Skip to main content
SpringerLink
Log in

Overexpression of a 10-deacetylbaccatin III-10 β-O-acetyltransferase gene leads to increased taxol yield in cells of Taxus chinensis

  • Original Paper
  • Published:
Plant Cell, Tissue and Organ Culture (PCTOC) Aims and scope Submit manuscript

Abstract

Taxus chinensis suspension cells were transformed by the Agrobacterium-mediated transformation method to overexpress the dbat gene coding for 10-deacetylbaccatin III-10 β-O-acetyltransferase, a key enzyme for taxol biosynthesis. A. tumefaciens strain LBA4404 harboring either pCAMBIA1303 or the recombinant plasmid p1303-SdbatN was used. Both plasmids harbored the hygromycin phosphotransferase gene (hptII) and gusA-mgfp5 gene as selectable markers, but the latter plasmid also harbored the dbat gene in the T-DNA region. The transgenic T. chinensis cells had been maintained in modified Gamborg’s B5 medium supplemented with hygromycin for more than 14 months and were subcultured at 4-week intervals. The selected transgenic cells were identified by PCR, Southern blot analysis, β-glucuronidase assay and western blot analysis, and the results showed that the transgenes were integrated in the chromosomal DNA of T. chinensis cells with single-copy style, and that the gusA-mgfp5 reporter gene was expressed in the transgenic cells. The dbat mRNA expression level in the p1303-SdbatN-transgenic T. chinensis cells tested by real-time quantitative PCR was 5.3 ± 0.6 times that of the non-transformed cells. Taxol yield of the p1303-SdbatN-transgenic T. chinensis cells was about 1.7 times that of the non-transformed cells. These results suggest that the overexpression of dbat gene in transgenic T. chinensis cells leads to increased taxol yield.

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

Similar content being viewed by others

Abbreviations

CaMV:

Cauliflower mosaic virus

NOS:

Nopaline synthase

GUS:

β-glucuronidase

MeJ:

Methyl jasmonate

dbat :

10-deacetylbaccatin III-10 β-O-acetyltransferase gene

hptII :

Hygromycin phosphotransferase gene

DW:

Dry weight

T-DNA:

Transfer DNA

PCR:

Polymerase chain reaction

HPLC:

High performance liquid chromatography

References

  • Banyai W, Kirdmanee C, Mii M, Supaibulwatana K (2010) Overexpression of farnesyl pyrophosphate synthase (FPS) gene affected artemisinin content and growth of Artemisia annua L. Plant Cell Tiss Organ Cult 103:255–265

    Article  CAS  Google Scholar 

  • Christen AA, Bland J, Gibson DM (1989) Cell cultures as a means to produce taxol. Proc Am Assoc Cancer Res 30:566

    Google Scholar 

  • Croteau R, Ketchum REB, Long RM, Kaspera R, Wildung MR (2006) Taxol biosynthesis and molecular genetics. Phytochem Rev 5:75–97

    Article  PubMed  CAS  Google Scholar 

  • Espasandin FD, Collavino MM, Luna CV, Paz RC, Tarrago JR, Ruiz OA, Mroginski LA, Sansberro PA (2010) Agrobacterium tumefaciens-mediated transformation of Lotus tenuis and regeneration of transgenic lines. Plant Cell Tiss Organ Cult 102:181–189

    Article  CAS  Google Scholar 

  • Gelvin SB (2009) Agrobacterium in the genomics age. Plant Physiol 150:1665–1676

    Article  PubMed  CAS  Google Scholar 

  • Guo BH, Kai GY, Jin HB, Tang KX (2006) Taxol synthesis. Afr J Biotechnol 5:15–20

    CAS  Google Scholar 

  • Han KH, Fleming P, Walker K, Loper M, Chilton WS, Mocek U, Gordon MP, Floss HG (1994) Genetic transformation of mature taxus: an approach to genetically control the in vitro production of the anticancer drug, taxol. Plant Sci 95:187–196

    Article  CAS  Google Scholar 

  • Ho CK, Chang SH, Lung JH, Tsai CJ, Chen KP (2005) The strategies to increase taxol production by using Taxus mairei cells transformed with TS and DBAT genes. Int J Appl Sci Eng 3:179–185

