Use of Physcomitrella patens actin 5′ regions for high transgene expression: importance of 5′ introns

We have isolated four actin (Act) genes from Physcomitrella patens and used their corresponding 5′ regions for recombinant expression of the human vascular endothelial growth factor (rhVEGF121) in transiently transformed Physcomitrella protoplasts and in stable transformed lines. In the transient system, we found up to 11-fold activity of the corresponding 5′ regions as compared with that of the plant constitutive 35S promoter. Moreover, the use of an optimised expression vector in which the human VEGF signal peptide was exchanged with a plant signal peptide resulted in an additional 7-fold increase in secreted rhVEGF. We found that the 5′ introns of PpAct1, PpAct5 and PpAct7 are essential for high expression. The enhancing mechanisms of the introns, however, seem to be different since in the case of PpAct1, the expression level is stimulated only in the presence of the endogenous promoter, whereas the 5′ introns of PpAct5 and PpAct7 stimulate expression also in combination with the 35S promoter. Beyond this, the isolated 5′ regions are shown to be useful for high expression levels in transgenic moss lines with values of secreted rhVEGF up to 96 μg g-1 dry weight.