Shahid Bahonar University of Kerman and Iranian Biotechnology Society Agricultural Biotechnology Journal 2228-6705 10 1 2018 06 22 Expression of recombinant human bone morphogenetic protein 2 (rhBMP2) in tobacco transgenic hairy roots Expression of recombinant human bone morphogenetic protein 2 (rhBMP2) in tobacco transgenic hairy roots 21 34 2065 10.22103/jab.2018.2065 FA Balal Dehghani M.Sc. Agriculture Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran Amir Mossavi Associated Professor, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran. Sadegh Hassannia Associated Professor, Tarbiat Modares University, Tehran, Iran Ali Hatef Professor, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran. Ali Sharafi Assistant Professor, School of Pharmacy, Zanjan University of Medical Sciences, Tehran, Iran. 0000-0002-6012-1424 Mohammadreza Eftekharian M.Sc. Plants Biology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran. Journal Article 2017 07 23 Today, demand for production of recombinant proteins in different tissues of transgenic plants is increasing day by day. Meanwhile, hairy roots are under consideration due to their high levels of biomass and high genetic stability as appropriate target tissue. Among pharmaceutical proteins, regulatory glycoproteins, are proposed as one of the most important growth factors with therapeutic and research applications in tissue engineering. Selection of proper host species, plant tissue and desirable gene, are of the most important factors to elevate the levels of recombinant protein production. In order to achieve this goal, in the present study, production of recombinant protein BMP2 in transgenic hairy roots of tobacco and canola were investigated. For optimal transgene expression, TMV (5’-UTR) and KOZAK sequences were inserted upstream the BMP2 gene to increase expression level, furthermore, six-histidine sequence for protein purification and KDEL sequence as ER retention signal protein were inserted downstream of the target gene. The transgene was designed to be under the control of CaMV 35S promoter. One-month old tobacco leaf explants and six-day-old cotyledons of canola were transformed with <em>Agrobacterium rhizogenes </em>carrying the construct. Transformed hairy roots that grew well in the medium were confirmed by gPCR analysis for transgene integration. Moreover, to verify mRNA transcription as well as protein production, RT-PCR, ELISA and western blot analyses were performed. The results of this study demonstrated the efficiency of the current systems for the production of rhBMP2 is tobacco hairy roots suggesting them as an efficient bioreactor. Today, demand for production of recombinant proteins in different tissues of transgenic plants is increasing day by day. Meanwhile, hairy roots are under consideration due to their high levels of biomass and high genetic stability as appropriate target tissue. Among pharmaceutical proteins, regulatory glycoproteins, are proposed as one of the most important growth factors with therapeutic and research applications in tissue engineering. Selection of proper host species, plant tissue and desirable gene, are of the most important factors to elevate the levels of recombinant protein production. In order to achieve this goal, in the present study, production of recombinant protein BMP2 in transgenic hairy roots of tobacco and canola were investigated. For optimal transgene expression, TMV (5’-UTR) and KOZAK sequences were inserted upstream the BMP2 gene to increase expression level, furthermore, six-histidine sequence for protein purification and KDEL sequence as ER retention signal protein were inserted downstream of the target gene. The transgene was designed to be under the control of CaMV 35S promoter. One-month old tobacco leaf explants and six-day-old cotyledons of canola were transformed with <em>Agrobacterium rhizogenes </em>carrying the construct. Transformed hairy roots that grew well in the medium were confirmed by gPCR analysis for transgene integration. Moreover, to verify mRNA transcription as well as protein production, RT-PCR, ELISA and western blot analyses were performed. The results of this study demonstrated the efficiency of the current systems for the production of rhBMP2 is tobacco hairy roots suggesting them as an efficient bioreactor. https://jab.uk.ac.ir/article_2065_0ae3f79a30234b6c45a6f7d298ba1310.pdf