T-DNA Activation Tagging as a Tool to Isolate Salvia miltiorrhiza Transgenic Lines for Higher Yields of Tanshinones

Abstract

The study of functional genomics has paved the way for directed approaches to the generation of genetically modified plants that produce novel and/or improved yields of pharmaceuticals. In the present study, an activation tagging mutagenesis (ATM) population of Salvia miltiorrhiza Bunge, a medicinal plant, was established by Agrobacterium-mediated transformation. The optimum conditions for Agrobacterium transformation were determined by the expression of green fluorescent protein. Under these optimized conditions, we isolated 1435 ATM cell lines with our initial antibiotic selection. Of these 1435 ATM cell lines, six lines (T1 - T6) showed a red color on a selective medium containing 4.5 μM 2,4-dichlorophenoxyacetic acid (2,4-D), which is used as a phenotypic model system to identify the accumulation of tanshinones. 700 out of 1435 ATM cell lines were tested with a β-glucuronidase (GUS) assay, 35 showed GUS activity. Southern blotting analysis revealed that the T1 - T7 ATM cell lines have a single copy of the T-DNA insertion. Comparative analysis by high-performance liquid chromatography of the tanshinones expressed by non-transformed and ATM-transformed calli revealed varying quantities of tanshinones. There were negligible tanshinones in non-transformed white calli induced with 2,4-D. ATM lines T1 - T6 showed significant increases in the yields of tanshinone-I (up to 43-fold), tanshinone-IIA (up to 26-fold) and cryptotanshinone (up to 104-fold) compared with those of the non-transgenic lines on 2,4-D medium. Interestingly, the yield of cryptotanshinone from line T4 on 2,4-D medium was two times higher than that of the non-transgenic lines on trans-zeatin riboside medium. To the best of our knowledge, this is the first report of a quantitative and qualitative improvement in quinoid diterpene production achieved in a medicinally important plant species by activation tagging.

activation tagging - Agrobacterium tumefaciens - gene trapping - green fluorescent protein - Salvia miltiorrhiza - Lamiaceae - tanshinones

