Cold inducible promoter driven Cre-lox system proved to be highly efficient for marker gene excision in transgenic barley

Csaba Éva, Flóra Téglás, Helga Zelenyánszki, Cecília Tamás, Angéla Juhász, Klára Mészáros, László Tamás

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

A Cre-lox based auto-excision strategy has been adapted for barley, capable of cre and selectable marker gene (SMG) removal. The cold inducible wheat promoter called wcs120 was utilised for driving Cre expression. The binary vector was carrying the transgene (uidA) and a so called ‘recombination cassette’ flanked by the lox sequences. This part included both the recombinase gene and the SMG (bar) under the control of a constitutive promoter. T0, T1 and T2 transgenic plants were subjected to low temperature (at 4 °C, 10 °C and 12 °C) at different developmental stages to induce recombination. The presence of uidA, cre, and bar genes and recombination footprints were studied by PCR and DNA sequencing, while cre transcription was followed by qRT-PCR. These analyses indicated that, cold treatment of the germinating seeds (4 °C for 3 days) followed by plant growing at higher temperature (24 °C) has been the most efficient (90–100%), and this treatment lead to heritable changes in the genome. Thermal separation of Cre accumulation (at low temperature) from Cre enzyme activity (at higher temperature) could have prevented the premature excision of its own encoding gene, and lead to high expression level thereby increasing recombination frequency.

Original languageEnglish
Pages (from-to)15-24
Number of pages10
JournalJournal of Biotechnology
Volume265
DOIs
Publication statusPublished - Jan 10 2018

Keywords

  • Auto-excision strategy
  • Cold induction
  • Site-specific recombination
  • Transgenic plants

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

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