Abstract
Inducible transgene expression provides a useful tool to analyze gene function. The moss Physcomitrella patens is a model basal land plant with well-developed research tools, including a high efficiency of gene targeting and substantial genomics resources. However, current systems for controlled transgene expression remain limited. Here we report the development of an estrogen receptor mediated inducible gene expression system, based on the system used in flowering plants. After identifying the appropriate promoters to drive the chimeric transducer, we succeeded in inducing transcription over 1,000-fold after 24 h incubation with β-estradiol. The P . patens system was also effective for high-level long-term induction of gene expression; transcript levels of the activated gene were maintained for at least seven days on medium containing β-estradiol. We also established two potentially neutral targeting sites and a set of vectors for reproducible expression of two transgenes. This β-estradiol-dependent system will be useful to test genes individually or in combination, allowing stable, inducible transgenic expression in P . patens .
Highlights
The moss Physcomitrella patens lineage diverged from the flowering plant lineage approximately 450 million years ago [1,2] and comparisons of its physiology and development to those in flowering plants are useful to elucidate generality, diversity, and evolution in land plants
We modified the XVE construct, pER8 ( [22]: Figure 1A) containing the following three regions: (1) the XVE chimeric gene encoding a DNA-binding domain of the bacterial repressor LexA (X [28,29]:), the transcriptional activation domain of VP16 (V [30]:), and the regulatory region of the human estrogen receptor (E [31]:), which is driven by the synthetic G10-90 promoter [32] and connected to Pisum sativum rbcS E9 terminator [33], (2) the HYGROMYCIN PHOSPHOTRANSFERASE (HPT [17]:) gene cassette composed of a nopaline synthase promoter (Pnos [17]:) and a nopaline synthase terminator (Tnos [17]:) for selection of transformants, and (3) eight copies of a bacterial LexA operator [22] connected to Pm35S [23], a multiple cloning site to introduce a transgene, and a Pisum sativum pea 3A terminator
The HPT cassette was replaced with the aphIV cassette, which uses a modified CaMV35S promoter and CaMV35S terminator that are efficient in P. patens [35,36]
Summary
The moss Physcomitrella patens lineage diverged from the flowering plant lineage approximately 450 million years ago [1,2] and comparisons of its physiology and development to those in flowering plants are useful to elucidate generality, diversity, and evolution in land plants. The tetracycline repression system can be used as an inducible expression system in P. patens [16], but maintaining repression of transgene expression requires cultivating the moss on medium supplemented with tetracycline, which causes growth retardation [13]. Another chemically inducible expression system is the GVG system [13,17], which uses the DNA binding domain of the yeast GAL4 transcription factor, the transcriptional activation domain of the herpes viral protein VP16, and the mammalian glucocorticoid receptor. Based on mammalian expression technologies several inducible and autoregulated promoters have been introduced into P. patens protoplasts [21] but have not been examined yet in stably transformed moss lines during the life cycle
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