An efficient transformation method for estrogen-inducible transgene expression in Catharanthus roseus hairy roots
- 815 Downloads
Efficient genetic engineering of the medicinal plant, Catharanthus roseus, is essential in improving the production of the pharmaceutically important anticancer compounds, vinblastine and vincristine. Here we optimize several steps in the Agrobacterium-mediated transformation of C. roseus, including antibiotic selection and Agrobacterium elimination parameters. With the optimized protocol, stable transgenic hairy root cultures expressing green fluorescent protein (GFP) under control of the estrogen-inducible XVE system were established with a transformation efficiency of 33 %. The estrogen-inducible system has not been previously tested in C. roseus hairy roots but offers several advantages as an inducible system. Prior to induction, low basal levels of Gfp are observed. Upon induction with 5 mM 17β-estradiol, Gfp transgene expression is highly and rapidly induced within 1 h, increasing after 24 h. Strongest GFP expression is observed in the meristematic root tips and is sustained over 5 days in the presence of estradiol. Neither the 17β-estradiol induction nor the estrogen-inducible system elicits a defense response in C. roseus hairy roots.
KeywordsCatharanthus roseus Agrobacterium-mediated transformation Genetic engineering Estrogen-inducible Hairy roots
This work was supported by the National Science Foundation (NSF CBET Award #1033889). The authors thank Dr. Nam-Hai Chua (The Rockefeller University, New York, NY) for providing the pER8-GFP vector and Dr. Jacqueline V. Shanks (Iowa State University, Ames, IA), Dr. Ka-Yiu San (Rice University, Houston, TX) for providing the wild-type C. roseus hairy root cultures.
- Lee SY, Kim SG, Song WS, Kim YK, Park NI, Park SU (2010) Influence of different strains of Agrobacterium rhizogenes on hairy root induction and production of alizarin and purpurin in Rubia akane Nakai. Romanian Biotechnol Lett 15:5406Google Scholar
- Menke FLH, Champion A, Kijne JW, Memelink J (1999) A novel jasmonate- and elicitor-responsive element in the periwinkle secondary metabolite biosynthetic gene Str interacts with a jasmonate- and elicitor-inducible AP2-domain transcription factor, ORCA2. EMBO J 18:4455–4463PubMedCentralPubMedCrossRefGoogle Scholar
- Peebles CA, Sander GW, Hughes EH, Peacock R, Shanks JV, San KY (2011) The expression of 1-deoxy-d-xylulose synthase and geraniol-10-hydroxylase or anthranilate synthase increases terpenoid indole alkaloid accumulation in Catharanthus roseus hairy roots. Metab Eng 13:234–240PubMedCrossRefGoogle Scholar
- Rizvi N, Goklany S, Cram EJ, Lee-Parsons CWT (2013) Rapid increases of key regulators precede the increased production of pharmaceutically valuable compounds in Catharanthus roseus. Pharm Eng 33:1–9Google Scholar
- Vanhala LH, Hilunen R, Oksman-Caldentey K-M (1995) Virulence of different Agrobacterium strains on hairy root formation of Hyoscyamus muticus. Plant Cell Rep 14:236–340Google Scholar
- Wang C-T, Liu H, Gao X-S, Zhang H-X (2010) Overexpression of G10H and ORCA3 in the hairy roots of Catharanthus roseus improves catharanthine production. Plant Cell Rep 29:887–894Google Scholar
- Zhou M-L, Zhu X-M, Shao J-R, Wu Y-M, Tang Y-X (2010) Transcriptional response of the catharanthine biosynthesis pathway to methyl jasmonate/nitric oxide elicitation in Catharanthus roseus hairy root culture. Appl Microbiol Biotechnol 88:737–750Google Scholar