Yang Jing, Xun HongWei, Niu Lu, He Hongli, Cheng Yunqing, Zhong Xiaofang, Zhao Qianqian, Xing Guojie, Liu Jianfeng, Yang Xiangdong
Jilin Provincial Key Laboratory of Agricultural Biotechnology, Jilin Academy of Agricultural Sciences, Changchun, 130033, China.
Jilin Normal University, Siping, 136000, China.
Transgenic Res. 2021 Oct;30(5):675-686. doi: 10.1007/s11248-021-00258-7. Epub 2021 May 8.
Soybean seeds are an ideal host for the production of recombinant proteins because of their high content of proteins, long-term stability of seed proteins under ambient conditions, and easy establishment of efficient purification protocols. In this study, a polypeptide fusion strategy was applied to explore the capacity of elastin-like polypeptide (ELP) and γ-zein fusions in increasing the accumulation of the recombinant protein in soybean seeds. Transgenic soybean plants were generated to express the γ-zein- or ELP-fused green fluorescent protein (GFP) under the control of the soybean seed-specific promoter of β-conglycinin alpha subunit (BCSP). Significant differences were observed in the accumulation of zein-GFP and GFP-ELP from that of the unfused GFP in transgenic soybean seeds based on the total soluble protein (TSP), despite the low-copy of T-DNA insertions and similar expression at the mRNA levels in selected transgenic lines. The average levels of zein-GFP and GFP-ELP accumulated in immature seeds of these transgenic lines were 0.99% and 0.29% TSP, respectively, compared with 0.07% TSP of the unfused GFP. In mature soybean seeds, the accumulation of zein-GFP and GFP-ELP proteins was 1.8% and 0.84% TSP, an increase of 3.91- and 1.82-fold, respectively, in comparison with that of the unfused GFP (0.46% TSP). Confocal laser scanning analysis showed that both zein-GFP and GFP-ELP were abundantly deposited in many small spherical particles of transgenic seeds, while there were fewer such florescence signals in the same cellular compartments of the unfused GFP-expressing seeds. Despite increased recombinant protein accumulation, there were no significant changes in the total protein and oil content in seeds between the transgenic and non-transformed plants, suggesting the possible presence of threshold limits of total protein accumulation in transgenic soybean seeds. Overall, our results indicate that γ-zein and ELP fusions significantly increased the accumulation of the recombinant protein, but exhibited no significant influence on the total protein and oil content in soybean seeds.
大豆种子是生产重组蛋白的理想宿主，因为其蛋白质含量高、种子蛋白在环境条件下具有长期稳定性，且易于建立高效的纯化方案。在本研究中，应用多肽融合策略来探索弹性蛋白样多肽（ELP）与γ-玉米醇溶蛋白融合体在增加重组蛋白在大豆种子中积累量方面的能力。利用大豆β-伴大豆球蛋白α亚基种子特异性启动子（BCSP）的控制，培育出表达γ-玉米醇溶蛋白或ELP融合绿色荧光蛋白（GFP）的转基因大豆植株。尽管转基因大豆种子中T-DNA插入拷贝数低且所选转基因株系在mRNA水平上表达相似，但基于总可溶性蛋白（TSP）观察到，玉米醇溶蛋白-GFP和GFP-ELP的积累量与未融合GFP的积累量存在显著差异。这些转基因株系未成熟种子中积累的玉米醇溶蛋白-GFP和GFP-ELP的平均水平分别为TSP的0.99%和0.29%，而未融合GFP为TSP的0.07%。在成熟大豆种子中，玉米醇溶蛋白-GFP和GFP-ELP蛋白的积累量分别为TSP的1.8%和0.84%，与未融合GFP（TSP的0.46%）相比，分别增加了3.91倍和1.82倍。共聚焦激光扫描分析表明，玉米醇溶蛋白-GFP和GFP-ELP都大量沉积在转基因种子的许多小球形颗粒中，而在表达未融合GFP的种子的相同细胞区室中，此类荧光信号较少。尽管重组蛋白积累增加，但转基因植株与非转基因植株种子中的总蛋白和油含量没有显著变化，这表明转基因大豆种子中总蛋白积累可能存在阈值限制。总体而言，我们的结果表明，γ-玉米醇溶蛋白和ELP融合体显著增加了重组蛋白的积累，但对大豆种子中的总蛋白和油含量没有显著影响。
