.

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Acknowledgments

This work was supported by National Natural Science Fund (81522049, 31270007, 31571735, 31501394), New Century Talent Project (NECT-13-0902), the State Key Laboratory of Phytochemistry and Plant Resources in West China (P2015-KF02), Shanghai Science and Technology Committee Project (15430502700).

Author contributions

GK conceived and designed the study. WZ, FH, CZ, MS, JC, SL and JW performed the research. WZ, CZ, MS and YW analyzed the data. WZ, CZ, GK, YW and HW wrote the paper. All authors read and approved the final manuscript.

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    Authors and Affiliations

    1. Laboratory of Plant Biotechnology, College of Life and Environment Sciences, Shanghai Normal University, 100 Guilin Road, Shanghai, 200234, People’s Republic of China

      Wei Zhou, Fenfen Huang, Congcong Zhou, Min Shi, Jing Wang, Yingjie Chen, Yao Wang & Guoyin Kai

    2. The Key Laboratory for Quality Improvement of Agricultural Products of Zhejiang Province, School of Agriculture and Food Science, Zhejiang A&F University, Linan, Hangzhou, 311300, Zhejiang, People’s Republic of China

      Wei Zhou, Shen Li & Yang Wang

    3. Zhejiang Provincial Key Laboratory for Genetic Improvement and Quality Control of Medicinal Plants, Hangzhou Normal University, Hangzhou, 310018, People’s Republic of China

      Huizhong Wang

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    1. Wei Zhou
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    Correspondence to Guoyin Kai.

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    The experiment conducted complies with the laws of China.

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    Wei Zhou and Fenfen Huang have contributed equally to this work.

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    Zhou, W., Huang, F., Li, S. et al. Molecular cloning and characterization of two 1-deoxy-d-xylulose-5-phosphate synthase genes involved in tanshinone biosynthesis in Salvia miltiorrhiza . Mol Breeding 36, 124 (2016). https://doi.org/10.1007/s11032-016-0550-3

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