纳米载体介导的植物遗传转化研究现状和前景

doi:10.13560/j.cnki.biotech.bull.1985.2020-0521

摘要/Abstract

摘要：

高效遗传转化技术是植物重要性状功能基因鉴定的前提和转基因育种的基础。随着纳米生物技术的发展,以纳米载体介导的植物转基因技术已显示出巨大的应用潜力。综述了国内外应用于植物纳米载体的类型、与外源基因的结合方式以及传输细胞的原理,重点阐述了影响纳米基因载体性能与转化效率的重要因素,以及纳米载体介导外源基因转化植物细胞的方法,分析了纳米载体介导法与其他转基因方法的特点和优势,并提出纳米载体介导的转化技术应加强稳定遗传转化、基因编辑与植物原位转化等方面探索研究,旨为植物遗传转化技术和方法提供新的思路。

关键词: 纳米粒子, 基因载体, 载体性能, 转化方法

Abstract:

Efficient genetic transformation technology is the prerequisite for the identification of functional genes for important traits in plants and the basis for transgenic breeding.With the development of nanotechnology,the genetic transformation technology of plant based on gene vector constructed by nano particle has shown great application potential. The type and properties of nano particle applied in gene vector of plant,its combination way with DNA and the basic principle of gene transfection technology were reviewed. In the meanwhile,the important factors that affect the performance and transformation efficiency of nano-gene vector,as well as the transformation methods by which nano-gene vectors mediate exogenous genes into plant cells,were expounded emphatically. The advantages of nano-gene vectors were analyzed compared with that of other vectors. Further researches are needed to improve the stable genetic transformation,gene editing and in planta transformation with nano-gene vector,which will provide new ideas for plant genetic transformation technology and methods.

Key words: nanoparticle, gene vector, vector performance, transformation method

孙敬爽, 胡瑞阳, 郑广顺, 麻文俊, 许言, 王军辉. 纳米载体介导的植物遗传转化研究现状和前景[J]. 生物技术通报, 2021, 37(2): 162-173.

SUN Jing-shuang, HU Rui-yang, ZHENG Guang-shun, MA Wen-jun, XU Yan, WANG Jun-hui. Research Progress and Prospect of Plant Genetic Transformation Mediated by Nano-gene Vector[J]. Biotechnology Bulletin, 2021, 37(2): 162-173.

