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Article

Increased Accumulation of Recombinant Proteins in Soybean Seeds via the Combination Strategy of Polypeptide Fusion and Suppression of Endogenous Storage Proteins

by
Jing Yang
†,
Yuanyu Zhang
†,
Guojie Xing
,
Jia Wei
,
Lu Niu
,
Qianqian Zhao
,
Qinan Cai
,
Xiaofang Zhong
* and
Xiangdong Yang
*
Jilin Provincial Key Laboratory of Agricultural Biotechnology, Jilin Academy of Agricultural Sciences, Changchun 130033, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agronomy 2023, 13(11), 2680; https://doi.org/10.3390/agronomy13112680
Submission received: 30 August 2023 / Revised: 20 October 2023 / Accepted: 24 October 2023 / Published: 25 October 2023
(This article belongs to the Special Issue Functional Genomics and Molecular Breeding of Soybeans)
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Abstract

:
Soybean seeds show great potential as a safe and cost-effective host for the large-scale production of biopharmaceuticals and industrially important macromolecules. However, the yields of desired recombinant proteins in soybean seeds are usually lower than the economic threshold for their potential commercialization. Our previous study demonstrated that polypeptide fusion such as maize γ-zein or elastin-like polypeptide (ELP) could significantly increase the accumulation of foreign proteins. In the present study, a recombination strategy of polypeptide fusions (γ-zein or ELP) and suppression of intrinsic storage proteins (glycinin or conglycinin) via RNA interference was further exploited to improve the yield of the target protein in soybean seeds. Transgenic soybean plants harboring both polypeptide-fused green fluorescent protein (GFP) and glycinin/conglycinin RNAi expression cassettes were generated and confirmed by molecular analysis. The results showed that on both the glycinin and conglycinin suppression backgrounds, the average accumulation levels of recombinant zein-GFP and GFP-ELP proteins were significantly increased as compared to that of their counterparts without such suppressions in our previous study. Moreover, zein-GFP and GFP-ELP accumulation was also remarkably higher than unfused GFP on the glycinin suppression background. However, no significant differences were detected in the glycinin or conglycinin suppression backgrounds for the same polypeptide fusion constructs, though suppression of one of the storage proteins in soybean seeds led to a significant increase in the other. Additionally, the increases in the recombinant protein yield did not affect the total protein content and the protein/oil ratio in soybean seeds. Taken together, the results indicate that both the fusion of the foreign protein with polypeptide tags together with the depletion of endogenous storage proteins contributed to a higher accumulation of the recombinant proteins without affecting the total protein content or the protein/oil ratio in soybean seeds.

1. Introduction

In recent decades, many plant systems have been explored as expression hosts for the cost-effective and scalable production of recombinant pharmaceutical proteins and industrial macromolecules [1,2,3]. Seed-based systems are particularly attractive because of their relatively high protein/mass ratios favorable for downstream processing, a 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 [4,5,6,7,8]. 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 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 [5]. Furthermore, high stability at ambient temperature, a simple proteome, and the relative homogeneity of soybean seeds can greatly facilitate the downstream processing and purification of recombinant proteins [9,10,11]. 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 [4,5]. To date, a number of recombinant pharmaceutical proteins have been successfully expressed in the seeds of transgenic soybeans [6,10,12,13,14,15,16]. However, the accumulation levels of some recombinant proteins in soybean seeds are usually less than 1% total soluble protein (TSP) of economic threshold [17,18], which affects its potential of application as a viable production platform.
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 [12,13,15,19]. 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 [12,13,15]. 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 [4,5]. 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 [12,13,15,19,20,21]. 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 (HFBI) has shown great potential [19,22,23,24,25,26]. 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 [19], which not only provided protection from proteolysis but also facilitated target protein purification [15,19].
Another factor to consider in using seed-based expression systems is endogenous storage proteins such as prolamin and/or glutelin, which hamper the accumulation of foreign proteins by competing for the biosynthesis and transport pathways and deposition space [27]. Yang et al. [28] have reported that a decrease in the expression of rice seed prolamins or glutelin enhanced the accumulation of recombinant human IL-10 by approximately 3 fold. Complete inhibition or a reduction in endogenous storage protein expression could even lead to changes in the localization of foreign proteins in rice seeds [29] or the formation of novel ER-derived PBs in soybean seeds [19,30,31]. These researches indicate that the trade-off between the foreign and native storage proteins can notably influence the accumulation of desirable proteins in plant seeds.
The main storage proteins accumulated in specialized PSVs of soybean seeds include 11S- and 7S-globulins (glycinin and conglycinin, respectively), which account for approximately 70% of soybean storage proteins [30]. 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.

2. Materials and Methods

2.1. Vector Constructs for Soybean Genetic Transformation

Expression vectors were constructed based on the binary vectors pTF101-zein-GFP and pTF101-GFP-ELP carrying the GFP-encoding gene fused with zein- or ELP-coding sequences under the control of soybean seed-specific promoter of β-conglycinin alpha subunit (BCSP) as described previously [19]. Another soybean seed-specific promoter Pgy1 of the glycinin subunit Gy1 was amplified by polymerase chain reaction (PCR) from the pCB2004B-Gy1 plasmid and inserted into the pHANNIBALG RNAi vector at SacI/XhoI sites to replace the native CaMV 35S promoter. A 723 bp tandem RNAi fragment Gy was synthesized based on the conserved sequences of five genes encoding soybean glycinin subunits A1aB2 (Gy1, X15121), A2B1a (Gy2, NM_001248881), A1bB1b (Gy3, X15123), A5A4B3 (Gy4, AB195712), and A3B4 (Gy5, FJ599666) (see Supplementary Figure S1), and sequentially inserted at the XhoI/KpnI and HindIII/XbaI sites of the pHANNIBALG-Pgy1 vector in the sense and antisense directions, respectively. The whole Gy RNAi cassette, including the Pgy1 promoter, two inverted Gy fragments separated by the pdk intron, and the octopine synthase terminator ocs, was then ligated into the corresponding binary vectors to generate constructs pGyless/zein-GFP and pGyless/GFP-ELP, respectively (Figure 1). For construction of the control vector pGyless/GFP, the Gy RNAi cassette was ligated into the pTF101-GFP vector, which contained the unfused gfp under the BCSP promoter as described previously [19]. All three vectors contained the phosphinothricin acetyltransferase marker gene (bar) for the selection of transgenic plants based on glufosinate resistance. Similarly, a 721 bp tandem RNAi fragment Cgy was synthesized based on the conserved sequences of three genes encoding soybean conglycinin subunits Cgy1 (GU723691), Cgy2 (AB237643), and Cgy3 (NM_001249943) (see Supplementary Figure S2), and sequentially inserted at the XhoI/KpnI and HindIII/XbaI sites of the pHANNIBALG-Pgy1 vector as described above. The whole Cgy RNAi cassette was then subcloned into the corresponding binary vectors to obtain pCgyless/zein-GFP and pCgyless/GFP-ELP constructs, respectively (Figure 1).

