BMB Reports

Korean Society for Biochemistry and Molecular Biology (생화학분자생물학회)
권호 표지
DOI QR코드

DOI QR Code

Molecular Cloning and Characterization of a New cDNA Encoding Hyoscyamine 6β-hydroxylase from Roots of Anisodus acutangulus

  • Kai, Guoyin (Laboratory of Plant Biotechnology, College of Life and Environment Sciences, Shanghai Normal University) ;
  • Chen, Junfeng (Laboratory of Plant Biotechnology, College of Life and Environment Sciences, Shanghai Normal University) ;
  • Li, Li (Laboratory of Plant Biotechnology, College of Life and Environment Sciences, Shanghai Normal University) ;
  • Zhou, Genyu (Laboratory of Plant Biotechnology, College of Life and Environment Sciences, Shanghai Normal University) ;
  • Zhou, Limin (Laboratory of Plant Biotechnology, College of Life and Environment Sciences, Shanghai Normal University) ;
  • Zhang, Lei (School of Pharmacy, Second Military Medical University) ;
  • Chen, Yuhui (Fudan-SJTU-Nottingham Plant Biotechnology R&D Center, School of Agriculture and Biology, Shanghai Jiaotong University) ;
  • Zhao, Linxia (Fudan-SJTU-Nottingham Plant Biotechnology R&D Center, School of Agriculture and Biology, Shanghai Jiaotong University)
  • Published : 2007.09.30
Download PDF
⟨ Previous Next ⟩

Abstract

A new full-length cDNA encoding hyoscyamine $6\beta$-hydroxylase (designated as aah6h, GenBank Accession No. EF187826), which catalyzes the last committed step in the scopolamine biosynthetic pathway, was isolated from young roots of Anisodus acutangulus by rapid amplification of cDNA ends (RACE) for the first time. The full-length cDNA of aah6h was 1380 bp and contained a 1035 bp open reading frame (ORF) encoding a deduced protein of 344 amino acid residues. The deduced protein had an isoelectric point (pI) of 5.09 and a calculated molecular mass of about 38.7 kDa. Sequence analyses showed that AaH6H had high homology with other H6Hs isolated from some scopolamine-producing plants such as Hyoscyamus niger, Datura metel and Atropa belladonna etc. Bioinformatics analyses results indicated AaH6H belongs to 2-oxoglutarate-dependent dioxygenase superfamily. Phylogenetic tree analysis showed that AaH6H had closest relationship with H6H from A. tanguticus. Southern hybridization analysis of the genomic DNA revealed that aah6h belonged to a multi-copy gene family. Tissue expression pattern analysis firstly founded that aah6h expressed in all the tested tissues including roots, stems and leaves and indicated that aah6h was a constitutive-expression gene, which was the first reported tissue-independent h6h gene compared to other known h6h genes.

