Abstract
BackgroundThe moss Physcomitrella patens is an attractive model system for plant biology and functional genome analysis. It shares many biological features with higher plants but has the unique advantage of an efficient homologous recombination system for its nuclear DNA. This allows precise genetic manipulations and targeted knockouts to study gene function, an approach that due to the very low frequency of targeted recombination events is not routinely possible in any higher plant.ResultsAs an important prerequisite for a large-scale gene/function correlation study in this plant, we are establishing a collection of Physcomitrella patens transformants with insertion mutations in most expressed genes. A low-redundancy moss cDNA library was mutagenised in E. coli using a derivative of the transposon Tn1000. The resulting gene-disruption library was then used to transform Physcomitrella. Homologous recombination of the mutagenised cDNA with genomic coding sequences is expected to target insertion events preferentially to expressed genes. An immediate phenotypic analysis of transformants is made possible by the predominance of the haploid gametophytic state in the life cycle of the moss. Among the first 16,203 transformants analysed so far, we observed 2636 plants ( = 16.2%) that differed from the wild-type in a variety of developmental, morphological and physiological characteristics.ConclusionsThe high proportion of phenotypic deviations and the wide range of abnormalities observed among the transformants suggests that mutagenesis by gene-disruption library transformation is a useful strategy to establish a highly diverse population of Physcomitrella patens mutants for functional genome analysis.
Highlights
The moss Physcomitrella patens is an attractive model system for plant biology and functional genome analysis
Results and Discussion cDNA library To establish a Physcomitrella cDNA library representing most genes expressed during vegetative growth before the onset of differentiation, RNA was extracted from protonemata cultured for different time periods in liquid culture, and a cDNA library in plasmid vectors was established after normalization to decrease redundancy [16]
Mass DNA sequencing and clustering of 57,000 EST sequences yielded 12,000 non-overlapping sequence clusters, and showed a low degree of clone redundancy in the cDNA library used. Sequence analysis of these contigs, together with a large number of additional EST sequences derived from other growth stages and tissues, suggest that the total number of coding sequences for the moss Physcomitrella patens and the flowering plant Arabidopsis thaliana is similar (Rensing et al, submitted), despite a three-fold larger genome size for the moss [12]
Summary
The moss Physcomitrella patens is an attractive model system for plant biology and functional genome analysis It shares many biological features with higher plants but has the unique advantage of an efficient homologous recombination system for its nuclear DNA. This allows precise genetic manipulations and targeted knockouts to study gene function, an approach that due to the very low frequency of targeted recombination events is not routinely possible in any higher plant. After the first demonstration of high-frequency recombination between chromosomal sequences and homologous DNA introduced by transformation [5], gene targeting in Physcomitrella was used successfully to study the function of several genes by creating functional knockouts [6,7,8,9]. The high specificity provided by homologous recombination even allows the specific targeting of single members of multi-gene families [10]
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