图/表 2

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纳米载体类型	纳米粒子	纳米粒子特性	功能修饰	转入植物细胞途径	转基因植株（组织或细胞）	转入基因	转基因植株表达特性	参考文献
无机物纳米材料	四氧化三铁（Fe₃O₄）	超顺磁性,外加磁场条件靶向性强	PEI修饰	外加磁场作用	棉花（Gossypium hirsutum）花粉	BtΔα·Cpti基因	稳定性表达,通过杂交获得抗虫转基因棉花	[25]
			未经修饰	悬浮细胞电击穿孔后直接转化	水稻悬浮细胞（Oryza sativa）	GFP质粒	瞬时表达	[56]
			PEI修饰	外加磁场作用	拟南芥（A. thaliana）原生质体	GFP质粒	瞬时表达	[62]
	磁性金纳米粒子（GNP）	超顺磁性,外加磁场条件靶向性强	异硫氰酸荧光素修饰（FITC）	超声波作用	油菜悬浮细胞和原生质体（Brassica napus var.Jec Neaf ）	GUS质粒	稳定表达	[63]
	介孔二氧化硅（MSN）	比表面积大,比孔容大,介孔孔径可调节	金纳米粒子包裹	基因枪	烟草（N. tabacum L.）子叶、玉米（Zea. mays）未成熟胚	GFP蛋白	稳定表达	[13]
			金纳米粒子覆盖	基因枪	洋葱（Allium cepa）表皮细胞	GFP蛋白和mCherry蛋白	瞬时表达	[64]
			金纳米粒子覆盖	基因枪	玉米（Z. mays）胚	DsRed2质粒和Loxp蛋白	瞬时表达	[65]
			TMAPS等有机物修饰	直接浸染	拟南芥（A. thaliana）根	mCherry质粒	46.5%瞬时表达	[66]
	单壁碳纳米管（SWNT）	高深径比,可塑性和生物兼容性强	有机物修饰	叶片组培孵育	烟草（N. tabacum L.）叶子	nptⅡ基因	与组培培养结合获得了转 nptⅡ基因植株,转化率6%	[67]
			纯化单壁碳纳米管	直接叶片浸染	烟草（N. tabacum L.）、芥菜（Eruca sativa）、小麦（Triticum aestivum）、棉花（G. hirsutum）	GFP-siRNA	实现在非模式植物基因的转录后沉默,瞬时沉默率95%;	[58]
			羧化壳聚糖（CS）等有机物的修饰	直接叶片浸染	芥菜（E. sativa）、豆瓣菜（Nasturtium officinale）、烟草（N. tabacum L.）、菠菜（Spinacia oleracea）	YFP质粒	实现外源基因转化叶绿体质体基因组的瞬时表达	[49]
			聚乙烯亚胺（PEI）功能修饰	直接叶片浸染	烟草（N. tabacum L.）、棉花（G.hirsutum）、小麦（T. aestivum）、芥菜（E. sativa）、	GFP质粒	实现多种非模式植物瞬时转染	[37,59]
	层状双氢氧化物（LDH-NS）	典型的层状结构,一般为六角形纳米片,厚度0.5-.0nm, 粒径30-60 nm	片层间为阴离子乳酸修饰	叶面喷洒	拟南芥（A. thaliana）和烟草（N. tabacum L.）	dsRNA	使PMMoV和CMV同源基因沉默,延长植物抗虫效果	[61]
	碳纳米点（CD）	类圆球形,<10 nm粒子	聚乙二醇（PEG）功能修饰	叶面喷洒	小麦（T. estivum）	GFP、Cas9和gRNA	SPO11 基因编辑,实现了作物的基因编辑	[30]
	ZnS量子点	粒径小仅3-5 nm	多聚赖氨酸（poly-L-lysine）修饰	超声波处理	烟草（N. tabacum L.）悬浮细胞	GUS质粒	GUS基因稳定表达	[68]
天然高分子有机物	淀粉纳米粒子（StNP）	50-100nm,生物降解与生物相容性好	多聚赖氨酸（poly-L-lysine）修饰	超声波处理悬浮细胞后转染	盾叶薯蓣（Dioscrea zigiberensis）悬浮细胞	GFP质粒	瞬时表达和稳定表达	[47]
			多聚赖氨酸（poly-L-lysine）和水溶性量子点CdSe）	超声波处理悬浮细胞后转染	麻枫树（J. curcas）悬浮细胞	GFP质粒	瞬时表达	[69]
			多聚赖氨酸（poly-L-lysine）和水溶性量子点CdSe）	与愈伤组织共孵育	麻枫树（Jatropha curcas）愈伤组织	GFP质粒	稳定表达	[70]
	壳聚糖（CS）	需要化学或生物学修饰提高在溶液中的稳定性	未修饰	共孵育	拟南芥（A. thaliana）原生质体	GFP质粒	瞬时表达	[71]
	细胞穿膜肽（CPPs）	由10-30个氨基酸组成的短肽,有较强的跨膜转运能力	聚阳离子和细胞穿膜肽融合	叶片浸染	烟草（N. tabacum L.）和拟南芥（A. thaliana）叶片	GFP和荧光素酶基因	瞬时表达	[29]
			聚阳离子和细胞穿膜肽融合	叶片浸染	拟南芥（A. thaliana）叶片	CHS siRNA	YFP和CHS瞬时沉默,抑制低温条件下叶花青素合成	[72]
			线粒体靶向肽和细胞穿膜肽融合	叶片浸染	拟南芥（A. thaliana）叶片	GFP质粒	GFP在表皮细胞的线粒体中表达	[73]
人工合成高分子有机物	甲基丙烯酸二甲氨基乙酯（DMAEM）聚合物	分支带正电荷,与DNA以静电作用结合	未修饰	PEG转化	烟草（N. tabacum L.）和角齿藓（Ceratodon purpureus）原生质体	YFP和GFP质粒	瞬时表达和稳定表达	[54]
	磷酸钙纳米粒子（CaPNPs）	20-50 nm,钙离子的渗透不平衡使负载DNA逃逸内含体,通过核孔。	未修饰	胚轴组培转染	芥菜（Brassica juncea L.）胚轴外植体培养物	GUS质粒	80.7%稳定转化	[74]
	阳离子荧光纳米粒子	水溶性并含有荧光发色团	未修饰	幼苗根浸染	拟南芥（A. thaliana）根和幼苗	dsRNA	SAM基因沉默表现型发生改变	[75]
	荧光共轭聚合物纳米粒子	水溶液稳定悬浮	未修饰	浸染	烟草（N. tabacum L.）原生质体	SiRNA	抑制CesA-1基因表达,丧失细胞壁再生能力	[76]

