Skip to main content

Advertisement

SpringerLink
Log in

Biosynthesis and bioactivity of trichosanthin in cultured crown gall tissues of Trichosanthes kirilowii Maximowicz

  • Genetic Transformation and Hybridization
  • Published:
Plant Cell Reports Aims and scope Submit manuscript

Abstract

Trichosanthin (TCS) from Trichosanthes kirilowii Maximowicz (T. kirilowii) can be used to treat choriocarcinoma. In this work, we established a novel system to produce TCS in crown gall tissues of T. kirilowii infected by Agrobacterium tumefaciens C58 (A. tumefaciens). In the crown gall tissues, a nopaline synthase (NOS) gene of A. tumefaciens was identified by polymerase chain reaction (PCR), and nopaline accumulation was confirmed by a high-voltage filter paper electrophoresis. Furthermore, we optimized conditions to culture the crown gall tissues able to grow fast and produce TCS in an auxin-free medium, and found that a fungal elicitor of Armillaria mellea was capable of stimulation of TCS secretion into the medium. Moreover, we identified that the TCS purified from the crown gall tissues could induce gastric cancer cell death. These data underscore the usefulness of our system as an inexpensive and virtually unlimited source of TCS.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

Abbreviations

TCS:

Trichosanthin

MS:

Murashige–Skoog

SDS-PAGE:

Polyacrylamide gel electrophoresis

MTT:

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide

RIP:

Ribosome-inactivating protein

DMSO:

Dimethyl sulfoxide

DAB:

3,3′-Diaminobezidine

PCR:

Polymerase chain reaction

ELISA:

Enzyme-linked immunosorbent assay

A. Mellea :

Armillaria mellea

A. tumefaciens :

Agrobacterium tumefaciens C58

NOS:

Nopaline synthase

T. kirilowii :

Trichosanthes kirilowii Maximowicz

PBS:

Phosphate-buffered saline

BSA:

Bovine serum albumin

Ti:

Tumor inducing

T-DNA:

Transferring DNA

References

  • Aloni R, Wolf A, Feigenbaum P, Avni A, Klee HJ (1998) The Never ripe mutant provides evidence that tumor-induced ethylene controls the morphogenesis of Agrobacterium tumefaciens-induced crown galls on tomato stems. Plant Physiol 117:841–849

    Article  PubMed  CAS  Google Scholar 

  • Baebler S, Camloh M, Kovac M (2002) Jasmonic acid stimulates taxane production in cell suspension culture of yew (Taxus media). Planta Med 68:475–476

    Article  PubMed  CAS  Google Scholar 

  • Chan WL, Shaw PC, Tam SC, Jacobsen C, Gliemann J, Nielsen MS (2000) Trichosanthin interacts with and enters cells via LDL receptor family members. Biochem Biophys Res Commun 270:453–457

    Article  PubMed  CAS  Google Scholar 

  • Chow TP, Feldman RA, Lovett M, Piatak M (1990) Isolation and DNA sequence of a gene encoding alpha-trichosanthin, a type I ribosome-inactivating protein. J Biol Chem 265:8670–8674

    PubMed  CAS  Google Scholar 

  • Collins EJ, Robertus JD, LoPresti M, Stone KL, Williams KR, Wu P, Hwang K, Piatak M (1990) Primary amino acid sequence of alpha-trichosanthin and molecular models for abrin A-chain and alpha-trichosanthin. J Biol Chem 265:8665–8669

    PubMed  CAS  Google Scholar 

  • Depicker A, Satchel S, Dhaese P (1982) Nopaline synthase: transcript mapping and DNA sequence. J Mol Appl Genet 1:561–573

    PubMed  CAS  Google Scholar 

  • Gelvin SB (2000) Agrobacterium and plant genes involved in T-DNA transfer and integration. Annu Rev Plant Physiol Plant Mol Biol 51:223–256

    Article  PubMed  CAS  Google Scholar 

  • Grant JE, Cooper PA, Dale TM (2004) Transgenic Pinus radiata from Agrobacterium tumefaciens-mediated transformation of cotyledons. Plant Cell Rep 22:899–915

    Article  Google Scholar 

  • Harlow E., Lane D (1988) Antibodies: a laboratory manual. Cold Spring Harbor Laboratory, New York

    Google Scholar 

  • Kumagai MH, Turpen TH, Weinzettl N, Della-Cioppa G, Turpen AM, Donson J, Hilf ME, Grantham GL, Dawson WO, Chow TP, Piatak M Jr, Grill LK (1993) {alpha} Rapid, high-level expression of biologically active {alpha}-trichosanthin in transfected plants by an RNA viral vector. Proc Natl Acad Sci USA 90:427–430

    Article  PubMed  CAS  Google Scholar 

  • Lei H, An P, Song S, Liu X, He L, Wu J, Meng L, Liu M, Yang J, Shou C (2002) A novel peptide isolated from a phage display library inhibits tumor growth and metastasis by blocking the binding of vascular endothelial growth factor to its kinase domain receptor. J Biol Chem 277:43137–43142