2.2. Generation of Transgenic Soybean Plants

In this study, the acceptor material was soybean Cultivar P03, belonging to Jilin Academy of Agricultural Sciences, and agreed to carry out scientific experiments. All experiments were completed at the Jilin Provincial Academy of Agricultural Sciences, Jilin Provincial Key Laboratory of Agricultural Biotechnology. For genetic transformation, mature seeds were sterilized with chlorine gas in a sealed chamber for 12–16 h and imbibed with three layers of filter paper soaked in sterile water for 12 h. Then, the seed coats and primary shoots were removed for explant preparation. Inoculation with Agrobacterium tumefaciens EHA101, decontamination of Agrobacterium with ticarcillin and cefotaxime, induction, elongation and rooting of resistant shoots were performed as described previously [32]. The regenerated plantlets resistant to glufosinate (6 mg/L) were transferred into a greenhouse with controlled temperature (25 °C) and light/dark cycle (16-h/8-h) for growth and seed setting. The primary transformants were detected using the LibertyLink® strip (EnviroLogix Inc., Portland, ME, USA) and PCR with gene-specific primers (Table S1). The progenies of transgenic plants in the subsequent generations (T1–T3) were further screened by glufosinate (1000 mg/L) spraying and PCR detection until the homozygous transgenic lines were obtained.

2.3. Transcription Analysis of Transgenic Soybean Seeds

To determine the expression of foreign genes in the T3 transgenic seeds at the transcription level, quantitative reverse transcription PCR (qRT-PCR) was carried out with GFP-specific primers (Table S1). Briefly, total RNA was extracted from immature seeds (~10 mm in length) at 45 days after flowering (DAF) using the EasyPure Plant RNA Kit (TransGen Biotech, Beijing, China). First-strand cDNA was synthesized using TransScript® One-Step gDNA Removal and cDNA Synthesis SuperMix (TansGen Biotech). qRT-PCR was performed using TransStart® Top Green qPCR SuperMix (TansGen Biotech) according to the manufacturer’s protocol under the following reaction conditions: uracil-N-glycosylase treatment at 50 °C for 2 min, denaturation at 95 °C for 10 min, and 35 cycles of denaturation at 95 °C for 30 s, annealing at 60 °C for 30 s, and extension at 72 °C for 30 s. We also analyzed the transcription of soybean glycinin (Gy1, Gy2, Gy3, Gy4, and Gy5) and conglycinin (Cgy1, Cgy2, and Cgy3) genes using specific primers (Table S1). The native soybean gene Gmactin6 (GenBank No. NM_001289231) was amplified as the internal control. Relative gene expression was calculated using the 2−ΔΔCt method as previously described [19]. All qRT-PCR experiments were conducted with three biological replicates for each sample.

2.4. Protein Expression and Quantification Analysis

The expression of recombinant GFP proteins was analyzed by Western blotting and enzyme-linked immunosorbent assay (ELISA). The accumulation of the fusion proteins in the T3 transgenic soybean seeds with the inhibited glycinin synthesis was analyzed using ELISA and transgenic soybean plants without such inhibition (DF8, DG44, and DE9) were used as controls as described in our previous study [19]. Immature seeds at 45 DAF were collected from the transgenic plants, grounded to fine powder in liquid nitrogen with a mortar and pestle. Each sample (100 mg) was used for total soluble protein extraction performed as previously described [19]. Total protein was quantified using the BCA Protein Quantification Kit (Yeasen Biotechnology, Wuhan, China) according to the manufacturer’s protocol. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in 12% gels, transferred to PVDF membranes (GE Healthcare, Chicago, IL, USA), and subjected to Western blotting using primary anti-GFP antibodies (1:500 dilution; Abcam, Cambridge, UK) and secondary horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:5000 dilution; Abcam) as previously described [19].
For quantification of recombinant protein expression, the total protein extracts of immature (45 DAF) and dry mature trangenic seeds were subjected to ELISA. Blocking, incubation with primary anti-GFP antibodies (1:500 dilution) and secondary HRP-conjugated secondary antibodies (1:500 dilution), washing, and reaction with the substrate 3,3,5,5-tetramethyl benzidine (TMB) were performed as previously described [19]. The reaction was terminated with 2 N H2SO4, and the optical density (OD) was determined at 450 nm using a plate reader (EL×800; BioTek, Winooski, VT, USA).

2.5. Analysis of Total Protein and Oil Content in Soybean Seeds

The protein content of soybean seeds was measured as described previously [33]. Briefly, mature soybean seeds were ground in a mortar, and 200 mg of the powder was used for analysis. Total nitrogen was determined in a LECO CHN 2000 analyzer (LECO, St. Joseph, MI, USA) as described by Hwang et al. [34]. Protein content was calculated as the amount of nitrogen multiplied by 6.25 based on the dry weight basis. Seed oil was extracted with methanol: chloroform (2:1 v/v), and total fatty acids were quantified by gas chromatography-mass spectrometry (GC-MS) after methyl esterification using 5% sulfuric acid in methanol. The oil content was calculated as the percentage of dry weight [33]. All experiments were conducted with three biological replacates.

2.6. Statistical Analysis

All statistical analyses were performed using SPSS software v.17.0 (SPSS Inc., Chicago, IL, USA). Differences in the average values of samples were tested by multiple comparisons of one-way ANOVA using the LSD test. The data were presented as the mean ± standard deviation (SD) of three biological replicates.

