Abstract
BackgroundThe mycobacteriophage large serine recombinase Bxb1 catalyzes site-specific recombination between its corresponding attP and attB recognition sites. Previously, we and others have shown that Bxb1 has catalytic activity in various eukaryotic species including Nicotiana tabacum, Schizosaccharomyces pombe, insects and mammalian cells.ResultsIn this work, the Bxb1 recombinase gene was transformed and constitutively expressed in Arabidopsis thaliana plants harboring a chromosomally integrated attP and attB-flanked target sequence. The Bxb1 recombinase successfully excised the target sequence in a conservative manner and the resulting recombination event was heritably transmitted to subsequent generations in the absence of the recombinase transgene. In addition, we also show that Bxb1 recombinase expressing plants can be manually crossed with att-flanked target transgenic plants to generate excised progeny.ConclusionThe Bxb1 large serine recombinase performs site-specific recombination in Arabidopsis thaliana germinal tissue, producing stable lines free of unwanted DNA. The precise site-specific deletion produced by Bxb1 in planta demonstrates that this enzyme can be a useful tool for the genetic engineering of plants without selectable marker transgenes or other undesirable exogenous sequences.
Highlights
The mycobacteriophage large serine recombinase Bxb1 catalyzes site-specific recombination between its corresponding attP and attB recognition sites
Experimental design To test for site-specific recombination, we initially sought to use a gain-of-function strategy whereby excision of a transgene would lead to promoter fusion with a previously distal marker [14]
Upon sitespecific excision of the recognition site-flanked DNA, the TR1 plants were back crossed to wild type plants and the BC1 progeny screened for segregants that retain the excision event but lack the recombinase gene
Summary
The mycobacteriophage large serine recombinase Bxb catalyzes site-specific recombination between its corresponding attP and attB recognition sites. Large serine recombinases act on two unique sequences, known as the recognition sites attP and attB, to yield the product sites known as attL and attR [4]. Several recombinase systems of this type including Bxb, TP901-1, U153 [5,6,7] and phiC31 [8], have been shown to function in eukaryotic cells. The Bxb recombinase is a 500 amino acid protein that binds minimal recognition sites attP and attB that are 39 bp and 34 bp, respectively and enzymatically executes uni-directional site-specific recombination [9]. Recombinases from the the small tyrosine family which have been shown to function in plants (i.e. Cre, FLP, R) all naturally perform bidirectional or fully reversible recombination reactions
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