| Microalgae are one of the main sources of natural carotenoids and can be an excellent model to study the carotenogenesis, which control mechanisms are not clear yet. Phytoene desaturase and phytoene synthase are key enzymes in the carotenogenic pathway. Their downregulation by homologous recombination would help to elucidate their paper in this metabolic pathway, but traditional gene inactivation by homologous recombination is not possible in microalgae or plants, in which exogenous genes are randomly inserted in the nuclear genome. Post-transcriptional gene silencing via siRNA has been achieved in Chlamydomonas with different degree of success and has helped to the functional characterization of a good number of genes. Here we show that transformation of Chlamydomonas reinhardtii with DNA encoding short hairpin RNA sequences targeting phytoene desaturase and with an antisense pds cDNA fragment causes underexpression of the target gene. Using Real-time PCR we analysed the level of pds expression in the 140 transfomants that had integrated the interference construction in their genome and isolated five of them in which the pds transcript level was under the 20% of its level in the control wild type. Interestingly the carotenoids profile in these transformants was practically the same of the wild-type untransformed cells. The existence of previous bottlenecks or the presence of more than one functional pds gene in Chlamydomonas genome, as has been previously proposed, could explain this fact. The effect of inverted repeated psy fragments on the expression of this enzyme is presently being studied.
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