3. Results

3.1. Generation and Molecular Analysis of Transgenic Soybeans

To suppress the expression of endogenous glycinin or conglycinin in soybean seeds, two tandem RNAi fragments based on conservative sequences of five glycinin subunit or three conglycinin subunit-encoding genes were synthesized and used for the construction of the expression vectors (see Supplementary Figures S1 and S2; Figure 1). To optimize the transcription of the foreign genes, two soybean seed-specific promoters, BCSP and Pgy1, were used to drive the expression of the gfp and Gy/Cgy RNAi fragments, respectively. A series of transformation experiments produced 84 transgenic soybean plants, including 12 Gyless/zein-GFP, 19 Gyless/GFP-ELP, 21 Gyless/GFP plants, 13 Cgyless/zein-GFP, and 19 Cgyless/GFP-GFP, as shown by LibertyLink® strip analysis (see Supplementary Figure S3). The presence of foreign gfp and RNAi fragments was further confirmed by PCR detection, with expected sizes of amplified fragments observed in these transgenic plants, but absent in the non-transformed (Nt) controls (see Supplementary Figure S4). Transgenic soybean lines from each construct were further advanced to the subsequent generations until homozygous lines were obtained using glufosinate spraying and PCR detection.
The relative mRNA expression of gfp in T3 transgenic soybean seeds was determined by qRT-PCR. Similar expression levels of the foreign genes were also observed at the same time point of seed development in some transgenic plants (Figure 2a). To avoid the influence of differential mRNA expression on the recombinant protein accumulation in seeds, only transgenic lines with similar expression levels were selected for further analysis, including three Gyless/zein-GFP (G45, G54, and G59), three Gyless/GFP-ELP (A44, A58, and A59), three Gyless/GFP (B14, B43, and B66), two Cgyless/zein-GFP (F24 and F32) plants, and three Cgyless/GFP-ELP (J41, J47, and J53).
Western blotting was further conducted to detect the expression of the recombinant proteins in the T3 transgenic seeds. The results showed that the bands representing zein-GFP, GFP-ELP, and GFP with the expected sizes of 26.8, 51.8, and 40.5 kDa, were observed in the corresponding transgenic seeds, confirming the accurate translation of these recombinant proteins (Figure 2b–f). However, the cleavage of the fusion proteins was also detected, as evidenced by the 26.8 kDa band corresponding to a GFP monomer detected in both zein-GFP and GFP-ELP seed samples (Figure 2c–f), which was consistent with the previous reports [19].

3.2. Suppression of Glycinin and Conglycinin Expression in the Transgenic Soybean Seeds

The expressions of glycinin and conglycinin family proteins were analyzed in immature seeds of the selected T3 transgenic plants at 45 DAF using primers specific for five glycinin (Gy1, Gy2, Gy3, Gy4, and Gy5) and three conglycinin (Cgy1, Cgy2, and Cgy3) subunit-encoding genes. The results showed that in the Gyless transgenic plants (B66, G45, and A58), the relative mRNA expressions of glycinin subunits (Gy1–Gy5) were markedly downregulated with 3.16–78.46-fold decreases compared with the Nt plants (Figure 3a). Similar to glycinin, transcriptional suppression was also observed for three conglycinin subunits (Cgy1–Cgy3) in the Cgyless transgenic plants (F24 and J41) with 3.75–8.42-fold decreases compared with the Nt control (Figure 3b).
Next, we analyzed the protein expression in immature seeds of transgenic plants at 45 DAF. The results showed that in Gyless/zein-GFP and Gyless/GFP-ELP plants carrying the Gy RNAi cassette, the glycinin proteins had a significantly decreased accumulation compared with Nt control or DG44 and DE9 plants, which harbored the same zein-gfp and gfp-elp cassettes but not the Gy RNAi cassette in our previous study [19] (Figure 3c). Meanwhile, conglycinin proteins were visibly increased as compared with the Nt plants in these soybean seeds with the suppressed glycinin expression. A similar effect was observed in Cgyless/zein-GFP and Cgyless/GFP-ELP plants with the downregulated conglycinin expression. In this case, the accumulation of conglycinin proteins was markedly decreased compared with Nt control or DG44 and DE9 plants, whereas that of glycinin proteins was markedly increased (Figure 3c). Taken together, these results indicated that the RNAi-mediated suppression of one of the storage proteins (glycinin or conglycinin) significantly inhibited its transcription and translation, and concomitantly led to a remarkable increase in the other endogenous storage proteins in soybean seeds.

3.3. Influence of RNAi-Mediated Suppression of Glycinin or Conglycinin Synthesis on the Accumulation of Recombinant Polypeptides

The increased accumulation of conglycinin to compensate for RNAi-mediated glycinin suppression suggested that a concomitant increase may be expected for the expression of foreign proteins. As expected, the results revealed a marked increase in the accumulation of the recombinant fusion proteins in the Gyless immature soybean seeds (Table 1). The average accumulation levels of zein-GFP and GFP-ELP were 2.69% and 2.30% TSP, which indicated 2.2- and 7.7-fold increases compared to those in control DG44 and DE9 plants without the suppression of both glycinin and conglycinin, respectively (Table 1). The accumulation of unfused GFP (1.02% TSP) on the Gyless background also showed a remarkable increase compared to its counterpart DF8 without such suppression (0.08% TSP) (Table 1). Similar observations were found in mature soybean seeds, which the accumulation of zein-GFP, GFP-ELP, and unfused GFP on the Gyless background reached the average levels of 3.49%, 4.31%, and 1.53% TSP, representing 1.68-, 4.89-, and 2.68-fold higher than those in their counterparts DG44, DE9, and DF8, respectively (Table 1). When compared with unfused GFP on the Gyless background, the average accumulation levels of zein-GFP and GFP-ELP were also increased by 2.28 and 3.29 fold, respectively (Table 1). These results indicated that both the suppression of endogenous glycinin and the fusion with zein or ELP increases the accumulation of the recombinant protein in soybean seeds, and the depletion of glycinin in soybean seeds could be compensated for by the increased abundance of both conglycinin and foreign proteins.
However, there appeared to be no significant difference in the accumulation of fused zein-GFP and GFP-ELP for the different backgrounds of glycinin or conglycinin suppression. In Cgyless transgenic plants with suppressed conglycinin expression, the average accumulation levels of zein-GFP and GFP-ELP in immature seeds (2.80% and 3.04%TSP, respectively) showed no significant difference with those in their Gyless counterparts (Table 1). Similar trends were observed in mature soybean seeds, in which the accumulation levels of zein-GFP and GFP-ELP were 4.49% and 5.03% TSP. Taken together, these results indicated that the suppression of both endogenous glycinin or conglycinin could lead to a marked increase in the accumulation of recombinant proteins in soybean seeds.

