Cloning and sequencing of Trichosanthin gene and its expression in Escherichia coli and tobacco plant

Yi Ming Bao, Rui Yin Chu, Jin Hua Han, Hui Zhang, Nai Sui Pan, Xiao Cheng Gu, Zhang Liang Chen

Research output: Contribution to journalArticlepeer-review

Abstract

A Trichosanthin gene was cloned from Trichosanthes kirilowii genomic DNA by polymerase chain reaction (PCR). Nucleotide sequence data indicated that we obtained the coding region of the mature Trichosanthin peptide as well as its signal peptide at the N-terminus. Comparisons of our sequence with the previously reported nucleotide sequences of this gene showed 99.25% homology, yet there were notable differences between the previously reported amino acid sequence and our deduced result. This gene was subcloned into a high-level expression plasmid (pJLA502) of E. coli under the control of a PRPL promoter, and we observed the gene product after temperature induction. The gene was further cloned into plant intermediate vector pE3 under the control of a CaMV 35S promoter, and transferred into a tobacco genome using the agrobacterium-mediated gene transfer system. Western blotting analysis of the protein extracted from Escherichia coli and transgenic tobacco plants proved that the Trichosanthin gene has been expressed in both organisms.

Original languageEnglish (US)
Pages (from-to)669-676
Number of pages8
JournalScience in China (Scientia Sinica) Series B
Volume36
Issue number6
StatePublished - Jun 1 1993
Externally publishedYes

Keywords

  • Trichosanthin
  • gene expression
  • nucleotide sequence analysis
  • polymerase chain reaction
  • transgenic plant

ASJC Scopus subject areas

  • Environmental Science(all)
  • Engineering(all)
  • Earth and Planetary Sciences(all)

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