Keywords

References

  1. Christen, P. M. F. R., Phillipson, D. and Evans, W. C. (1993) Alkaloids of Erythroxylum zambesiacum stem-bark. Phytochemistry 34, 1147-1151. https://doi.org/10.1016/S0031-9422(00)90733-7
  2. Endo, T., Hamaguchi, N., Eriksson, T. and Yamada, Y. (1991) Alkaloid biosynthesis in somatic hybrids of Duboisia leichhardtii F. Muell and Nicotiana tabacum L. Planta 183, 505-510.
  3. Evans, W. C. (1979) Tropane alkaloids in the Solanaceae; in The Biology and Taxonomy of the Solanaceae, Hawkes, J. G., Lester, R. N. and Skelding, A. D. (eds.), pp. 241-254, Linnean Society Symposium Series, Academic Press, London, UK.
  4. Geourjon, C. and Deleage, G. (1995) SOPMA: Significant improvement in protein secondary structure prediction by consensus prediction from multiple alignments. Cabios 11, 681-684.
  5. Hakkinen, S. T., Moyano, E., Cusido, R. M., Palazon, J., Pinol, M. T. and Oksman-Caldentey, K. M. (2005) Enhanced secretion of tropane alkaloids in Nicotiana tabacum hairy roots expressing heterologous hyoscyamine-$6{\beta}$-hydroxylase. J. Exp. Bot. 420, 2611-2618.
  6. Hashimoto, T., Hayashi, A., Amano, Y., Kohno, J., Iwanari, H., Usuda, S. and Yamada, Y. (1991) Hyoscyamine $6{\beta}$-hydroxylase, an enzyme involved in tropane alkaloid biosynthesis, is localized at the pericycle of the root. J. Biol. Chem. 266, 4648-4653.
  7. Hashimoto, T., Nakajima, K., Ongena, G. and Yamada, Y. (1992) Two tropinone reductases with distinct stereospecifities from cultured roots of hyoscyamus niger. Plant Physiol. 100, 836-845. https://doi.org/10.1104/pp.100.2.836
  8. Hashimoto, T. and Yamada, Y. (1994) Alkaloid biogenesis: molecular aspects. Annu. Rev. Plant Physiol. Plant Mol. Biol. 45, 257-285. https://doi.org/10.1146/annurev.pp.45.060194.001353
  9. Hashimoto, T., Yun, D. J. and Yamada, Y. (1993) Production of tropane alkaloids in genetically engineered root cultures. Phytochemistry 32, 713-718. https://doi.org/10.1016/S0031-9422(00)95159-8
  10. Hsiao, P. K., Cheng, H. K. and Ho, L. Y. (1973) The occurrence of some important tropane alkaloids in Chinese solanaceous plants. J. Integr. Plant Biol. 15, 187-194.
  11. Huang, F., Dai, X. D., Hu, Y. L., Chen, C. Y. and Zhu, G. Z. (2005) Progress in synthesis of tropane alkaloids. Chem. Reagent 27,141-144.
  12. Jouhikainen, K., Lindgren, L., Jokelainen, T., Hiltunen, R., Teeri, T. H. and Oksman-Caldentey, K. M. (1999) Enhancement of scopolamine production in Hyoscyamus muticus L. hairy root cultures by genetic engineering. Planta 208, 545-551. https://doi.org/10.1007/s004250050592
  13. Kai, G. Y., Jiang, J. H., Zhao, D. L., Zhao, L. X., Zhang, L., Li, Z. G., Guo, B. H., Sun, X. F., Miao, Z. Q. and Tang, K. (2006b) Isolation and expression profile analysis of a new cDNA encoding 5-alpha-taxadienol-10-beta-hydroxylase from Taxus media. J. Plant Biochem. Biotechnol. 15, 1-5. https://doi.org/10.1007/BF03321893
  14. Kai, G. Y., Miao, Z. Q., Qiu, C. Y., Zhang, L., Zhao, L. X., Li, Z. J., Xu, T. F., Zhang, L. D., Gong, Y. F., Zhao, D. L., Liu, D. H., Sun, X. F. and Tang, K. (2004) Molecular cloning and characterization of a taxadienol acetyl transferase cDNA from Taxus media. Plant science 167, 759-764. https://doi.org/10.1016/j.plantsci.2004.05.008
  15. Kai, G. Y., Miao, Z. Q., Zhang, L., Zhao, D. L., Liao, Z. H., Sun, X. F., Zhao, L. X. and Tang, K. (2006a) Molecular cloning and expression analyses of a new gene encoding 3-hydroxy-3-methylglutaryl-CoA synthase from Taxus x media. Biol. Plant 50, 359-366. https://doi.org/10.1007/s10535-006-0050-0
  16. Kai, G. Y., Zhao, L. X., Zhang, L., Li, Z. G., Guo, B. H., Zhao, D. L., Sun, X. F., Miao, Z. Q. and Tang, K. (2005) Characterization and expression profile analysis of a new cDNA encoding taxadiene synthase from Taxus media. J. Biochem. Mol. Biol. 38, 675-685. https://doi.org/10.5483/BMBRep.2005.38.6.668