3.4. Increased Accumulation of Recombinant Proteins Did Not Affect Total Protein and Oil Contents in Soybean Seeds

Total protein and oil accumulation in transgenic soybean seeds showed that despite protein profile changes by introduction of foreign protein and exchanges of glycinin/conglycinin, no significant changes were observed in the total protein and protein/oil ratio in seeds between the transgenic and non-transformed plants (Table 2). The results were consistent with the previous reports that limited plasticity of total protein content resulted from the loss of one major storage proteins compensated by the increase in accumulation of foreign and other heterologous proteins, possibly through the rebalancing of protein synthesis in the remodeled seeds to maintain stable levels of protein accumulation [19,30,35].

4. Discussion

The applicability of plant systems to large-scale production of the pharmaceutical and industrial products of significance critically depends on the high protein yield and the ease of downstream recovery and purification [36,37]. Several strategies have been exploited to enhance the production of recombinant proteins in plant seeds, among which intracellular targeting and polypeptide fusion play an important role in determining the protein yield [12,13,15,16,19,20,21]. Our previous studies demonstrated that polypeptide fusion with ELP or γ-zein significantly increases recombinant protein accumulation in soybean seeds [19], which might result from increased transportation and deposition in cell compartments such as PBs or PSVs, and protection from proteolytic digestion. Furthermore, ELP and γ-zein can be used as tags to facilitate the recovery and purification of heterologous proteins from plant materials. In this study, we further explored the capability of the combination strategy of specific suppression of endogenous storage proteins (glycinin or conglycinin) and polypeptide fusion in increasing the yield of recombinant protein production in soybean seeds. Our results indicated that the accumulation of the foreign polypeptide (zein or ELP) fused GFP proteins (zein-GFP and GFP-ELP) was significantly increased in soybean seeds with suppressed glycinin or conglycinin biosynthesis compared to their counterparts without such suppression. The results demonstrate that both depletion of endogenous storage proteins (glycinin or conglycinin) and polypeptide fusion contribute to the increase in the yield of foreign protein accumulation, suggesting a potential of the developed combination strategy for cost-effective large-scale production of industrially important proteins in soybean seeds.
An interesting question is the influence of endogenous proteins on the accumulation of recombinant proteins in plant seeds, especially in soybean seeds which naturally accumulate higher amounts of proteins than most of other crops. It has been reported that the mutation or suppression of endogenous storage proteins in seeds increases the expression of other seed proteins at the transcriptional and translational levels [38,39,40,41,42,43]. Kawakatsu et al. [40] have shown that in transgenic rice seeds, the downregulation of one or several storage proteins such as glutelins, prolamins, and globulins significantly affects the expression of other seed proteins and PB formation, which could be due to a compensatory mechanism to maintain seed protein homoeostasis. In this study, we also observed that the suppression of one major storage protein (glycinin) resulted in the upregulation of another endogenous protein (conglycinin). The observed compensatory effect and/or the extra deposition space created by the loss of an endogenous protein may promote concomitantly the marked increase in the other native storage proteins and foreign proteins in soybean seeds [35]. The intrinsic plasticity of seed protein composition also provides a possibility for further enhancement of the foreign protein yield by the simultaneous inhibition of the major endogenous storage proteins (i.e., glycinin and conglycinin) in seeds.
However, a question arises whether the increased accumulation of recombinant proteins could affect the total protein and oil content in soybean seeds. It has been shown that the suppression of the α and α′ subunits of β-conglycinin does not influence the total oil and protein content and ratio of soybean seeds, and the decrease in β-conglycinin protein can be compensated by increased accumulation of glycinin [19,30]. Consistent with previous reports, our results indicated that no significant differences were observed in the total protein content and the protein/oil ratio among transgenic and non-transformed plants, suggesting that the loss of one endogenous protein was compensated by increased synthesis of foreign and other endogenous storage proteins, so that the total protein content in soybean seeds was stably maintained by proteome rebalancing, despite the changes in seed protein composition.

5. Conclusions

In the present study, a combination strategy was explored for its capability of increasing the accumulation of foreign proteins in soybean seeds, based on polypeptide fusion and native storage protein suppression. On both glycinin and conglycinin suppression backgrounds, the accumulation of recombinant proteins zein-GFP, GFP-ELP and unfused GFP was significantly increased, compared with those of their counterparts without such suppression. This suggested that inhibition of the native storage protein can be partly compensated by the increased expression of foreign proteins. On the other hand, higher accumulation levels of both zein-GFP and GFP-ELP were observed than that of unfused GFP on the glycinin suppression background, indicating that polypeptide fusion could also contribute to recombinant protein accumulation in soybean seeds. However, no significant differences were observed in the accumulation levels of the foreign proteins under different suppression backgrounds. Moreover, both introduction of the foreign proteins and inhibition of the intrinsic storage proteins showed no remarkable influences on the total protein and protein/oil ratios in soybean seeds. Conclusively, this protein-enhancing combination strategy can result in a more accumulation of foreign proteins, which promises a great potential for soybean seeds as a viable platform for the production of high-value pharmaceutical proteins and industrial macromolecules in future.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy13112680/s1, Figure S1: Tandem RNAi fragment (723 bp) based on the conservative sequences of five soybean glycinin subunit-encoding genes (Gy1, Gy2, Gly3, Gy4, and Gy5); Figure S2: Tandem RNAi fragment (721 bp) based on the conservative sequences of thee soybean conglycinin subunit-encoding genes (Cgy1, Cgy12, and Cgy13); Figure S3: LibertyLink® strip analysis of T0 transgenic soybean plants; Figure S4: Verification of T1 transgenic soybean plants by PCR to confirm the presence of foreign genes (a) and Gy or Cgy RNAi fragments (b); Table S1: Primers used for PCR and RT-qPCR analysis in this study.

Author Contributions

Conceptualization, X.Y. and X.Z.; methodology, J.Y.; software, Y.Z.; validation, G.X. and J.W.; formal analysis, L.N.; investigation, Q.Z. and Q.C.; resources, X.Y.; data curation, J.Y. and Y.Z.; writing—original draft preparation, J.Y. and G.X; writing—review and editing, J.W.; visualization, L.N.; supervision, X.Y.; project administration, J.Y.; funding acquisition, X.Y. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Jilin Provincial Agricultural Science & Technology Innovation Project (CXGC2021TD015) and the National Natural Science Foundation of China (31671764). These funds played an important role in experimental reagents and consumables, and also provided strong support for preparing the manuscript.

