Background: In addition to studies of plant gene function and developmental analyses, plant biotechnological use is largely dependent upon transgenic technologies. The moss Physcomitrella patens has become an exciting model system for studying plant molecular processes due to an exceptionally high rate of nuclear gene targeting by homologous recombination compared with other plants. However, its use in transgenic approaches requires expression vectors that incorporate sufficiently strong promoters. To satisfy this requirement, a set of plant expression vectors was constructed and equipped with either heterologous or endogenous promoters.
The moss Physcomitrella patens is an excellent tool to study plant gene-function relationships due to its high rate of homologous recombination (HR). It has also been shown to be very useful in the production of recombinant proteins which are secreted into a simple medium. Thus, there is a need for suitable promoters functional in this well established model organism. We isolated genomic flanking regions of the beta-tubulin gene family from Physcomitrella, concentrating on those family members showing high transcript abundance integrated over gametophytic tissues. Using a novel, fast and reliable quantification assay based on the transient expression and secretion of a recombinant human protein, three genomic upstream regions were characterised in serial deletion constructs. Expression rates were up to three times higher than those obtained with the 35S cauliflower mosaic virus (35S) promoter, which served as a reference.
Using primers annealing to S locus sequences the cleaved amplified polymorphic sequences (CAPS) method was applied to develop a marker and to characterize different alleles at the self-incompatibility locus in Brassica napus. A segregating F 2 population from a cross of a self-incompatible (SI) and a self-compatible parent, as well as seven SI lines representing four different S alleles were used. Several primers specific to the S locus in B. oleracea and B. campestris, chosen from the literature, allow polymerase chain reaction (PCR) amplification of genomic DNA. However, only one primer pair amplified a single specific and reproducible PCR fragment of the expected length in B. napus. Digestion with restriction endonucleases revealed polymorphisms for two CAPS markers absolutely linked to the S locus. Using the codominant marker efMboI it was possible to discriminate all three F 2 genotypes. With this marker and an additional marker using another primer pair it was possible to distinguish between three of the four different S alleles and five of the seven SI lines, respectively.
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