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T‐DNA recombination and replication in maize cells
Author(s) -
Zhao Xiaoxia,
Coats Isabelle,
Fu Ping,
GordonKamm Bill,
Lyznik L. Alexander
Publication year - 2003
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1046/j.1365-313x.2003.016016.x
Subject(s) - recombination , homologous recombination , in vitro recombination , biology , dna , site specific recombination , recombinant dna , dna replication , genetic recombination , microbiology and biotechnology , genetics , replication protein a , cre lox recombination , gene , molecular cloning , recombinase , dna binding protein , transgene , genetically modified mouse , transcription factor , peptide sequence
Summary T‐DNA recombination and replication was analyzed in ‘black mexican sweet’ (BMS) cells transformed with T‐DNAs containing the replication system from wheat dwarf virus (WDV). Upon recombination between the T‐DNA ends, a promoterless marker gene ( gusA ) was activated. Activation of the recombination marker gene was delayed and increased exponentially over time, suggesting that recombination and amplification of the T‐DNA occurred in maize cells. Mutant versions of the viral initiator gene ( rep ), known to be defective in the replication function, failed to generate recoverable recombinant T‐DNA molecules. Circularization of T‐DNA by the FLP/ FRT site‐specific recombination system and/or homologous recombination was not necessary to recover circular T‐DNAs. However, replicating T‐DNAs appeared to be suitable substrates for site‐specific and homologous recombination. Among 33 T‐DNA border junctions sequenced, only one pair of identical junction sites was found implying that the population of circular T‐DNAs was highly heterogenous. Since no circular T‐DNA molecules were detected in treatments without rep , it suggested that T‐DNA recombination was linked to replication and might have been stimulated by this process. The border junctions observed in recombinant T‐DNA molecules were indicative of illegitimate recombination and were similar to left‐border recombination of T‐DNA into the genome after Agro‐mediated plant transformation. However, recombination between T‐DNA molecules differed from T‐DNA/genomic DNA junction sites in that few intact right borders were observed. The replicating T‐DNA molecules did not enhance genomic random integration of T‐DNA in the experimental configuration used for this study.