  17. Kanegae, T., Kajiya, H., Amano, Y., Hashimoto, T. And Yamada, Y. (1994). Species-dependent expression $6{\beta}-Hydroxylase$ gene in of the Hyoscyamine the pericycle. Plant Physiol. 105, 483-490. https://doi.org/10.1104/pp.105.2.483
  18. Kumar, S., Tamura, K., Jakobsen, I. B. and Nei, M. (2001) MEGA2: molecular evolutionary genetics analysis software. Bioinformatics 12, 1244-1245.
  19. Liu, T., Zhu, P., Cheng, K. D., Meng, C. and He, H. X. (2005) Molecular cloning, expression and characterization of hyoscyamine 6beta-hydroxylase from hairy roots of Anisodus tanguticus. Planta Med. 71, 249-253. https://doi.org/10.1055/s-2005-837825
  20. Mateus, L., Chekaoui, S., Christen, P. and Oksman-Caldentey, K. M. (2000) Simultaneous determination of scopolamine, hyoscyamine and littorine in plants and different hairy root clones of Hyoscyamus muticus by micellar electrokinetic chromatography. Phytochemistry 54, 517-523. https://doi.org/10.1016/S0031-9422(00)00134-5
  21. Matsuda, J., Okabe, S., Hashimoto, T. and Yamada, Y. (1991) Molecular cloning of hyoscyamine $6{\beta}-hydroxylase$, a 2-oxoglutarate-dependent dioxygenase, from cultured roots of Hyoscyamus niger. J. Biol. Chem. 266, 9460-9464.
  22. Nakajima, K., Hashimoto, T. and Yamada, Y. (1993) Two tropinone reductases with different stereo specific cities are short-chain dehydrogenases evolved from a common ancestor. Proc. Natl. Acad. Sci. USA 20, 9591-9595.
  23. Sambrook, J., Fritsh, E. F. and Maniatis, T. (1989) Molecular cloning: A laboratory manual, Cold Spring Harbor Laboratory Press, New York, USA.
  24. Suzuki, K., Yun, D. J., Chen, X. Y., Yamada, Y. and Hashimoto, T. (1999) An Atropa belladonna hyoscyamine 6-hydroxylase gene is differentially expressed in the root pericycle and anthers. Plant Mol. Biol. 40, 141-152. https://doi.org/10.1023/A:1026465518112
  25. Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. and Higgins, D. G. (1997) The CLUSTAL X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucl. Acids Res. 25, 4876-4882. https://doi.org/10.1093/nar/25.24.4876
  26. Wu, D. Q., Wang, F. C., Chen, Z. R., Yang, J. S. and Huang, Q. L. (1962) Production of atropine sulfate and chemical analysis of Anisodus acutangulus in Yunnan. Medicine and Pharmacy of Yunnan. 3, 69-70.
  27. Yun, D. J., Hashimoto, T. and Yamada, Y. (1992) Metabolic engineering of medicinal plants: transgenic Atropa belladonna with an improved alkaloid composition. Proc. Natl. Acad. Sci. USA 89, 11799-11803. https://doi.org/10.1073/pnas.89.24.11799
  28. Zhang, L., Kai, G. Y., Lu, B. B., Zhang, H. M., Tang, K., Jiang, J. H. and Chen, W. S. (2005) Metabolic engineering of tropane alkaloid biosynthesis in plants. J. Integr. Plant Biol. 47, 136-143. https://doi.org/10.1111/j.1744-7909.2005.00024.x
  29. Zhang, L., Ding, R. X., Chai, Y. R., Bonfill, M., Pinol, M. T., Xu, T. F., Pi, Y., Wang, Z. N., Zhang, H. M., Kai, G. Y., Liao, Z. H., Sun, X. F. and Tang, K. (2004) Engineering tropane biosynthetic pathway in Hyoscyamus niger hairy root cultures. Proc. Natl. Acad. Sci. USA 101, 6786-6791. https://doi.org/10.1073/pnas.0401391101
  30. Zheng, G. Z. and Liang, Z. (1976) Study on tissue culture of medicinal plants I. Callus cultures of Anisodus acutangulus for the production of hyoscyamine and scopolamine. J. Integr. Plant Biol. 18,163-169.
  31. Zarate, R., El Jaber-Vazdekis, N., Medina, B. and Ravelo, A. G. (2006) Tailoring tropane alkaloid accumulation in transgenic hairy roots of atropa baetica by over-expressing the gene encoding hyoscyamine 6a-hydroxylase. Biotechnol. Lett. 28, 1271-1277. https://doi.org/10.1007/s10529-006-9085-8
  32. Zeng, Y. L. (1962) Pharmaceutical study and investigation of Anisodus acutangulus in Yunnan. Medicine and Pharmacy of Yunnan. 3, 66-68.

