Over the past few decades, many plant systems have been explored as expression hosts for the cost-effective and scalable production of recombinant pharmaceutical proteins and industrial macromolecules [1, 2]. Seed-based systems are particularly attractive because of their relatively high protein/mass ratios favorable for downstream processing, stable biochemical environment optimized for long-term storage, and abundant seed-specific organelles such as protein storage vacuoles (PSVs), or endoplasmic reticulum (ER)-derived protein bodies (PBs) specialized for protein accumulation [3, 4]. Compared with the existing expression systems, soybean seeds provide a practical and promising production platform, as they naturally accumulate large amounts of protein (approximately 40% of dry mass) and represent one of the most protein-rich plant seeds. Such a high protein/mass ratio even makes it possible to produce over 1 mg of recombinant proteins in a single soybean seed, which is a much higher yield than could be reached in the other plant or non-plant host systems . Furthermore, high stability at ambient temperature, a simple proteome, and relative homogeneity of soybean seeds can greatly facilitate the downstream processing and purification of recombinant proteins [5–7]. Additional advantages such as low production costs, easy scalability, well-established processing procedures, and the possibility of oral delivery of the expressed proteins and vaccines make soybean seeds a very attractive system for the industrial-scale production of recombinant proteins [3, 4].
To date, a number of recombinant pharmaceutical proteins have been successfully expressed in the seeds of transgenic soybeans [6, 8–12]. However, the accumulation levels of some recombinant proteins in soybean seeds are usually less than 1% total soluble protein (TSP) of economic threshold [13, 14]. Significant efforts have been made to improve the yield of target proteins in soybean seeds up to the level acceptable for practical application, including the use of constitutive or seed-specific promoters, codon optimization, and subcellular targeting of the synthesized proteins by signal peptides, ER retention signals (H/KDEL), or polypeptide partners [8, 9, 11, 15]. The selection of an appropriate promoter is essential to achieve the optimal transcription level of heterologous proteins, and strong soybean seed-specific promoters such as β-conglycinin and glycinin are usually used to increase the yield of foreign proteins expressed in soybean seeds [8, 9, 11].
The targeting of the expressed proteins to the subcellular compartments such as the ER or PSVs can not only influences their high-level accumulation in cells, but also protein stability and post-translational modifications [3, 4]. Increased accumulation of recombinant proteins in soybean and other crop seeds has been achieved through manipulations of the secretory pathway and protein targeting to different subcellular locations [8, 9, 11, 15–17]. Among the strategies used to promote protein accumulation, fusion with small polypeptides such as 27 kDa maize γ-zein, 13.56 kDa human elastin-like polypeptide (ELP), and 12 kDa fungal hydrophobin I have shown great potential [15, 18–22]. Our previous studies in soybean seeds indicated that fusion with γ-zein or ELP could significantly increase the accumulation of recombinant proteins in ER-derived vesicles , which not only provided protection from proteolysis but also facilitated target protein purification [11, 15].
Another factor to consider in using seed-based expression systems is endogenous storage proteins such as prolamins or glutelins, which hamper the accumulation of foreign proteins by competing for the biosynthesis and transport pathways and deposition space . Yang et al.  have reported that a decrease in the expression of rice seed prolamins or glutelins increases the accumulation of recombinant human IL-10 by approximately 3-fold. Complete inhibition or reduction of endogenous storage protein expression could even lead to changes in the localization of foreign proteins in rice seeds  or the formation of novel ER-derived PBs in soybean seeds [15, 29, 30].
The main storage proteins accumulated in specialized PSVs of soybean seeds are 11S- and 7S-globulins (glycinin and conglycinin, respectively), which account for approximately 70% of soybean storage proteins . It is reasonable to redirect the intrinsically high transcriptional and translational activity in soybean seeds towards heterologous proteins by suppressing the synthesis of these storage proteins. In the present study, a recombination strategy of the polypeptide (γ-zein or ELP) fusions and specific suppression of endogenous storage proteins (glycinin or conglycinin) by RNA interference (RNAi) was exploited to explore its potential in increasing the accumulation of foreign proteins in soybean seeds. Our results indicated that the suppression of glycinin or conglycinin synthesis led to a significant increase in the expression of zein- or ELP-fused green fluorescent protein (GFP) in soybean seeds without affecting their total protein content and protein/oil ratio.