Shahid Bahonar University of Kerman and Iranian Biotechnology Society Agricultural Biotechnology Journal 2228-6705 10 3 2018 12 22 Designing of polyoleosin-proinsulin fusion gene and its transformation to rapeseed (Brassica napus L.) Designing of polyoleosin-proinsulin fusion gene and its transformation to rapeseed (Brassica napus L.) 1 16 2204 10.22103/jab.2018.2204 FA Najmeh Jami M.Sc. Student, Department of Plant Production and Genetics, Agricultural Sciences and Natural Resources University of Khouzestan, Khouzestan, Iran. Esmael Ghasemi Assistant Professors, Department of Plant Production and Genetics, Agricultural Sciences and Natural Resources University of Khouzestan, Khouzestan, Iran. Alireza Shafei Assistant Professors, Department of Plant Production and Genetics, Agricultural Sciences and Natural Resources University of Khouzestan, Khouzestan, Iran. Payam Pourmohammadi Assistant Professors, Department of Plant Production and Genetics, Agricultural Sciences and Natural Resources University of Khouzestan, Khouzestan, Iran. 0000-0003-2292-6069 Journal Article 2018 06 19 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. 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. https://jab.uk.ac.ir/article_2204_4b4edc2630fe75800ddc29a7b4070add.pdf