جداسازي ترادف نشانه زيرواحد كوچك آنزيم روبيسكواز گياه كلزا و ارزيابي عملكرد آن در گياه تراريخت توتون‌

Isolation of transit peptide from small subunit of rubisco enzyme from rapeseed and its analysis in Ttransgenic tobacco plants

Rubisco is the key enzyme in photosynthesis reactions in chloroplast stroma. The small subunit of this enzyme which is encoded by the nuclear genome is targeted into the chloroplast by the activity of its signal peptide. In order to utilize the efficiency of this transit peptides in specific targeting of foreign proteins to chloroplast, the genomic DNA from rapeseed (Brassica napus L.) leaves was isolated. By using the specific primers and PCR methods, the signal sequance (170bp) was isolated and cloned into pUC18 as a cloning and pBI121 as a plant expression vectors. The constract was transfered into tobaccosterile explants via Agrobacterium tumefaciens (strain LBA4404) mediated transformation procedure.The selection of transformed cells were done on kanamycine containing media. PCR with specific primers on the DNA isolated from transgenic plant and GUS staining on leaves of the same samples confirm the presence of the recombinant construct and its activity.Further more, the GUS staining on isolated chloroplast from putative transgenic and control plants show that the product of gus gene targeted to chloroplast succesfully. These results show the general ability of rubisco small subunit transit peptide from Brassica napus in targeting of the heterologus protein (GUS reporter gene) to chloroplast of a heterologus host (tobacco).