介孔硅纳米粒作为植物细胞转基因载体的研究

doi:10.13560/j.cnki.biotech.bull.1985.2021-1040

摘要/Abstract

摘要：

随着纳米生物技术的快速发展,以纳米材料作为基因载体在植物细胞转导研究中取得了初步进展。本研究制备了两种尺寸的介孔硅纳米颗粒,氨基修饰后对其进行表征,并负载含smGFP基因的质粒DNA对拟南芥原生质体进行细胞转导。结果表明,直径约20 nm的氨基化介孔硅纳米颗粒（Am-MSN-20）呈类球形,花形结构的氨基化介孔硅纳米颗粒（Am-MSN-50）直径约50 nm,两者均带正电荷。Am-MSN-50结合DNA的能力高于Am-MSN-20,两种纳米载体都表现出了良好的稳定性,能够保护负载的pDNA不被细胞核酸酶降解,并且对原生质体没有毒害作用。与Am-MSN-20相比较,Am-MSN-50具备更高的转导效率。本研究表明,氨基修饰的MSNs有望成为一种安全高效的新型植物基因载体。

关键词: 介孔硅纳米粒, 氨基化, 遗传转化, 载体, 植物细胞

Abstract:

With the rapid development of nanobiotechnology,nanoparticles have been applied as vectors for gene transformation in plant cells. In this study,mesoporous silica nanoparticles（MSNs）with different particle sizes were synthesized and then amine-functionalized. The transformation efficiencies of amine-functionalized MSNs（Am-MSNs）for delivering plasmid DNA encoding green fluorescent protein（smGFP）were investigated in the protoplasts of Arabidopsis thaliana. As results,the Am-MSN-20 exhibited monodispersed spheroids with average particle size of 20 nm,while Am-MSN-50 showed a flower-like structure and had an average particle size of 50 nm. Both amine-functionalized MSNs were positively charged. Am-MSN-50 had higher binding capacity of pDNA than that of Am-MSN-20. The Am-MSNs/pDNA complex showed good stability,which suggested that both Am-MSNs protected bounded pDNA efficiently against degradation by cellular nucleases. The MSNs also exhibited no cytotoxic effects on the protoplasts of A. thaliana. The Am-MSN-50 enabled much higher transformation efficiency of the pDNA encoding smGFP compared to that of Am-MSN-20. The results suggest that Am-MSNs can be employed as promising carriers for safe and effective gene delivery.

Key words: mesoporous silica nanoparticles, amine-functionalized, gene transfer, carrier, plant cells

陆新华, 孙德权, 张秀梅. 介孔硅纳米粒作为植物细胞转基因载体的研究[J]. 生物技术通报, 2022, 38(7): 194-204.

LU Xin-hua, SUN De-quan, ZHANG Xiu-mei. Genetic Transformation of Plant Cells Mediated by Mesoporous Silica Nanoparticles[J]. Biotechnology Bulletin, 2022, 38(7): 194-204.

图/表 8

图1 不同纳米材料透射电镜图 A：直径为20 nm MSN;B：氨基修饰的直径为20 nm的MSN;C：直径为50 nm MSN;D：氨基修饰的直径为50 nm的MSN

Fig.1 TEM images of different nanoparticles A：MSN-20;B：Am-MSN-20;C：MSN-50;D：Am-MSN-50

图2 不同纳米材料红外光谱图 A：直径为20 nm MSN;B：氨基修饰直径为20 nm的MSN;C：直径为50 nm MSN;D：氨基修饰的直径为50 nm的MSN

Fig.2 FTIR spectra of of different nanoparticles A：MSN-20;B：Am-MSN-20;C：MSN-50;D：Am-MSN-50

图3 不同纳米材料的Zeta电位

Fig.3 Zeta potential of different nanoparticles

图4 两种Am-MSNs与DNA结合的凝胶阻滞实验 A：Am-MSN-20/pDNA;B：Am-MSN-50/pDNA。1：纯pDNA（阳性对照）;2：Am-MSNs（阴性对照）,3-8：分别代表Am-MSNs与pDNA的质量比为5∶1,10∶1,20∶1,30∶1,40∶1和 50∶1时的DNA结合情况

Fig.4 Agarose gel electrophoresis assay of Am-MSNs/DNA complexes A：Am-MSN-20/pDNA;B：Am-MSN-50/pDNA;1：free pDNA（positive CK）;2：Am-MSNs（negative CK）;3-8 presented that the mass ratios of Am-MSNs to pDNA are 5∶1,10∶1,20∶1,30∶1,40∶1 and 50∶1,respectively

图5 氨基化硅纳米粒对pDNA的酶切保护 1：pDNA;2：pDNA + DNase I;3：Am-MSN-20/pDNA;4：Am-MSN-20/pDNA + DNase I;5：Am-MSN-50/pDNA;6：Am-MSN-50/pDNA + DNase I

Fig.5 Protection of Am-MSNs against pDNA digestion 1：free pDNA;2：free pDNA + DNase I;3：Am-MSN-20/pDNA;4：Am-MSN-20/pDNA + DNase I;5：Am-MSN-50/pDNA;6：Am-MSN-50/pDNA + DNase I

图6 氨基化硅纳米粒对拟南芥原生质体的细胞毒性评价 A：氨基修饰的直径为20 nm的MSN;B：氨基修饰的直径为50 nm的MSN

Fig.6 Assessment of Am-MSNs cytotoxicity to A. thaliana mesophyll protoplasts A：Am-MSN-20;B：Am-MSN-50

图7 氨基化硅纳米粒介导的smGFP质粒在拟南芥原生质体中的瞬时表达 A：质粒DNA;B：氨基修饰的MSN;C：质粒DNA与PEG混合同;D：氨基修饰直径20 nm MSN与质粒DNA混合;E：氨基修饰直径20 nm MSN与质粒DNA混合;比例尺= 5 µm

Fig.7 Am-MSN-mediated transient smGFP expression in the protoplasts of A. thaliana A：pDNA only;B：Am-MSNs;C：pDNA/PEG;D：pDNA/Am-MSN-20;E：pDNA/Am-MSN-50. Scale bars=5 µm

图8 转化48 h后 smGFP在拟南芥原生质体中的基因表达评价图中误差线表示标准偏差。不同小写字母表示差异达到显著水平（P<0.05）

Fig.8 Assessment of smGFP gene expression in the proto-plasts of A. thaliana 48 h post-transformation The error line in the figure refers to the standard deviation. Different lowercase letters indicate a significant difference（P < 0.05）

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