Data Availability Statement

The data we use are publicly available, and the sources have been noted in the article. No data from the store are required. All authors agree to publish without conflict of interest.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Ma, J.K.C.; Christou, P.; Chikwamba, R.; Haydon, H.; Paul, M.; Ferrer, M.P.; Ramalingam, S.; Rech, E.; Rybicki, E.; Wigdorowitz, A. Realising the value of plant molecular pharming to benefit the poor in developing countries and emerging economies. Plant Biotechnol. J. 2013, 11, 1029–1033. [Google Scholar] [CrossRef] [PubMed]
  2. Stoger, E.; Fischer, R.; Moloney, M.; Ma, J.K.-C. Plant Molecular Pharming for the Treatment of Chronic and Infectious Diseases. Annu. Rev. Plant Biol. 2014, 65, 743–768. [Google Scholar] [CrossRef] [PubMed]
  3. Shi, Y.; Habibi, P.; Haq, A.N.U.; Saeed, M.; Gulghutay Amjad, N.; Khan, I. Seed-Based System for Cost-Effective Production of Vaccine Against Chronic Respiratory Disease in Chickens. Mol. Biotechnol. 2023, 65, 570–580. [Google Scholar] [CrossRef] [PubMed]
  4. Vianna, G.R.; Cunha, N.B.; Murad, A.M.; Rech, E.L. Soybeans as bioreactors for biopharmaceuticals and industrial proteins. Genet. Mol. Res. 2011, 10, 1733–1752. [Google Scholar] [CrossRef] [PubMed]
  5. Hudson, L.C.; Garg, R.; Bost, K.L.; Piller, K.J. Soybean Seeds: A Practical Host for the Production of Functional Subunit Vaccines. Biomed. Res. Int. 2014, 2014, 340804. [Google Scholar] [CrossRef] [PubMed]
  6. Queiroz, L.N.; Maldaner, F.R.; Mendes, É.A.; Sousa, A.R.; D’Allastta, R.C.; Mendonça, G.; Mendonça, D.B.S.; Aragão, F.J.L. Evaluation of lettuce chloroplast and soybean cotyledon as platforms for production of functional bone morphogenetic protein 2. Transgenic Res. 2019, 28, 213–224. [Google Scholar] [CrossRef] [PubMed]
  7. Vianna, G.R.; Cunha, N.B.; Rech, E.L. Soybean seed protein storage vacuoles for expression of recombinant molecules. Curr. Opin. Plant Biol. 2023, 71, 102331. [Google Scholar] [CrossRef]
  8. Khan, M.S.; Joyia, F.A.; Mustafa, G. Seeds as Economical Production Platform for Recombinant Proteins. Protein Pept. Lett. 2020, 27, 89–104. [Google Scholar] [CrossRef]
  9. Müntz, K. Deposition of storage proteins. Plant Mol. Biol. 1998, 38, 77–99. [Google Scholar] [CrossRef]
  10. Cunha, N.B.; Murad, A.M.; Ramos, G.L.; Maranhão, A.Q.; Brígido, M.M.; Araújo, A.C.G.; Lacorte, C.; Aragão, F.J.L.; Covas, D.T.; Fontes, A.M. Accumulation of functional recombinant human coagulation factor IX in transgenic soybean seeds. Transgenic Res. 2011, 20, 841–855. [Google Scholar] [CrossRef]
  11. Morandini, F.; Avesani, L.; Bortesi, L.; Van, D.B.; De Wilde, K.; Arcalis, E.; Bazzoni, F.; Santi, L.; Brozzetti, A.; Falorni, A.; et al. Non-food/feed seeds as biofactories for the high-yield production of recombinant pharmaceuticals. Plant Biotechnol. J. 2011, 9, 911–921. [Google Scholar] [CrossRef] [PubMed]
  12. Ding, S.H.; Huang, L.Y.; Wang, Y.D.; Sun, H.C.; Xiang, Z.H. High-level Expression of Basic Fibroblast Growth Factor in Transgenic Soybean Seeds and Characterization of its Biological Activity. Biotechnol. Lett. 2006, 28, 869–875. [Google Scholar] [CrossRef] [PubMed]
  13. Moravec, T.; Schmidt, M.A.; Herman, E.M.; Woodford-Thomas, T. Production of Escherichia coli heat labile toxin (LT) B subunit in soybean seed and analysis of its immunogenicity as an oral vaccine. Vaccine 2007, 25, 1647–1657. [Google Scholar] [CrossRef] [PubMed]
  14. Yamada, Y.; Nishizawa, K.; Yokoo, M.; Zhao, H.; Onishi, K.; Teraishi, M.; Utsumi, S.; Ishimoto, M.; Yoshikawa, M. Anti-hypertensive activity of genetically modified soybean seeds accumulating novokinin. Peptides 2008, 29, 331–337. [Google Scholar] [CrossRef] [PubMed]
  15. Cunha, N.B.; Murad, A.M.; Cipriano, T.M.; Araújo, A.C.; Aragão, F.J.; Leite, A.; Vianna, G.R.; McPhee, T.R.; Souza, G.H.; Waters, M.J.; et al. Expression of functional recombinant human growth hormone in transgenic soybean seeds. Transgenic Res. 2011, 20, 811–826. [Google Scholar] [CrossRef]
  16. O’Keefe, B.R.; Murad, A.M.; Vianna, G.R.; Ramessar, K.; Saucedo, C.J.; Wilson, J.; Buckheit, K.W.; da Cunha, N.B.; Araújo, A.C.; Lacorte, C.C.; et al. Engineering soya bean seeds as a scalable platform to produce cyanovirin-N, a non-ARV microbicide against HIV. Plant Biotechnol. J. 2015, 13, 884–892. [Google Scholar] [CrossRef]
  17. Sirko, A.; Vaněk, T.; Góra-Sochacka, A.; Redkiewicz, P. Recombinant Cytokines from Plants. Int. J. Mol. Sci. 2011, 12, 3536–3552. [Google Scholar] [CrossRef]
  18. Kusnadi, A.R.; Nikolov, Z.L.; Howard, J.A. Production of recombinant proteins in transgenic plants: Practical considerations. Biotechnol. Bioeng. 1997, 56, 473–484. [Google Scholar] [CrossRef]
  19. Yang, J.; Xun, H.; Niu, L.; He, H.; Cheng, Y.; Zhong, X.; Zhao, Q.; Xing, G.; Liu, J.; Yang, X. Elastin-like polypeptide and γ-zein fusions significantly increase recombinant protein accumulation in soybean seeds. Transgenic Res. 2021, 30, 675–686. [Google Scholar] [CrossRef]
