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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.

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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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Southern blot analysis of gene-disruption library transformants. 1 μg of genomic DNA isolated from two representative Physcomitrella transformants (BC344 and BC3238) was digested to completion with PvuII, which has a recognition site within the nptII coding sequence, or one of seven other restriction enzymes that do not cut within nptII and only rarely in Physcomitrella cDNAs. Fragments carrying nptII -sequences were detected by a DIG-labelled probe after electrophoresis and transfer to a nylon membrane.
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Figure 6: Southern blot analysis of gene-disruption library transformants. 1 μg of genomic DNA isolated from two representative Physcomitrella transformants (BC344 and BC3238) was digested to completion with PvuII, which has a recognition site within the nptII coding sequence, or one of seven other restriction enzymes that do not cut within nptII and only rarely in Physcomitrella cDNAs. Fragments carrying nptII -sequences were detected by a DIG-labelled probe after electrophoresis and transfer to a nylon membrane.

Mentions: For molecular analysis, genomic DNA was isolated from randomly chosen transformants and analysed in Southern blot experiments. To obtain an estimate for the number of gene-disruption constructs integrated in the transformed moss plants, this DNA was digested with PvuII, which has a single recognition site within the nptII coding sequence. This results in the formation of two fragments detected by a nptII-derived probe for each copy of the selection cassette. The length of these fragments differs depending on the adjacent sequences, which can be either derived from genomic sequences or from cDNA contributed by the transforming DNA. On Southern blots of 55 transformants, 19.3 ± 3.6 (mean ± s. d.) bands were detected, suggesting an average insertion number of about 10 nptII-cassettes per transformant (data not shown). To discriminate between independent integration events at different genomic positions and co-integrations at the same locus, genomic DNA of 16 transformants was digested with several restriction enzymes that cut genomic Physcomitrella DNA frequently but do not cut within the nptII cassette, and only rarely within the cDNA sequences carried by the transforming DNA (Fig. 6). Most of the genomic DNA was cut by these enzymes into fragments ranging from 1 to 15 kb, as judged by ethidium-bromide staining after gel electrophoresis. However, the majority of fragments detected after hybridisation with the disruption-construct specific nptII-probe were more than 20 kb in size; only few distinct nptII-hybridising bands were observed. Digestion of the same DNA with PvuII, which cuts within the nptII selection cassette, resulted in several nptII-hybridising fragments of smaller size ranging between 1 and 10 kb (Fig. 6). This suggests that most of the transforming DNA molecules are co-integrated in close vicinity to each other, without intervening genomic sequences, at few genomic loci. PCR amplification of transgene sequences between the nptII-insertions using nptII-primers and sequence analysis of the obtained products confirmed tandem integrations of different gene disruption constructs in close vicinity (data not shown). Similar integration patterns of transforming DNA molecules in Physcomitrella have been observed previously [5,15].


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)

Southern blot analysis of gene-disruption library transformants. 1 μg of genomic DNA isolated from two representative Physcomitrella transformants (BC344 and BC3238) was digested to completion with PvuII, which has a recognition site within the nptII coding sequence, or one of seven other restriction enzymes that do not cut within nptII and only rarely in Physcomitrella cDNAs. Fragments carrying nptII -sequences were detected by a DIG-labelled probe after electrophoresis and transfer to a nylon membrane.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC117800&req=5

Figure 6: Southern blot analysis of gene-disruption library transformants. 1 μg of genomic DNA isolated from two representative Physcomitrella transformants (BC344 and BC3238) was digested to completion with PvuII, which has a recognition site within the nptII coding sequence, or one of seven other restriction enzymes that do not cut within nptII and only rarely in Physcomitrella cDNAs. Fragments carrying nptII -sequences were detected by a DIG-labelled probe after electrophoresis and transfer to a nylon membrane.
Mentions: For molecular analysis, genomic DNA was isolated from randomly chosen transformants and analysed in Southern blot experiments. To obtain an estimate for the number of gene-disruption constructs integrated in the transformed moss plants, this DNA was digested with PvuII, which has a single recognition site within the nptII coding sequence. This results in the formation of two fragments detected by a nptII-derived probe for each copy of the selection cassette. The length of these fragments differs depending on the adjacent sequences, which can be either derived from genomic sequences or from cDNA contributed by the transforming DNA. On Southern blots of 55 transformants, 19.3 ± 3.6 (mean ± s. d.) bands were detected, suggesting an average insertion number of about 10 nptII-cassettes per transformant (data not shown). To discriminate between independent integration events at different genomic positions and co-integrations at the same locus, genomic DNA of 16 transformants was digested with several restriction enzymes that cut genomic Physcomitrella DNA frequently but do not cut within the nptII cassette, and only rarely within the cDNA sequences carried by the transforming DNA (Fig. 6). Most of the genomic DNA was cut by these enzymes into fragments ranging from 1 to 15 kb, as judged by ethidium-bromide staining after gel electrophoresis. However, the majority of fragments detected after hybridisation with the disruption-construct specific nptII-probe were more than 20 kb in size; only few distinct nptII-hybridising bands were observed. Digestion of the same DNA with PvuII, which cuts within the nptII selection cassette, resulted in several nptII-hybridising fragments of smaller size ranging between 1 and 10 kb (Fig. 6). This suggests that most of the transforming DNA molecules are co-integrated in close vicinity to each other, without intervening genomic sequences, at few genomic loci. PCR amplification of transgene sequences between the nptII-insertions using nptII-primers and sequence analysis of the obtained products confirmed tandem integrations of different gene disruption constructs in close vicinity (data not shown). Similar integration patterns of transforming DNA molecules in Physcomitrella have been observed previously [5,15].

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