基于长期马铃薯病毒 X (PVX) 的重组 GFP 蛋白在本氏烟草体外培养中的瞬时表达,Plants

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基于长期马铃薯病毒 X (PVX) 的重组 GFP 蛋白在本氏烟草体外培养中的瞬时表达
Plants ( IF 4.0 ) Pub Date : 2021-10-15 , DOI: 10.3390/plants10102187
Yana Sindarovska ₁ , Mykola Kuchuk ₁

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植物分子农业具有生产有价值蛋白质的巨大潜力。瞬时表达技术可在温室种植的植物中提供高产量的重组蛋白，但每株植物都必须进行人工农杆菌渗透，而且开放式温室系统的控制较少。在这里，我们建议在控制良好的封闭体外系统中繁殖具有高效长期自我复制瞬时基因表达的无农杆菌植物。本氏烟草通过从受基于 PVX 的载体感染的叶外植体的芽诱导获得具有重组 GFP 瞬时表达的体外植物组织培养物。由于携带靶基因的病毒 RNA 的自然全身分布，瞬时表达发生在新组织和再生体中。在体外生长的植物中基因沉默被延迟，并且在植物中检测到 GFP 持续了五到六个月。不含农杆菌、表达 GFP 的植物可以在体外微繁殖（避免农杆菌渗透步骤），通过再生（保持培养多年）“恢复活力”，或转移到土壤中。再生体中的平均 GFP 为总可溶性蛋白 (TSP) 的 18% (0.52 mg/g 鲜叶重 (FW)。最高值达到 47% TSP (2 mg/g FW)。

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Long-Term Potato Virus X (PVX)-Based Transient Expression of Recombinant GFP Protein in Nicotiana benthamiana Culture In Vitro

Plant molecular farming has a great potential to produce valuable proteins. Transient expression technology provides high yields of recombinant proteins in greenhouse-grown plants, but every plant must be artificially agroinfiltrated, and open greenhouse systems are less controlled. Here, we propose to propagate agrobacteria-free plants with high-efficient long-term self-replicated transient gene expression in a well-controlled closed in vitro system. Nicotiana benthamiana plant tissue culture in vitro, with transient expression of recombinant GFP, was obtained through shoot induction from leaf explants infected by a PVX-based vector. The transient expression occurs in new tissues and regenerants due to the natural systemic distribution of viral RNA carrying the target gene. Gene silencing was delayed in plants grown in vitro, and GFP was detected in plants for five to six months. Agrobacteria-free, GFP-expressing plants can be micropropagated in vitro (avoiding an agroinfiltration step), “rejuvenated” through regeneration (maintaining culture for years), or transferred in soil. The mean GFP in the regenerants was 18% of the total soluble proteins (TSP) (0.52 mg/g of fresh leaf weight (FW). The highest value reached 47% TSP (2 mg/g FW). This study proposes a new method for recombinant protein production combining the advantages of transient expression technology and closed cultural systems.

更新日期：2021-10-15

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