Journal of Medicinal Plants Research Vol. 4(17), pp. 1708-1713, 4 September, 2010 Available online at http://www.academicjournals.org/JMPR DOI: 10.5897/JMPR10.269 ISSN 1996-0875 ©2010 Academic Journals Full Length Research Paper Promoting scopolamine accumulation in transgenic plants of Atropa belladonna generated from hairy roots with over expression of pmt and h6h gene Xiaoqiang Liu1, Chunxian Yang1, Min Chen2, Mingyang Li3, Zhihua Liao1* and Kexuan Tang4* 1Laboratory of Natural Products and Metabolic Engineering, Key Laboratory of Eco-environments in Three Gorges Reservoir Region (Ministry of Education), School of Life Sciences, Southwest University, Chongqing 400715, People’s Republic of China. 2School of Pharmaceutical Sciences, Southwest University, Chongqing 400715, People’s Republic of China. 3Institute of Ornamental Plants Research, Southwest University, Chongqing 400715, People’s Republic of China. 4Plant Biotechnology Research Center, School of Agriculture and Biology, Fudan-SJTU-Nottingham Plant Biotechnology R and D Center, Shanghai Jiao Tong University, Shanghai 200030, People’s Republic of China. Accepted 29 June, 2010 Atropa belladonna is the most important commercial source for obtaining pharmaceutical tropane alkaloids such as scopolamine and hyoscyamine. In the present study, two rate-limiting enzyme genes including putrescine N-methyltransferase and hyoscyamine 6β-hydroxylase were introduced into A. belladonna, and integration of the pmt and h6h genes into the genomic DNA of transgenic plants were confirmed by genomic polymerase chain reaction (PCR) analysis. The scopolamine content of transgenic lines was increased to 1.3 - 2.5 folds than that in wild type, which was caused by over expression of the pmt and h6h genes in the transgenic plant lines of A. belladonna. The current study provides a more effective approach for commercially large-scale production of scopolamine by cultivating A. belladonna plants in large fields but not using the hairy root systems as bioreactors. Key words: Atropa belladonna, pmt, h6h, scopolamine, transformation, regeneration. INTRODUCTION Atropa belladonna, commonly known as belladonna or tropane alkaloids especially scopolamine in natural deadly nightshades, is a perennial herbaceous plant and plants, there has been a long-standing interest in most importantly commercial source of pharmaceutical increasing the content of alkaloids, especially the much tropane alkaloids in the family of Solanaceae. The plant is more valuable scopolamine in cultivated medicinal plants. of interest due to its production of bioactive tropane Over the past years, extracting these secondary alkaloids, including scopolamine and hyoscyamine, which metabolites from whole plants was viewed as the only are wildly used as antagonists of acetylcholine in both the economical method for the production of these pharma- autonomic and central nervous system (Guggisberg and ceuticals. However, in natural plants, the low endogenous Hesse, 1983). For medicinal purposes, scopolamine is content of these chemicals remains as the limitation for much more useful and valuable because of its higher mass production. Although, other options are available for physiological activity and fewer side-effects (Evans, tropane alkaloid production, they are not currently 1996). However, because of the low abundance of available. For example, chemical synthesis remains extremely expensive due to the existence of multiple chiral centers. Also, incomplete characterization of the pathways required for tropane alkaloid production makes *Corresponding author. E-mail: [email protected], it impossible to acquire them by means of alternative [email protected]. Tel: 86-21-62932002. Fax: 86-21- biosystems such as microorganisms (Verpoorte, 2000). 62824073. Nowadays, with the development in plant cell cultures, Liu et al. 1709 tissue cultures and genetic engineering, it becomes Researchers were able to elevate scopolamine levels by feasible to manipulate the biosynthetic pathways of plant over expressing h6h. Yun et al. (1992) reported that h6h secondary metabolisms such as tropane alkaloids at the from H. niger was constitutively expressed in A. level of metabolic and enzyme level to increase the phar- Belladonna plants, including stems and leaves where maceutical tropane alkaloids content in the cultivated A. endogenous h6h is not expressed. Hashimoto et al. belladonna (Oksman-Caldentey, 2004; Trethewey, 2004). (1993b) reported a five fold increase in the scopolamine Putrescine is a common precursor of both polyamines content of transgenic A. Belladonna hairy roots over such as spermidine and spermine and tropane/pyridine expressing the same h6h gene. Over expression of h6h alkaloids (Guggisberg and Hesse, 1983; Hashimoto et