  20. He, Y.; Ning, T.; Xie, T.; Qiu, Q.; Zhang, L.; Sun, Y.; Jiang, D.; Fu, K.; Yin, F.; Zhang, W.; et al. Large-scale production of functional human serum albumin from transgenic rice seeds. Proc. Natl. Acad. Sci. USA 2011, 108, 19078–19083. [Google Scholar] [CrossRef]
  21. Zhang, D.; Lee, H.F.; Pettit, S.C.; Zaro, J.L.; Huang, N.; Shen, W.C. Characterization of transferrin receptor-mediated endocytosis and cellular iron delivery of recombinant human serum transferrin from rice (Oryza sativa L. ). BMC Biotechnol. 2012, 12, 92. [Google Scholar]
  22. Scheller, J.; Leps, M.; Conrad, U. Forcing single-chain variable fragment production in tobacco seeds by fusion to elastin-like polypeptides. Plant Biotechnol. J. 2006, 4, 243–249. [Google Scholar] [CrossRef] [PubMed]
  23. Torrent, M.; Llompart, B.; Lasserre-Ramassamy, S.; Llop-Tous, I.; Ludevid, M.D. Eukaryotic protein production in designed storage organelles. BMC Biol. 2009, 7, 5. [Google Scholar] [CrossRef] [PubMed]
  24. Hofbauer, A.; Peters, J.; Arcalis, E.; Rademacher, T.; Lampel, J.; Eudes, F.; Vitale, A.; Stoger, E. The Induction of Recombinant Protein Bodies in Different Subcellular Compartments Reveals a Cryptic Plastid-Targeting Signal in the 27-kDa γ-Zein Sequence. Front. Bioeng. Biotechnol. 2014, 2, 67. [Google Scholar] [CrossRef]
  25. Phan, H.T.; Hause, B.; Hause, G.; Arcalis, E.; Stoger, E.; Maresch, D.; Altmann, F.; Joensuu, J.; Conrad, U. Influence of elastin-like polypeptide and hydrophobin on recombinant hemagglutinin accumulations in transgenic tobacco plants. PLoS ONE 2014, 9, e99347. [Google Scholar] [CrossRef]
  26. Mainieri, D.; Marrano, C.A.; Prinsi, B.; Maffi, D.; Tschofen, M.; Espen, L.; Stöger, E.; Faoro, F.; Pedrazzini, E.; Vitale, A. Maize 16-kD γ-zein forms very unusual disulfide-bonded polymers in the endoplasmic reticulum: Implications for prolamin evolution. J. Exp. Bot. 2018, 69, 5013–5027. [Google Scholar] [CrossRef]
  27. Hegedus, D.D.; Baron, M.; Labbe, N.; Coutu, C.; Lydiate, D.; Lui, H.; Rozwadowski, K. A strategy for targeting recombinant proteins to protein storage vacuoles by fusion to Brassica napus napin in napin-depleted seeds. Protein Expr. Purif. 2014, 95, 162–168. [Google Scholar] [CrossRef]
  28. Yang, L.; Hirose, S.; Takahashi, H.; Kawakatsu, T.; Takaiwa, F. Recombinant protein yield in rice seed is enhanced by specific suppression of endogenous seed proteins at the same deposit site. Plant Biotechnol. J. 2012, 10, 1035–1045. [Google Scholar] [CrossRef]
  29. Kurokawa, S.; Kuroda, M.; Mejima, M.; Nakamura, R.; Takahashi, Y.; Sagara, H.; Takeyama, N.; Satoh, S.; Kiyono, H.; Teshima, R.; et al. RNAi-mediated suppression of endogenous storage proteins leads to a change in localization of overexpressed cholera toxin B-subunit and the allergen protein RAG2 in rice seeds. Plant Cell Rep. 2014, 33, 75–87. [Google Scholar] [CrossRef]
  30. Kinney, A.J.; Jung, R.; Herman, E.M. Cosuppression of the alpha subunits of beta-conglycinin in transgenic soybean seeds induces the formation of endoplasmic reticulum-derived protein bodies. Plant Cell. 2001, 13, 1165–1178. [Google Scholar]
  31. Tada, Y.; Utsumi, S.; Takaiwa, F. Foreign gene products can be enhanced by introduction into low storage protein mutants. Plant Biotechnol. J. 2003, 1, 411–422. [Google Scholar] [CrossRef] [PubMed]
  32. Yang, X.; Niu, L.; Zhang, W.; He, H.; Yang, J.; Xing, G.; Guo, D.; Du, Q.; Qian, X.; Yao, Y.; et al. Robust RNAi-mediated resistance to infection of seven potyvirids in soybean expressing an intron hairpin NIb RNA. Transgenic Res. 2017, 26, 665–676. [Google Scholar] [CrossRef] [PubMed]
  33. Yang, J.; Xing, G.; Niu, L.; He, H.; Guo, D.; Du, Q.; Qian, X.; Yao, Y.; Li, H.; Zhong, X.; et al. Improved oil quality in transgenic soybean seeds by RNAi-mediated knockdown of GmFAD2-1B. Transgenic Res. 2018, 27, 155–166. [Google Scholar] [CrossRef] [PubMed]
  34. Hwang, E.Y.; Song, Q.; Jia, G.; Specht, J.E.; Hyten, D.L.; Costa, J.; Cregan, P.B. A genome-wide association study of seed protein and oil content in soybean. BMC Genomics 2014, 15, 1. [Google Scholar] [CrossRef]
  35. Schmidt, M.A.; Herman, E.M. Proteome rebalancing in soybean seeds can be exploited to enhance foreign protein accumulation. Plant Biotechnol. J. 2008, 6, 832–842. [Google Scholar] [CrossRef]
  36. Gutiérrez, S.P.; Saberianfar, R.; Kohalmi, S.E.; Menassa, R. Protein body formation in stable transgenic tobacco expressing elastin-like polypeptide and hydrophobin fusion proteins. BMC Biotechnol. 2013, 13, 40. [Google Scholar] [CrossRef]
  37. Coates, R.J.; Young, M.T.; Scofield, S. Optimising expression and extraction of recombinant proteins in plants. Front. Plant Sci. 2022, 13, 1074531. [Google Scholar] [CrossRef]
  38. Iida, S.; Amano, E.; Nishio, T. A rice (Oryza sativa L.) mutant having a low content of glutelin and a high content of prolamine. Theor. Appl. Genet. 1993, 87, 374–378. [Google Scholar] [CrossRef]
