Regulatory sequences from a cyanobacteria-infecting phage are functional for transgene expression in tobacco chloroplasts

MIRKIN, FEDERICO GABRIEL; BOCCARDO, NOELIA; BRAVO-ALMONACID, FERNANDO FELIX; SEGRETIN, MARÍA EUGENIA

Foz de Iguazú

Congreso; 11th International Plant Molecular Biology Congress IPMB2015; 2015

The expression of heterologous proteins in chloroplasts is an attractive biotechnological tool because of the large number of genome copies, lack of position and silencing effects, and increased protein accumulation levels (up to 70% of total soluble proteins). Also, maternal inheritance of plastids in many plant species introduces a barrier for horizontal transgene transfer via pollen. To optimize protein expression levels in chloroplasts, we analyzed the functionality of heterologous sequences obtained from the cyanophage (virus infecting cyanobacteria) S-PM2. Chloroplasts share many aspects of transcriptional and translational mechanisms with cyanobacteria due to their endosymbiotic origin. Moreover, some cyanophage genes are homologous to cyanobacteria genes, possibly as a consequence of horizontal gene transfer. Among these genes, cyanophage S-PM2 psbA (psbAS-PM2) encodes a D1 protein that functionally replaces cyanobacteria D1 proteins in photosystem II assembly during infection. We hypothesize that cyanophage regulatory sequences that are functional in cyanobacteria are also functional in chloroplasts. We selected the promoter and 5´UTR sequences of two genes from S-PM2 cyanophage: psbAS-PM2 and the gene encoding the putative s-layer protein. In cyanobacteria, both genes are expressed at high levels throughout the entire infection cycle. The functionality of these sequences was analyzed by inserting them upstream of the uidA reporter gene into a chloroplast transformation vector previously developed in our laboratory. We obtained transplastomic tobacco plants, whose molecular characterization is underway. Preliminar histochemical assays revealed β-glucuronidase activity in these transplastomic plants, suggesting that the viral sequences of cyanophage S-PM2 used in this study are functional in the genetic context of the plastid.