    Article  CAS  Google Scholar 

  • Li MX, Yeung HW, Pan LP, Chan SI (1991) Trichosanthin, a potent HIV-1 inhibitor, can cleave supercoiled DNA in vitro. Nucleic Acids Res 19:6309–6312

    PubMed  CAS  Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissues culture. Plant Physiol 15:473–497

    Article  CAS  Google Scholar 

  • Otten LABM, Shitperoort RA (1978) A rapid micro scale method for the detection of lysoping and nopalin dehydrogenas activities. Biochem Biophys Acta 78:497–604

    Google Scholar 

  • Shaw PC, Yung MH, Zhu RH, Ho WK, Ng TB, Yeung HW (1991) Clone of trichosanthin cDNA and its expression in Escherichia coli. Gene 97:267–272

    Article  PubMed  CAS  Google Scholar 

  • Sigee DC (1993) Bacterial plant pathology: cell and molecular aspects. Cambridge University Press, Cambridge

    Google Scholar 

  • Song JY, Qi JJ, Lei H, Ren CL, Fu J, Zhang YL (2000) Effect of Armillaria mellea elicitor on accumulation of tanshinones in crown gall cultures of Salvia miltiorrhiza. Acta Bot Sin 42:316–320

    CAS  Google Scholar 

  • Tinland B (1996) The integration of T-DNA into plant genomes. Trends Plant Sci 1:178–184

    Article  Google Scholar 

  • Tsugawa H, Kagami T, Suzuki M (2004) High-frequency transformation of Lobelia erinus L. by Agrobacterium-mediated gene transfer. Plant Cell Rep 22:759–764

    Article  PubMed  CAS  Google Scholar 

  • Wang C (2000) The tian-hua-feng protein. Science Publisher, Beijing

    Google Scholar 

  • Wang Y, Qian RQ, Gu ZW, Jin SW, Zhang LQ, Xia ZX, Tian GY, Ni CZ (1986) Pure Appl Chem 58:789–798

    CAS  Google Scholar 

  • Weiler EW, Spanier K (1981) Phytohormones in the formation of crown gall tumors. Planta 153:326–337

    Article  CAS  Google Scholar 

  • Zambryski P, Tempé J, Schell J (1989) Transfer and function of T-DNA genes from Agrobacterium Ti and Ri plasmids in plants. Cell 56:193–201

    Article  PubMed  CAS  Google Scholar 

  • Zhang YL, Song JY, Zhao BH, Liu HL (1995) Crown gall culture and production of tanshinine in salvia miltiorrhiza. Chin J Biotechnol 11:137–141

    PubMed  CAS  Google Scholar 

  • Zhang YL, Zhu M, Zhao BH (1992) The effect of Arimillaria mellea elicior on alkaloid production in tissue culture of Corydalis yanhusuo. Acta Bot Sin 34:658–661

    CAS  Google Scholar 

  • Zhao J, Ben LH, Wu YL, Hu W, Ling K, Xin SM, Nie HL, Ma L, Pei G (1999) Anti-HIV agent trichosanthin enhances the capabilities of chemokines to stimulate chemotaxis and G protein activation, and this is mediated through interaction of trichosanthin and chemokine receptors. J Exp Med 190:101–111

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by the Science Foundation of Chinese Academy of Medical Sciences and Peking Union Medical College, and a National Key Project of China in the 9th 5-year-plan (# 96-903-02-02). We thank Dr. Qiu De You (Peking University, P.R. China) for the gift of a TCS standard, Dr. Shou Cheng Chao (Peking University, P.R. China) for gastric cancer cells, Prof. Ye He Qun and Prof. Zhao De Xiu (Institute of Plant Research, Academy of Chinese Sciences, P.R. China) for generously providing the bioreactor, and Dr. Giulio Romeo (Harvard Medical School, Boston, USA) for reading the manuscript. We also thank Dr. Wang Guo Ying (Chinese Agricultural University, P. R. China) for the gift of A. tumefaciens C58.

Author information

Authors and Affiliations

  1. Institute of Medicinal Plant Development, Chinese Academy of Medical Science & Peking Union Medical College, Xi-Bei-Wang, Hai-Dian District, Beijing, 100094, P. R. China

    Hetian Lei, Jianjun Qi, Jingyuan Song, Yinling Zhang & Junshan Yang

  2. Department of Chemistry and Life Science, Xiangnan College, Chenzhou City, Hunan Province, 423000, P. R. China

    Dejun Yang

  3. Lucheng Middle School, Daxing District, Beijing, 100260, P. R. China

    Yanzhi Wang

Authors
  1. Hetian Lei
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jianjun Qi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jingyuan Song
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Dejun Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yanzhi Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Yinling Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Junshan Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Hetian Lei.

Additional information

Communicated by D. Dudits

Rights and permissions

Reprints and permissions

About this article

Cite this article

Lei, H., Qi, J., Song, J. et al. Biosynthesis and bioactivity of trichosanthin in cultured crown gall tissues of Trichosanthes kirilowii Maximowicz. Plant Cell Rep 25, 1205–1212 (2006). https://doi.org/10.1007/s00299-006-0187-5

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00299-006-0187-5

Keywords

Search

Navigation