| Human Osteogenic Protein 1(hOP-1), also known as Bone Morphogenetic Protein-7 (BMP7), is a member of TGF-βsuperfamily. HOP-1 can induce IOPC (inducible osteogenic precursor cells) cells into osteoblasts and have broad clinical application. However due to limited source of human bones, the production of hOP-1 were restricted, which affected the clinical application. With the development of genetic engineering, heterologous expression of osteogenic protein is possible.Plant bio-reactor has been widely used to produce different kinds of target moleculars, such as recombinant pharmaceutical proteins, lipids, and secondary metabolites. Compared with other bio-reactor systems, plant bio-reactor can safely express the foreign proteins with a low cost. Oleosins are proteins localized on the surface of oil bodies in seeds. Oleosin contains an N-terminal hydrophilic region of variable length and a central conserved hydrophobic domain of about 70 amino acid residues, a C-terminal amphipathic region of variable length. When foreign protein fused with C or N-terminal of oleosin, the localization and expression of the oleosin was not affcted. The co-expressed foreign protein could also accummulate in a high level. In addition, the fused foreign protein can be easily isolated by flotation centrifugation due to the hydrophobic and oil body localized nature of oleosin. It has great advantages using peanut seeds to produce foreign proteins such as human osteogenic protein by oleosin expression system.Peanut (Arachis hypogaea L.) is one of the most important crops both for vegetable oil and protein sources and was wildly grown in China. Peanut oleosins are highly express and accumulated in seeds. It has great advantage to use peanut seeds and oleosin expression system to produce target proteins. In this study, the oil body-oleosin expression system has been established which could successfully express human osteogenic protein 1 in the transgenic tobacco seeds. This study laid a foundation for producing hOP-1 and other exogenous proteins in peanut using oil body-oleosin system. The following results have been achieved:The codon optimization of hOP-1: According to the expression of plant codon preference, we optimized the human osteogenic protein -1 gene cDNA sequence. The optimized sequence was synthesis for efficient expression in plants. The prokaryotic expression of hOP-1: Inserting hOP-1 full-length mature peptide gene cDNA sequence (420 bp) into prokaryotic expression vector PET28a and transformed into Escherichia coli (DE3). The results show that hOP-1 was correctly expressed with the expected molecular weight of about 15.7 KD.The cloning of peanut oleosin gene and promoter: According to the peanut oleosin gene sequences in NCBI, we cloned the full length of peanut oleosin gene and its promoter from Luhua-14 peanut.Construction of plant expression vectors: A vector of peanut oleosin promoter-driven"peanut oleosin - enteropeptidase - hOP-1"was constructed. The pCAMBIA1301 and pCAMBIA1302 vectors were modified by replacing the hygromycin (Hyg) resistance gene with Kanamycin resistance gene.Transformation and express of hOP-1 in plants: The modified plant expression vectors were transferred into Agrobacterium tumefaciens C58C1. Tobacco leaf disc was transformed by Agrobacterium tumefaciens. PCR amplification was used to detect the presence of the transgene in the regenerated plant and the expression of hOP-1 in transgenic tobacco was detected by RT-PCR. hOP-1 mRNA was not detected in leaves and flowers of the transgenic plants. However, hOP-1 was highly expressed in transgenic tobacco seeds. The accumulation of hOP-1 at the proten level will be analyzed either by GUS staining or western blot.
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