gene in H. muticus hairy roots produced over 100-fold al., 1989; Hibi et al., 1994). higher level of scopolamine and hyoscyamine compared Putrescine N-methyltransferase (PMT) is the first with controls (Jouhikainen et al., 1999). alkaloid-specific enzyme committed for the biosynthesis Over expressing multiple key-enzyme genes or mani- of tropane alkaloids, because it involves in the removal of pulating regulatory genes in the target bioengineering putrescine from the polyamine pool and then catalyzes pathway is a promising way to manipulate the biosyn- the N-methylation of this diamine to form N- thetic pathways of tropane alkaloids since the single rate- methylputrescine (Hibi et al., 1992), the product N- limiting step of most biosynthetic pathways for secondary methylputrescine is the first specific metabolite on the metabolites may not exist (Sato et al., 2001). A good route to nicotine, tropane and nortropane alkaloids example is simultaneous introduction and over (Biastoff et al., 2009). The pmt cDNAs were already expression of genes encoding the rate-limiting upstream cloned from tobacco (Hibi et al., 1994) and Hyoscyamus enzyme PMT and the downstream key enzyme H6H of niger (Suzuki et al., 1999). Regulation of the expression scopolamine biosynthesis in transgenic H. niger hairy of pmt gene has been shown to be crucial for alkaloid root cultures (Zhang et al., 2004). Transgenic hairy root production in several cases. After introducing the tobacco lines expressing both pmt and h6h produced significantly pmt gene into the genome of Datura meteland and higher levels of scopolamine compared with both the wild Hyoscyamus muticus respectively, it was found that both type and the transgenic lines harboring a single gene hyoscyamine and scopolamine production were improved (pmt or h6h). The best line produced more than nine folds in hairy root cultures of D. meteland, whereas in H. scopolamine than that in the wild type and more than muticus only hyoscyamine contents were increased twice the amount in the highest scopolamine-producing (Moyano et al., 2002). h6h single-gene transgenic line. It indicated that the However, in many cases, the over expression of expression of h6h played an important role in exogenous pmt can not improve alkaloid production as scopolamine biosynthesis, while the contribution to the expected. For example, over expression of pmt in A. increase of scopolamine production result from the belladonna did not affect tropane alkaloid levels either in activity of pmt is limited. transgenic plants or in hairy roots (Sato et al., 2001); In order to investigate the secondary metabolism Moyano et al. (2002) inserted the tobacco pmt gene into response to pmt and h6h over expression in A. the hairy roots of a hybrid of Duboisia and improved the belladonna, the Nicotiana tabacum pmt gene and the H. N-methylputrescine level of the transgenic hairy roots, but niger h6h gene were introduced into A. belladonna in the there was no significant increase in either tropane or present work. The regenerated plants from transgenic pyridine-type alkaloids. Rothe et al. (2003) also found hairy roots were obtained, which is different from previous that increased pmt expression alone was not sufficient to studies, the phenotype and scopolamine production of increase alkaloid production in A. belladonna plants and transgenic plant lines were investigated after planted in in root cultures. All of these reports indicated that the the field, with the blank-transformed plant lines and the regulation of tropane alkaloids synthetic pathways varied wild-type plants used as controls respectively. with different plant species, and the effect of over expression of a single enzyme may be inadequate. Although, genetic manipulation on the upstream MATERIALS AND METHODS pathway of tropane alkaloid in some plant species was unsuccessful, efforts closer to the final products have Strains and plasmids been effective. Hyoscyamine 6β-hydroxylase (H6H; EC 1.14.11.11) is a bifunctional enzyme, which catalyzes the The bivalent plant expression plasmid pXI containing both pmt and h6h genes, constructed by Zhang et al. (2004), were used in the last two oxidative reactions of tropane alkaloids study. The pXI contained two separate expression cassettes for pmt biosynthetic pathway, converting hyoscyamine to the and h6h, both driven by the CaMV 35S promoter and the nptII more pharmaceutically valuable tropane alkaloid cassette for conferring kanamycin resistance. Plasmid pXI was scopolamine (Hashimoto et al., 1993a; Matsuda et al., isolated from Escherichia coli strain DH5α and transformed into 1991; Jaber-Vazdekis et al., 2009). In the case of h6h disarmed Agrobacterium tumefaciens strain C58C1 containing Agrobacterium rhizogenes Ri plasmid pRiA4.