Abstract：Soybean (Glycine max) seeds provide an ideal host for the production of foreign recombinant proteins because of their robust capability for biosynthesis and accumulation of the native proteins. To further enhance the expression of foreign proteins in soybean seeds, a polypeptide fusion strategy was utilized to investigate the influence of the maize (Zea mays) γ-Zein fusion tag on accumulation of the foreign recombinant protein. The GFP (green fluorescent protein) and Zein-GFP driven by the soybean seed-specific promoter BCSP were individually introduced into the cultivated soybean genotype by Agrobacterium tumefaciens-mediated transformation, respectively. Totally 41 Zein-GFP and 46 GFP transgenic plants were obtained in this study. Reverse transcription PCR (RT-PCR) and Western blot analysis confirmed transcript and translation of the foreign genes in the transgenic soybean seeds. The transgenic lines with similar expression of Zein-GFP and GFP at mRNA levels in seeds were selected and accumulation of the recombinant proteins was quantified by enzyme linked immunosorbent assay (ELISA). Average accumulation level of the fusion protein Zein-GFP reached to 1.51% TSP (total soluble protein) ranging from 1.37% to 1.60% TSP in the transgenic seeds, increased by 10.78 fold compared with 0.14% TSP of the unfused GFP (0.13%~0.15% TSP). Subcellular targeting analysis by laser scanning confocal microscopy showed that the γ-Zein fused GFP were present in the protein bodies (PBs), while the unfused GFP was mainly distributed in the cytoplasm. Taken together, the results showed that the γ-Zein polypeptide fusion induced the formation of PBs and significantly enhanced accumulation of the foreign recombinant protein in the transgenic soybean seeds, and thus provide the basis for application of the soybean-based bio-reactor.