  39. Iida, S.; Kusaba, M.; Nishio, T. Mutants lacking glutelin subunits in rice: Mapping and combination of mutated glutelin genes. Theor. Appl. Genet. 1997, 94, 177–183. [Google Scholar] [CrossRef]
  40. Kawakatsu, T.; Hirose, S.; Yasuda, H.; Takaiwa, F. Reducing rice seed storage protein accumulation leads to changes in nutrient quality and storage organelle formation. Plant Physiol. 2010, 154, 1842–1854. [Google Scholar] [CrossRef]
  41. He, W.; Wang, L.; Lin, Q.; Yu, F. Rice seed storage proteins: Biosynthetic pathways and the effects of environmental factors. J. Integr. Plant Biol. 2021, 63, 1999–2019. [Google Scholar] [CrossRef] [PubMed]
  42. Kim, H.J.; Lee, J.Y.; Yoon, U.H.; Lim, S.H.; Kim, Y.M. Effects of reduced prolamin on seed storage protein composition and the nutritional quality of rice. Int. J. Mol. Sci. 2013, 14, 17073. [Google Scholar] [CrossRef] [PubMed]
  43. Takaiwa, F.; Yang, L.; Wakasa, Y.; Ozawa, K. Compensatory rebalancing of rice prolamins by production of recombinant prolamin/bioactive peptide fusion proteins within ER-derived protein bodies. Plant Cell Rep. 2018, 37, 209–223. [Google Scholar] [CrossRef] [PubMed]
Agronomy 13 02680 g001
Figure 1. Schematic maps of the vector constructs used for soybean genetic transformation. All these constructs contained the glycinin (Gy) or conglycinin (Cgy) RNAi cassette and gfp (zein- or elp-fused, and unfused) expression cassettes. BCSP and Pgy1, soybean seed-specific promoters of β-conglycinin alpha subunit and glycinin Gy1 subunit genes, respectively; GFP, green fluorescent protein; zein, maize 27 kDa γ-zein; ELP, a 13.56 kDa elastin-like polypeptide; D, enterokinase cleavage site (DDDDK); K, endoplasmic reticulum (ER) localization signal; nos, nopaline synthase terminator; Gyi-S and Gyi-R, tandem RNAi fragments based on glycinin subunit-encoding genes (Gy1–Gy5); Cgyi-S and Cgyi-R, tandem RNAi fragments based on conglycinin subunit-encoding genes (Cgy1–Cgy3); ocs, octopine synthase terminator; pdk, a pdk intron spacer from Flaveria trinervia; LB, left border; RB, right border.
Figure 1. Schematic maps of the vector constructs used for soybean genetic transformation. All these constructs contained the glycinin (Gy) or conglycinin (Cgy) RNAi cassette and gfp (zein- or elp-fused, and unfused) expression cassettes. BCSP and Pgy1, soybean seed-specific promoters of β-conglycinin alpha subunit and glycinin Gy1 subunit genes, respectively; GFP, green fluorescent protein; zein, maize 27 kDa γ-zein; ELP, a 13.56 kDa elastin-like polypeptide; D, enterokinase cleavage site (DDDDK); K, endoplasmic reticulum (ER) localization signal; nos, nopaline synthase terminator; Gyi-S and Gyi-R, tandem RNAi fragments based on glycinin subunit-encoding genes (Gy1–Gy5); Cgyi-S and Cgyi-R, tandem RNAi fragments based on conglycinin subunit-encoding genes (Cgy1–Cgy3); ocs, octopine synthase terminator; pdk, a pdk intron spacer from Flaveria trinervia; LB, left border; RB, right border.
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Agronomy 13 02680 g002
Figure 2. Expression analysis of the foreign genes in transgenic soybean plants. (a) Quantification of green fluorescent protein (gfp) expression in immature T3 transgenic seeds at 45 days after flowering (DAF) using qRT-PCR (n = 3). Variability in the mRNA levels of the foreign genes was observed among the independent transgenic plants, and only the plants with similar expression of the transgenes were selected and presented. (b–f) Western blotting analysis of the GFP expression in T3 soybean seeds at 45 DAF. The bands represent the unfused GFP (26.8 kDa), maize 27 kDa γ-zein fused GFP (zein-GFP, 51.8 kDa), and elastin-like polypeptide fused GFP (GFP-ELP, 40.5 kDa). Ct+, GFP standard; Nt, non-transformed plant.
Figure 2. Expression analysis of the foreign genes in transgenic soybean plants. (a) Quantification of green fluorescent protein (gfp) expression in immature T3 transgenic seeds at 45 days after flowering (DAF) using qRT-PCR (n = 3). Variability in the mRNA levels of the foreign genes was observed among the independent transgenic plants, and only the plants with similar expression of the transgenes were selected and presented. (b–f) Western blotting analysis of the GFP expression in T3 soybean seeds at 45 DAF. The bands represent the unfused GFP (26.8 kDa), maize 27 kDa γ-zein fused GFP (zein-GFP, 51.8 kDa), and elastin-like polypeptide fused GFP (GFP-ELP, 40.5 kDa). Ct+, GFP standard; Nt, non-transformed plant.
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Figure 3. Expression analysis of endogenous storage proteins glycinin and conglycinin in transgenic soybean seeds carrying the Gy or Cgy RNAi cassettes (glycinin-suppressed, Gyless, or conglycinin-suppressed, Cgyless). qRT–PCR analysis of glycinin (a) and conglycinin (b) gene expression. Nt, non-transformed plants; B66, Gyless/GFP plants; G45, Gyless/zein–GFP plants; A58, Gyless/GFP-ELP plants; F24, Cgyless/zein–GFP plants; J41, Cgyless/GFP–ELP plants. (c) Protein expression profiles analyzed using SDS-PAGE. M, protein marker; Nt, non-transformed plants; DG44, zein–GFP expressing plants without suppression; DE9, GFP-ELP expressing plants without suppression; G45, G54, and G59, Gyless/zein–GFP plants; F24 and F32, Cgyless/zein–GFP plants; A44, A58, and A59, Gyless/GFP–ELP plants; J41, J47, and J53, Cgyless/GFP–ELP plants.