References

• 1 Rogers D T, Falcone D L, Littleton J M. A functional genomics study for plant drug discovery.  PharmaGenomics. 2003;  3 26-34
• 2 Yagi A, Fujimoto K, Tanonaka K, Hirai K, Takeo S. Possible active components of tan-shen (Salvia miltiorrhiza) for protection of the myocardium against ischemia-induced derangements.  Planta Med. 1989;  55 51-4
• 3 Kim S Y, Moon T C, Chang H W, Son K H, Kang S S, Kim H P. Effects of tanshinone-I isolated from Salvia miltiorrhiza Bunge on arachidonic acid metabolism and in vivo inflammatory responses.  Phytother Res. 2002;  16 616-20
• 4 Sung H J, Choi S M, Yoon Y, An K S. Tanshinone-IIA, an ingredient of Salvia miltiorrhiza Bunge, induces apoptosis in human leukemia cell lines through the activation of caspase-3.  Exp Mol Med. 1999;  31 174-8
• 5 Wang N, Luo H W, Niwa M, Ji J. A new platelet aggregation inhibitor from Salvia miltiorrhiza. .  Planta Med. 1989;  55 390-1
• 6 Bruneton J. Pharmacognosy, photochemistry, medicinal plants. Andover; Intercept 1995: 522
• 7 Hu Z B, Alfermann A W. Diterpenoid production in hairy roots cultures of Salvia miltiorrhiza.  Phytochemistry. 1993;  32 99-103
• 8 Gao S, Zhu D, Cai Z, Xu D. Autotetraploid plants from colchicine-treated bud culture of Salvia miltiorrhiza Bge.  Plant Cell Tissue Organ Cult. 1996;  47 73-7
• 9 Chen H, Yuan J P, Chen F, Zhang Y L, Song J Y. Tanshinone production in Ti-transformed Salvia miltiorrhiza cell suspension cultures.  J Biotechnol. 1997;  58 147-56
• 10 Wu C T, Mulabagal V, Nalawade S M, Chen C L, Yang T F, Tsay H S. Isolation and quantitative analysis of cryptotanshinone, an active quinoid diterpene formed in callus of Salvia miltiorrhiza Bunge.  Biol Pharm Bull. 2003;  26 845-8
• 11 Ge X, Wu J. Induction and potentiation of diterpenoid tanshinone accumulation in Salvia miltiorrhiza hairy roots by beta-aminobutyric acid.  Appl Microbiol Biotechnol. 2005;  68 183-8
• 12 Yan Q, Hu Z, Tan R X, Wu J. Efficient production and recovery of diterpenoid tanshinones in Salvia miltiorrhiza hairy root cultures with in situ adsorption, elicitation and semi-continuous operation.  J Biotechnol. 2005;  119 416-24
• 13 Furini A, Koncz C, Salamini F, Bartels D. High level transcription of a member of a repeated gene family confers dehydration tolerance to callus tissue of Craterostigma plantagineum. .  Embo J. 1997;  16 3599-608
• 14 Kakimoto T. CKI1, a histidine kinase homolog implicated in cytokinin signal transduction.  Science. 1996;  274 982-5
• 15 Neff M M, Nguyen S M, Malancharuvil E J, Fujioka S, Noguchi T, Seto H. et al . BAS1: A gene regulating brassinosteroid levels and light responsiveness in Arabidopsis.  Proc Natl Acad Sci USA. 1999;  96 15 316-23
• 16 Weigel D, Ahn J H, Blazquez M A, Borevitz J O, Christensen S K, Fankhauser C. et al . Activation tagging in Arabidopsis.  Plant Physiol. 2000;  122 1003-13
• 17 Mathews H, Clendennen S K, Caldwell C G, Liu X L, Connors K, Matheis N. et al . Activation tagging in tomato identifies a transcriptional regulator of anthocyanin biosynthesis, modification, and transport.  Plant Cell. 2003;  15 1689-703
• 18 Busov V B, Meilan R, Pearce D W, Ma C, Rood S B, Strauss S H. Activation tagging of a dominant gibberellin catabolism gene (GA 2-oxidase) from poplar that regulates tree stature.  Plant Physiol. 2003;  132 1283-91
• 19 Murashige T, Skoog F. Revised medium for rapid growth and bioassays with tobacco tissue cultures.  Physiol Plant. 1962;  15 473-97
• 20 Jefferson R A, Kavanagh T A, Bevan M W. GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants.  Embo J. 1987;  6 3901-7
• 21 Ishida Y, Saito H, Ohta S, Hiei Y, Komari T, Kumashiro T. High efficiency transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens.  Nat Biotechnol. 1996;  14 745-50
• 22 Hiei Y, Ohta S, Komari T, Kumashiro T. Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA.  Plant J. 1994;  6 271-82
• 23 Wu H, Sparks C, Amoah B, Jones H D. Factors influencing successful Agrobacteriummediated genetic transformation of wheat.  Plant Cell Rep. 2003;  21 659-68
• 24 van der Fits L, Hilliou F, Memelink J. T-DNA activation tagging as a tool to isolate regulators of a metabolic pathway from a genetically non-tractable plant species.  Transgenic Res. 2001;  10 513-21
• 25 Jeon J S, Lee S, Jung K H, Jun S H, Jeong D H, Lee J. et al . T-DNA insertional mutagenesis for functional genomics in rice.  Plant J. 2000;  22 561-70
• 26 Jeong D H, An S, Kang H G, Moon S, Han J J, Park S. et al . T-DNA insertional mutagenesis for activation tagging in rice.  Plant Physiol. 2002;  130 1636-44
• 27 Gantet P, Memelink J. Transcription factors: tools to engineer the production of pharmacologically active plant metabolites.  Trends Pharmacol Sci. 2002;  23 563-9

1 J. J. W. C. and H.-S.T. contributed equally to this work and are joint corresponding authors.