Characterizing the Physcomitrella patens ecotype Reute
Ecotype collections are used for several plant models to unravel the molecular causes of phenotypic differences and to investigate effects of environmental adaptation and acclimation. For the model moss Physcomitrella patens, collections of accessions are available and have been used e.g. for phylog...
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|Summary:||Ecotype collections are used for several plant models to unravel the molecular causes of phenotypic differences and to investigate effects of environmental adaptation and acclimation. For the model moss Physcomitrella patens, collections of accessions are available and have been used e.g. for phylogenetic and taxonomic studies, but few were investigated further for phenotypic differences. My thesis focuses on the accession found in Reute close to Freiburg im Breisgau, Germany. My publication shows that the standard laboratory strain Gransden produces fewer sporophytes than Reute or Villersexel K3, although gametangia develop in the same time course and do not show evident morphological differences. My work provides expression profiling and comparative developmental data for several stages of sporophyte development, as well as information on genetic variation from genomic sequencing. There is variation in the expression profiles of several genes between Gransden and Reute, as well as genome segments that are variation hotspots. With my work I propose that Reute is considered a P. patens ecotype and suggest its use for investigations that involve progression through the life cycle and generational succession.
In my experiments I used the P. patens ecotype from Reute in molecular biology experiments introducing fluorescent proteins via chemical protoplast transformation to study codon usage bias and select a strong endogenous promoter. The 35S promoter, which is commonly used in plant systems, is only suitable to a limited extent in Physcomitrella patens due to mediocre and non-constitutive activity. Based on a broad range of Gransden and Reute microarray experiments we selected an LHCS gene that is highly expressed in most tissues and treatments. To measure expression strength I developed a novel fluorescence readout system, utilizing a microplate reader and an internal fluorescence control for normalization. The results from my publications demonstrate that the selected promoter is more active than the double 35S and Actin promoters. Deletion constructs were generated to develop a shorter promoter that retains the high activity of the full-length promoter. In parallel, codon bias within P. patens was analysed and demonstrated that the use of several codons is biased and correlates with expression strength. Two GFP variants were synthesized with different sets of codons and compared, showing that optimized codon usage increases the amount of protein under the control of strong promoters.
In summary the findings from my publications characterize the P. patens ecotype from Reute and demonstrate that it will be an useful tool in reverse genetic studies.|
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