Figure 3. Expression analysis of endogenous storage proteins glycinin and conglycinin in transgenic soybean seeds carrying the Gy or Cgy RNAi cassettes (glycinin-suppressed, Gyless, or conglycinin-suppressed, Cgyless). qRT–PCR analysis of glycinin (a) and conglycinin (b) gene expression. Nt, non-transformed plants; B66, Gyless/GFP plants; G45, Gyless/zein–GFP plants; A58, Gyless/GFP-ELP plants; F24, Cgyless/zein–GFP plants; J41, Cgyless/GFP–ELP plants. (c) Protein expression profiles analyzed using SDS-PAGE. M, protein marker; Nt, non-transformed plants; DG44, zein–GFP expressing plants without suppression; DE9, GFP-ELP expressing plants without suppression; G45, G54, and G59, Gyless/zein–GFP plants; F24 and F32, Cgyless/zein–GFP plants; A44, A58, and A59, Gyless/GFP–ELP plants; J41, J47, and J53, Cgyless/GFP–ELP plants.
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Table 1. Recombinant protein content in soybean seeds with the suppressed expression of glycinin and conglycinin.
Table 1. Recombinant protein content in soybean seeds with the suppressed expression of glycinin and conglycinin.
TransgenesLinesImmature Seeds Mature Seeds
% TSPMean ± SD% TSPMean ± SD
Gyless/zein-GFPG452.83 ± 0.112.69 ± 0.20 A3.15 ± 0.213.49 ± 0.33 A
G542.78 ± 0.213.80 ± 0.26
G592.46 ± 0.263.51 ± 0.11
Cgyless/zein-GFPF242.72 ± 0.272.80 ± 0.12 A3.22 ± 0.254.49 ± 1.80 A
F322.89 ± 0.105.76 ± 1.36
Zein-GFPDG44 1.20 ± 0.22 B 2.08 ± 0.21 B
Gyless/GFP-ELPA441.84 ± 0.112.30 ± 0.55 A2.78 ± 0.244.31 ± 1.50 A
A582.90 ± 0.494.37 ± 0.89
A592.14 ± 0.175.78 ± 1.58
Cgyless/GFP-ELPJ41 2.96 ± 0.043.04 ± 0.08 A3.66 ± 0.145.03 ± 1.9 A
J473.04 ± 0.025.74 ± 1.22
J533.12 ± 0.285.69 ± 0.41
GFP-ELPDE9 0.30 ± 0.12 B 0.88 ± 0.19 B
Gyless/GFPB141.03 ± 0.271.02 ± 0.08 A1.94 ± 0.841.53 ± 0.37 A
B430.94 ± 0.151.21 ± 1.1
B661.09 ± 0.181.45 ± 0.98
Unfused GFPDF8 0.08 ± 0.03 B 0.57 ± 0.18 B
Total soluble protein (TSP) was extracted from immature seeds at 45 days after flowering (DAF) and mature dry seeds, and recombinant green fluorescent protein (GFP) expression was quantified by ELISA using purified GFP as the standard. Three biological replicates were conducted for each sample. The average accumulation levels of the foreign proteins (zein-GFP, GFP-ELP, and unfused GFP) on the glycinin (Gyless) or conglycinin (Cgyless) backgrounds were compared with their counterparts in the plants DG44, DE9 and DF8 without such suppressions, respectively. Different uppercase letters denote a significant difference at the level of 0.01 (LSD).
Table 2. Protein and oil content (%) in mature soybean seeds.
Table 2. Protein and oil content (%) in mature soybean seeds.
TransgenesLinesTotal Protein (%)Total Oil (%)Protein/Oil Ratio
Gyless/zein-GFPG4540.76 ± 0.1518.61 ± 0.152.19
G5441.55 ± 0.2518.72 ± 0.042.22
G5941.56 ± 0.1719.15 ± 0.222.17
Cgyless/zein-GFPF2440.74 ± 0.7319.58 ± 0.052.08
F3240.60 ± 0.0619.80 ± 0.302.05
Gyless/GFP-ELPA4439.93 ± 0.6519.97 ± 0.622.00
A5841.19 ± 1.1818.55 ± 1.012.22
A5941.72 ± 0.4418.12 ± 0.242.30
Cgyless/GFP-ELPJ4141.61 ± 0.6520.00 ± 0.122.08
J4741.16 ± 0.7719.88 ± 0.432.07
J5341.08 ± 0.3519.94 ± 0.782.06
Gyless/GFPB1440.25 ± 0.0418.90 ± 0.072.13
B4340.24 ± 1.0719.35 ± 0.442.08
B6641.15 ± 0.3917.89 ± 0.112.30
Non-transformed controlP0340.60 ± 0.0219.71 ± 0.142.06
Total protein contents were determined based on dry weight, and the protein/oil ratios were calculated based on the total protein and oil contents in soybean seeds. No significant differences were found in the total protein and protein/oil ratio among testes samples (p > 0.05, LSD).
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Yang, J.; Zhang, Y.; Xing, G.; Wei, J.; Niu, L.; Zhao, Q.; Cai, Q.; Zhong, X.; Yang, X. Increased Accumulation of Recombinant Proteins in Soybean Seeds via the Combination Strategy of Polypeptide Fusion and Suppression of Endogenous Storage Proteins. Agronomy 2023, 13, 2680. https://doi.org/10.3390/agronomy13112680

AMA Style

Yang J, Zhang Y, Xing G, Wei J, Niu L, Zhao Q, Cai Q, Zhong X, Yang X. Increased Accumulation of Recombinant Proteins in Soybean Seeds via the Combination Strategy of Polypeptide Fusion and Suppression of Endogenous Storage Proteins. Agronomy. 2023; 13(11):2680. https://doi.org/10.3390/agronomy13112680

Chicago/Turabian Style

Yang, Jing, Yuanyu Zhang, Guojie Xing, Jia Wei, Lu Niu, Qianqian Zhao, Qinan Cai, Xiaofang Zhong, and Xiangdong Yang. 2023. "Increased Accumulation of Recombinant Proteins in Soybean Seeds via the Combination Strategy of Polypeptide Fusion and Suppression of Endogenous Storage Proteins" Agronomy 13, no. 11: 2680. https://doi.org/10.3390/agronomy13112680

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