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High frequency of phenotypic deviations in Physcomitrella patens plants transformed with a gene-disruption library.

Egener T, Granado J, Guitton MC, Hohe A, Holtorf H, Lucht JM, Rensing SA, Schlink K, Schulte J, Schween G, Zimmermann S, Duwenig E, Rak B, Reski R - BMC Plant Biol. (2002)

Bottom Line: The resulting gene-disruption library was then used to transform Physcomitrella.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Plant Biotechnology, Freiburg University, Sonnenstrasse 5, D-79104 Freiburg/Br, Germany. tanja.egener@biologie.uni-freiburg.de

ABSTRACT

Background: 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.

Results: As 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.

Conclusions: The 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.

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Shuttle transposon mutagenesis of Physcomitrella patens cDNA clones. The structure of a representative moss cDNA clone (ID: S_PP015059353; 808 bp) in the pUCMinIV minimal vector is shown. This defined plasmid was subjected to shuttle mutagenesis, and the transposon insertion sites for 72 resulting clones were mapped by DNA sequencing to assess the distribution of insertions. 41 insertions in "forward" orientation (nptII resistance marker on transposon and bla marker on vector transcribed in same orientation) are indicated by blue lines within the circle, 31 "reverse" insertions by red lines outside. Most of the insertions (66 / 72, corresponding to 92%) occurred throughout the cDNA, without apparent strong bias for insertion site or orientation. For production of the gene-disruption library used afterwards for the moss transformation, cDNA clones were mutagenised in pools; here about 70% of the mutagenised plasmids had insertions in the cDNA.
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Figure 2: Shuttle transposon mutagenesis of Physcomitrella patens cDNA clones. The structure of a representative moss cDNA clone (ID: S_PP015059353; 808 bp) in the pUCMinIV minimal vector is shown. This defined plasmid was subjected to shuttle mutagenesis, and the transposon insertion sites for 72 resulting clones were mapped by DNA sequencing to assess the distribution of insertions. 41 insertions in "forward" orientation (nptII resistance marker on transposon and bla marker on vector transcribed in same orientation) are indicated by blue lines within the circle, 31 "reverse" insertions by red lines outside. Most of the insertions (66 / 72, corresponding to 92%) occurred throughout the cDNA, without apparent strong bias for insertion site or orientation. For production of the gene-disruption library used afterwards for the moss transformation, cDNA clones were mutagenised in pools; here about 70% of the mutagenised plasmids had insertions in the cDNA.

Mentions: To create a gene-disruption library of cDNA clones carrying insertion mutations, cDNA clones were subjected to shuttle mutagenesis in E. coli. First, the normalised cDNA pool from the amplified protonema library was subcloned into the minimal vector pUCMinIV (Fig. 2). This plasmid is a 1.7 kb derivative of pUC19 from which we have deleted most non-essential DNA sequences to remove insertion targets within the vector sequence. Pooled minimal vectors with target cDNAs were introduced into a donor E. coli strain carrying an inducible transposase gene (tnpA) and a conjugative plasmid with a derivative of transposon Tn1000. This mini-transposon carries a modified nos-promoter driven nptII expression cassette encoding resistance against the antibiotic G418 as selectable plant marker gene between the border repeat sequences of Tn1000 required for transposition [17]. Induction of transposase activity by IPTG results in transposition and the formation of a cointegrate between conjugative plasmid and cDNA clone. Resolution of the cointegrates was achieved by conjugative transfer into a recipient strain overexpressing the tnpR resolvase gene, resulting in the release of a copy of the cDNA-carrying minimal vector with an insertion of the mini-Tn1000::nptII. These plasmids were isolated and retransformed into E. coli. Analysis of individual clones from this gene-disruption library, as well as model experiments using defined cDNA clone targets showed that in most of the resulting tagged plasmids the transposon had inserted into the moss cDNA, with no apparent preference for target sequence or insertion orientation (Fig. 2).