    Google Scholar 

  • Holton RA, Somoza C, Kim H-B, Liang F, Biediger RJ, Boatman PD, Shindo M, Smith CC, Kim S, Nadizadeh H, Suzuki Y, Tao C, Vu P, Tang S, Zhang P, Murthi KK, Gentile LN, Liu JH (1995) The total synthesis of paclitaxel starting with camphor. ACS Symp Ser 583:288–301

    Article  CAS  Google Scholar 

  • Jefferson RA, Kavanagh TA, Bevan MW (1987) Gus fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901–3907

    PubMed  CAS  Google Scholar 

  • Jennewein S, Croteau R (2001) Taxol: biosynthesis, molecular genetics, and biotechnological applications. Appl Microbiol Biotechnol 57:13–19

    Article  PubMed  CAS  Google Scholar 

  • Jennewein S, Rithner CD, Williams RM, Croteau R (2003) Taxoid metabolism: taxoid 14b-hydroxylase is a cytochrome P450-dependent monooxygenase. Arch Biochem Biophys 413:262–270

    Article  PubMed  CAS  Google Scholar 

  • Ji Y, Bi J-N, Yan B, Zhu X-D (2006) Taxol-producing fungi: a new approach to industrial production of taxol. Chin J Biotechnol 22:1–6

    Article  Google Scholar 

  • Kamath KR, Barry JJ, Miller KM (2006) The Taxus™ drug-eluting stent: A new paradigm in controlled drug delivery. Adv Drug Deliv Rev 58:412–436

    Article  PubMed  CAS  Google Scholar 

  • Ketchum REB, Gibson DM (1996) Paclitaxel production in cell suspension cultures of Taxus. Plant Cell Tiss Organ Cult 46:9–16

    Article  CAS  Google Scholar 

  • Ketchum REB, Gibson DM, Croteau RB, Shuler ML (1999) The kinetics of taxoid accumulation in cell suspension cultures of Taxus following elicitation with methyl jasmonate. Biotechnol Bioeng 62:97–105

    Article  PubMed  CAS  Google Scholar 

  • Ketchum REB, Wherland L, Croteau RB (2007) Stable transformation and long-term maintenance of transgenic Taxus cell suspension cultures. Plant Cell Rep 26:1025–1033

    Article  PubMed  CAS  Google Scholar 

  • Kim CH, Kim KI, Chung IS (2000) Expression of modified green fluorescent protein in suspension culture of Taxus cuspidata. J Microbiol Biotechnol 10:91–94

    CAS  Google Scholar 

  • Li M, Li H, Jiang H, Pan X, Wu G (2008) Establishment of an Agrobacteriuim-mediated cotyledon disc transformation method for Jatropha curcas. Plant Cell Tiss Organ Cult 92:173–181

    Article  CAS  Google Scholar 

  • Li L-Q, Fu CH, Zhao CF, Xia J, Wu W-J, Yu L-J (2009) Efficient extraction of RNA and analysis of gene expression in a long-term Taxus cell culture using Real-Time RT–PCR. Z Naturforsch 64c:125–130

    Google Scholar 

  • Ma X, Yu C, Tang S, Guo S, Zhang R, Wang Y, Zhu A, Zhu S, Xiong H (2010) Genetic transformation of the bast fiber plant ramie (Boehmeria nivea Gaud.) via Agrobacterium tumefaciens. Plant Cell Tiss Organ Cult 100:165–174

    Article  Google Scholar 

  • Nakamura T, Ishikawa M (2006) Transformation of suspension cultures of bromegrass (Bromus inermis) by Agrobacterium tumefaciens. Plant Cell Tiss Organ Cult 84:293–299

    Article  Google Scholar 

  • Nicolaou KC, Ueno H, Liu J-J, Nantermet PG, Yang Z, Renaud J, Paulvannan K, Chadha R (1995) Total synthesis of Taxol. 4. The final stages and completion of the synthesis. J Am Chem Soc 117:653–659

    Article  CAS  Google Scholar 

  • Raffeiner B, Serek M, Winkelmann T (2009) Agrobacterium tumefaciens-mediated transformation of Oncidium and Odontoglossum orchid species with the ethylene receptor mutant gene etr1-1. Plant Cell Tiss Organ Cult 98:125–134

