Designing of polyoleosin-proinsulin fusion gene and its transformation to rapeseed (Brassica napus L.)

Objective The use of plant Oleosin gene is a recommended method for large-scale, easier and cheaper production of recombinant protein. Attachment of oleosin gene to target gene cause to accumulate recombinant protein on seed oil bodies surface. Recent researches indicate that application of tandem Oleosin Genes (polyoleosin) fused to the target gene to facilitate recombinant protein purification is more efficient than single Oleosin. Materials and methods In present research, we designed a synthetic fusion fragment containing 5´- Kozak sequence - His-tags, three Oleosin Genes, a proteolytic site for a peptidase enzyme, Proinsulin protein, and a tetranucleotide stop codon. This synthetic sequence was cloned in binary vector pBI121by enzymatic digestion and ligation method. Then for transformation of cotyledon and hypocotyl explants of Canola, confirmed recombinant construct is transformed to Agrobacterium LBA4404 strain. Probable transgenic shoots were regenerated on selective medium containing kanamycin after 4-6 weeks. Results The analysis of transgenic shoots at DNA level was performed by PCR and amplification of a 120-bp fragment indicate integration of fusion gene in transgenic plant genome. Finally, the Proinsulin gene transcription was also confirmed by RT- PCR.