High frequency of phenotypic deviations in Physcomitrella patens plants transformed with a gene-disruption library.

Egener T, Granado J, Guitton MC, Hohe A, Holtorf H, Lucht JM, Rensing SA, Schlink K, Schulte J, Schween G, Zimmermann S, Duwenig E, Rak B, Reski R - BMC Plant Biol. (2002)

Shuttle transposon mutagenesis of Physcomitrella patens cDNA clones. The structure of a representative moss cDNA clone (ID: S_PP015059353; 808 bp) in the pUCMinIV minimal vector is shown. This defined plasmid was subjected to shuttle mutagenesis, and the transposon insertion sites for 72 resulting clones were mapped by DNA sequencing to assess the distribution of insertions. 41 insertions in "forward" orientation (nptII resistance marker on transposon and bla marker on vector transcribed in same orientation) are indicated by blue lines within the circle, 31 "reverse" insertions by red lines outside. Most of the insertions (66 / 72, corresponding to 92%) occurred throughout the cDNA, without apparent strong bias for insertion site or orientation. For production of the gene-disruption library used afterwards for the moss transformation, cDNA clones were mutagenised in pools; here about 70% of the mutagenised plasmids had insertions in the cDNA.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC117800&req=5

Figure 2: Shuttle transposon mutagenesis of Physcomitrella patens cDNA clones. The structure of a representative moss cDNA clone (ID: S_PP015059353; 808 bp) in the pUCMinIV minimal vector is shown. This defined plasmid was subjected to shuttle mutagenesis, and the transposon insertion sites for 72 resulting clones were mapped by DNA sequencing to assess the distribution of insertions. 41 insertions in "forward" orientation (nptII resistance marker on transposon and bla marker on vector transcribed in same orientation) are indicated by blue lines within the circle, 31 "reverse" insertions by red lines outside. Most of the insertions (66 / 72, corresponding to 92%) occurred throughout the cDNA, without apparent strong bias for insertion site or orientation. For production of the gene-disruption library used afterwards for the moss transformation, cDNA clones were mutagenised in pools; here about 70% of the mutagenised plasmids had insertions in the cDNA.
Mentions: To create a gene-disruption library of cDNA clones carrying insertion mutations, cDNA clones were subjected to shuttle mutagenesis in E. coli. First, the normalised cDNA pool from the amplified protonema library was subcloned into the minimal vector pUCMinIV (Fig. 2). This plasmid is a 1.7 kb derivative of pUC19 from which we have deleted most non-essential DNA sequences to remove insertion targets within the vector sequence. Pooled minimal vectors with target cDNAs were introduced into a donor E. coli strain carrying an inducible transposase gene (tnpA) and a conjugative plasmid with a derivative of transposon Tn1000. This mini-transposon carries a modified nos-promoter driven nptII expression cassette encoding resistance against the antibiotic G418 as selectable plant marker gene between the border repeat sequences of Tn1000 required for transposition [17]. Induction of transposase activity by IPTG results in transposition and the formation of a cointegrate between conjugative plasmid and cDNA clone. Resolution of the cointegrates was achieved by conjugative transfer into a recipient strain overexpressing the tnpR resolvase gene, resulting in the release of a copy of the cDNA-carrying minimal vector with an insertion of the mini-Tn1000::nptII. These plasmids were isolated and retransformed into E. coli. Analysis of individual clones from this gene-disruption library, as well as model experiments using defined cDNA clone targets showed that in most of the resulting tagged plasmids the transposon had inserted into the moss cDNA, with no apparent preference for target sequence or insertion orientation (Fig. 2).

Bottom Line: The resulting gene-disruption library was then used to transform Physcomitrella.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Plant Biotechnology, Freiburg University, Sonnenstrasse 5, D-79104 Freiburg/Br, Germany. tanja.egener@biologie.uni-freiburg.de

ABSTRACT

Background: 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.

Results: As 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.

Conclusions: The 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.

Show MeSH
Related in: MedlinePlus