    Article  CAS  Google Scholar 

  • Silva TER, Cidade LC, Alvim FC, Cascardo JCM, Costa MGC (2009) Studies on genetic transformation of Theobroma cacao L.: evaluation of different polyamines and antibiotics on somatic embryogenesis and the efficiency of uidA gene transfer by Agrobacterium tumefaciens. Plant Cell Tiss Organ Cult 99:287–298

    Article  CAS  Google Scholar 

  • Stierle A, Stroble G, Stierle D (1993) Taxol and taxane production by Taxomyces andreanae, an endophytic fungus of pacific yew. Science 260:214–216

    Article  PubMed  CAS  Google Scholar 

  • Tabata H (2004) Paclitaxel production by plant-cell-culture technology. Adv Biochem Eng Biotechnol 87:1–23

    PubMed  CAS  Google Scholar 

  • Travella S, Ross SM, Harden J, Everett C, Snape JW, Harwood WA (2005) A comparison of transgenic barley lines produced by particle bombardment and Agrobacterium-mediated techniques. Plant Cell Rep 23:780–789

    Article  PubMed  CAS  Google Scholar 

  • Wakler K, Croteau R (2000) Molecular cloning of a 10-deacetylbaccatin III-10-O-acetyl transferase cDNA from Taxus and functional expression in Escherichia coli. Proc Natl Acad Sci USA 97:583–587

    Article  Google Scholar 

  • Walker K, Croteau R (2001) Taxol biosynthetic genes. Phytochemistry 58:1–7

    Article  PubMed  CAS  Google Scholar 

  • Wani MC, Taylor HL, Wall ME, Coggon P, McPhail AT (1971) Plant antitumor agents. VI. Isolation and structure of taxol, a novel antileukemic and antitumor agent from Taxus brevifolia. J Am Chem Soc 93:2325–2327

    Article  PubMed  CAS  Google Scholar 

  • Zhang CH, Mei XG, Liu L, Yu LJ (2000) Enhanced paclitaxel production induced by the combination of elicitors in cell suspension cultures of Taxus chinensis. Biotechnol Lett 22:1561–1564

    Article  CAS  Google Scholar 

  • Zia M, Mirza B, Malik SA, Chaudhary MF (2010) Expression of rol genes in transgenic soybean (Glycine max L.) leads to changes in plant phenotype, leaf morphology, and flowering time. Plant Cell Tiss Organ Cult 103:227–236

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This research work was supported by a National Natural Science Foundation of China (grant number 20776058), a New Century Talents Support Program by the Ministry of Education of China in 2006, and a National “11th Five-Year Plan” to Support Science and Technology Project of China (2008BAI63B04). The authors would like to thank Dr Gu Xiao-man and Dr Cheng Hong of the Analytical and Testing Center in Huazhong University of Science and Technology for HPLC–MS analysis.

Author information

Authors and Affiliations

  1. Institute of Resource Biology and Biotechnology, Department of Biotechnology, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, 430074, Peoples Republic of China

    Peng Zhang, Shu-Tao Li, Ting-Ting Liu, Chun-Hua Fu, Peng-Peng Zhou, Chun-Fang Zhao & Long-Jiang Yu

  2. Key Laboratory of Molecular Biophysics Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China

    Peng Zhang, Shu-Tao Li, Ting-Ting Liu, Chun-Hua Fu, Peng-Peng Zhou, Chun-Fang Zhao & Long-Jiang Yu

Authors
  1. Peng Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Shu-Tao Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ting-Ting Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Chun-Hua Fu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Peng-Peng Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Chun-Fang Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Long-Jiang Yu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Long-Jiang Yu.

Additional information

Peng Zhang and Shu-Tao Li contributed equally to this work.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Zhang, P., Li, ST., Liu, TT. et al. Overexpression of a 10-deacetylbaccatin III-10 β-O-acetyltransferase gene leads to increased taxol yield in cells of Taxus chinensis . Plant Cell Tiss Organ Cult 106, 63–70 (2011). https://doi.org/10.1007/s11240-010-9894-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11240-010-9894-2

Keywords

Search

Navigation