Cited by

  1. Production of tropane alkaloids in diploid and tetraploid plants and in vitro hairy root cultures of Egyptian henbane (Hyoscyamus muticus L.) vol.110, pp.1, 2012, https://doi.org/10.1007/s11240-012-0127-8
  2. Transcriptome exploration for further understanding of the tropane alkaloids biosynthesis in Anisodus acutangulus vol.290, pp.4, 2015, https://doi.org/10.1007/s00438-015-1005-y
  3. Molecular cloning and expression analysis of a Cu/Zn SOD gene (BcCSD1) from Brassica campestris ssp. chinensis vol.186, 2015, https://doi.org/10.1016/j.foodchem.2014.07.121
  4. Functional characterisation of a tropine-forming reductase gene from Brugmansia arborea, a woody plant species producing tropane alkaloids vol.127, 2016, https://doi.org/10.1016/j.phytochem.2016.03.008
  5. Optimization of induction and culture conditions and tropane alkaloid production in hairy roots of Anisodus acutangulus vol.13, pp.5, 2008, https://doi.org/10.1007/s12257-008-0035-2
  6. Enhancing the production of tropane alkaloids in transgenic Anisodus acutangulus hairy root cultures by over-expressing tropinone reductase I and hyoscyamine-6β-hydroxylase vol.8, pp.11, 2012, https://doi.org/10.1039/c2mb25208b
  7. Effects of different elicitors on yield of tropane alkaloids in hairy roots of Anisodus acutangulus vol.39, pp.2, 2012, https://doi.org/10.1007/s11033-011-0912-1
  8. Cloning, characterization and analysis of expression profiles of a cDNA encoding a hyoscyamine 6β-hydroxylase (H6H) from Atropa baetica Willk vol.47, pp.1, 2009, https://doi.org/10.1016/j.plaphy.2008.09.004
  9. Molecular cloning and transgenic characterization of the genes encoding chalcone synthase and chalcone isomerase from the Tibetan herbal plantMirabilis himalaica vol.63, pp.3, 2016, https://doi.org/10.1002/bab.1376
  10. Biochemical and structural characterization of recombinant hyoscyamine 6β-hydroxylase from Datura metel L. vol.48, pp.12, 2010, https://doi.org/10.1016/j.plaphy.2010.09.003
  11. Enhancing the scopolamine production in transgenic plants of Atropa belladonna by overexpressing pmt and h6h genes vol.143, pp.4, 2011, https://doi.org/10.1111/j.1399-3054.2011.01506.x
  12. Functional identification of hyoscyamine 6β-hydroxylase from Anisodus acutangulus and overproduction of scopolamine in genetically-engineered Escherichia coli vol.33, pp.7, 2011, https://doi.org/10.1007/s10529-011-0575-y
  13. Expression of hyoscyamine 6β-hydroxylase in the root pericycle cells and accumulation of its product scopolamine in leaf and stem tissues of Datura metel L. vol.178, pp.2, 2010, https://doi.org/10.1016/j.plantsci.2009.11.004
  14. Medicinally important secondary metabolites in recombinant microorganisms or plants: Progress in alkaloid biosynthesis vol.4, pp.12, 2009, https://doi.org/10.1002/biot.200900229
  15. Co-expression of AaPMT and AaTRI effectively enhances the yields of tropane alkaloids in Anisodus acutangulus hairy roots vol.11, pp.1, 2011, https://doi.org/10.1186/1472-6750-11-43
  16. Chromium-induced tropane alkaloid production and H6H gene expression in Atropa belladonna L. (Solanaceae) in vitro-propagated plantlets vol.52, 2012, https://doi.org/10.1016/j.plaphy.2011.12.003
  17. Functional characterization of recombinant hyoscyamine 6β-hydroxylase from Atropa belladonna vol.20, pp.14, 2012, https://doi.org/10.1016/j.bmc.2012.05.042
  18. Molecular cloning and characterization of two tropinone reductases inAnisodus acutangulusand enhancement of tropane alkaloid production in AaTRI-transformed hairy roots vol.54, pp.3, 2009, https://doi.org/10.1042/BA20090171
  19. An atypical pattern of accumulation of scopolamine and other tropane alkaloids and expression of alkaloid pathway genes in Hyoscyamus senecionis vol.70, 2013, https://doi.org/10.1016/j.plaphy.2013.05.007
  20. Functional diversity of 2-oxoglutarate/Fe(II)-dependent dioxygenases in plant metabolism vol.5, 2014, https://doi.org/10.3389/fpls.2014.00524
  21. Molecular characterization and expression analysis of two distinct putrescineN-methyltransferases from roots ofAnisodus acutangulus vol.135, pp.2, 2009, https://doi.org/10.1111/j.1399-3054.2008.01178.x
  22. Recent advances in plant biotechnology and genetic engineering for production of secondary metabolites vol.44, pp.1, 2010, https://doi.org/10.3103/S009545271001010X