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Cloning and characterization of a squalene synthase gene from a petroleum plant, Euphorbia tirucalli L.

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Abstract

Euphorbia tirucalli L., which is also known as a petroleum plant, produces a large amount of phytosterols and triterpenes. During their biosynthesis, squalene synthase converts two molecules of the hydrophilic substrate farnesyl diphosphate into a hydrophobic product, squalene. An E. tirucalli cDNA clone of a putative squalene synthase gene (EtSS) was isolated by RT-PCR followed by 5′- and 3′-RACE. The restriction fragment polymorphisms revealed by Southern blot analysis suggest that EtSS is a single copy gene. The glycine at the 287th residue from the N-terminal end of domain C has replaced alanine, which is conserved among all the other SS sequences deposited in the Genbank database. The N-terminal 380 residues of the hydrophilic sequence was expressed as a peptide-tagged protein in E. coli, and the resultant bacterial crude extract was incubated with farnesyl diphosphate and NADPH. GC-MS analysis showed that squalene was detected in the in vitro reaction mixture. E. tirucalli transgenic callus lines, in which EtSS was overexpressed, accumulated increased amounts of phytosterols as compared with that of wild type callus. RT-PCR analysis of wild type E. tirucalli plants revealed that the EtSS transcript accumulated in almost equal amounts in the stems and the leaves with a stalk, while a lower amount was detected in the roots. In situ hybridization analysis revealed that prominent antisense-probe signal was detected in the cambia within bundle sheathes. These results indicate that EtSS functions prominently in cambia, which are located adjacent to conductive tubes, and that this gene plays important roles in phytosterol accumulation in petroleum plants.

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Abbreviations

35S:

Cauliflower mosaic virus 35S promoter

DIG:

Digoxigenin

FPP:

Farnesyl diphosphate

Et :

Euphorbia tirucalli

GA:

Gibberellic acid

GC-MS:

Gas chromatograph-mass spectrometer

GC-EIMS:

Gas chromatography-electron impact mass spectrometry

Nus:

N utilization substance protein

SC:

Sesquiterpene cyclase

SC :

Sesquiterpene cyclase gene

SE:

Squalene epoxidase

SE :

Squalene epoxidase gene

SS:

Squalene synthase

SS :

Squalene synthase gene

TIC:

Total ion chromatogram

Tnos:

Nopalin synthase gene terminator

Trx:

Thioredoxin

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Acknowledgments

The authors thank Drs. Takahiko Hayakawa, Nobuya Koizuka, Mr. Yasuyuki Hayashi, and Ms. Mio Watanabe, from Plantech Research Institute, Yokohama, for the PCR primer design. The authors also thank Dr. Masashi Suzuki, from RIKEN Plant Science Center, Dr. Tatsuro Hamada and Ms. Akiko Nakade, from Ishikawa Prefectural University, Dr. Soichi Nakamura, from University of Ryukyus, for supporting this work. This work was performed as one of the technology development projects of the “Green Biotechnology Program” and was supported by a NEDO (New Energy and Industrial Technology Development Organization) grant donated to K. O., Ishikawa Prefectural University.

Author information

Correspondence to Kanji Ohyama.

Additional information

Nucleotide sequence accession numbers are EtSS cDNA: AB433916, EtSS genome: AB433917.

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Uchida, H., Yamashita, H., Kajikawa, M. et al. Cloning and characterization of a squalene synthase gene from a petroleum plant, Euphorbia tirucalli L.. Planta 229, 1243–1252 (2009). https://doi.org/10.1007/s00425-009-0906-6

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Keywords

  • Bundle sheath
  • Cambium
  • Euphorbia
  • In vitro enzyme reaction
  • In situ hybridization
  • Mevalonate pathway
  • Petroleum plant
  • Phytosterol
  • Squalene synthase