纳米载体类型	纳米粒子	纳米粒子特性	功能修饰	转入植物细胞途径	转基因植株（组织或细胞）	转入基因	转基因植株表达特性	参考文献
无机物纳米材料	四氧化三铁（Fe₃O₄）	超顺磁性,外加磁场条件靶向性强	PEI修饰	外加磁场作用	棉花（Gossypium hirsutum）花粉	BtΔα·Cpti基因	稳定性表达,通过杂交获得抗虫转基因棉花	[25]
			未经修饰	悬浮细胞电击穿孔后直接转化	水稻悬浮细胞（Oryza sativa）	GFP质粒	瞬时表达	[56]
			PEI修饰	外加磁场作用	拟南芥（A. thaliana）原生质体	GFP质粒	瞬时表达	[62]
	磁性金纳米粒子（GNP）	超顺磁性,外加磁场条件靶向性强	异硫氰酸荧光素修饰（FITC）	超声波作用	油菜悬浮细胞和原生质体（Brassica napus var.Jec Neaf ）	GUS质粒	稳定表达	[63]
	介孔二氧化硅（MSN）	比表面积大,比孔容大,介孔孔径可调节	金纳米粒子包裹	基因枪	烟草（N. tabacum L.）子叶、玉米（Zea. mays）未成熟胚	GFP蛋白	稳定表达	[13]
			金纳米粒子覆盖	基因枪	洋葱（Allium cepa）表皮细胞	GFP蛋白和mCherry蛋白	瞬时表达	[64]
			金纳米粒子覆盖	基因枪	玉米（Z. mays）胚	DsRed2质粒和Loxp蛋白	瞬时表达	[65]
			TMAPS等有机物修饰	直接浸染	拟南芥（A. thaliana）根	mCherry质粒	46.5%瞬时表达	[66]
	单壁碳纳米管（SWNT）	高深径比,可塑性和生物兼容性强	有机物修饰	叶片组培孵育	烟草（N. tabacum L.）叶子	nptⅡ基因	与组培培养结合获得了转 nptⅡ基因植株,转化率6%	[67]
			纯化单壁碳纳米管	直接叶片浸染	烟草（N. tabacum L.）、芥菜（Eruca sativa）、小麦（Triticum aestivum）、棉花（G. hirsutum）	GFP-siRNA	实现在非模式植物基因的转录后沉默,瞬时沉默率95%;	[58]
			羧化壳聚糖（CS）等有机物的修饰	直接叶片浸染	芥菜（E. sativa）、豆瓣菜（Nasturtium officinale）、烟草（N. tabacum L.）、菠菜（Spinacia oleracea）	YFP质粒	实现外源基因转化叶绿体质体基因组的瞬时表达	[49]
			聚乙烯亚胺（PEI）功能修饰	直接叶片浸染	烟草（N. tabacum L.）、棉花（G.hirsutum）、小麦（T. aestivum）、芥菜（E. sativa）、	GFP质粒	实现多种非模式植物瞬时转染	[37,59]
	层状双氢氧化物（LDH-NS）	典型的层状结构,一般为六角形纳米片,厚度0.5-.0nm, 粒径30-60 nm	片层间为阴离子乳酸修饰	叶面喷洒	拟南芥（A. thaliana）和烟草（N. tabacum L.）	dsRNA	使PMMoV和CMV同源基因沉默,延长植物抗虫效果	[61]
	碳纳米点（CD）	类圆球形,<10 nm粒子	聚乙二醇（PEG）功能修饰	叶面喷洒	小麦（T. estivum）	GFP、Cas9和gRNA	SPO11 基因编辑,实现了作物的基因编辑	[30]
	ZnS量子点	粒径小仅3-5 nm	多聚赖氨酸（poly-L-lysine）修饰	超声波处理	烟草（N. tabacum L.）悬浮细胞	GUS质粒	GUS基因稳定表达	[68]
天然高分子有机物	淀粉纳米粒子（StNP）	50-100nm,生物降解与生物相容性好	多聚赖氨酸（poly-L-lysine）修饰	超声波处理悬浮细胞后转染	盾叶薯蓣（Dioscrea zigiberensis）悬浮细胞	GFP质粒	瞬时表达和稳定表达	[47]
			多聚赖氨酸（poly-L-lysine）和水溶性量子点CdSe）	超声波处理悬浮细胞后转染	麻枫树（J. curcas）悬浮细胞	GFP质粒	瞬时表达	[69]
			多聚赖氨酸（poly-L-lysine）和水溶性量子点CdSe）	与愈伤组织共孵育	麻枫树（Jatropha curcas）愈伤组织	GFP质粒	稳定表达	[70]
	壳聚糖（CS）	需要化学或生物学修饰提高在溶液中的稳定性	未修饰	共孵育	拟南芥（A. thaliana）原生质体	GFP质粒	瞬时表达	[71]
	细胞穿膜肽（CPPs）	由10-30个氨基酸组成的短肽,有较强的跨膜转运能力	聚阳离子和细胞穿膜肽融合	叶片浸染	烟草（N. tabacum L.）和拟南芥（A. thaliana）叶片	GFP和荧光素酶基因	瞬时表达	[29]
			聚阳离子和细胞穿膜肽融合	叶片浸染	拟南芥（A. thaliana）叶片	CHS siRNA	YFP和CHS瞬时沉默,抑制低温条件下叶花青素合成	[72]
			线粒体靶向肽和细胞穿膜肽融合	叶片浸染	拟南芥（A. thaliana）叶片	GFP质粒	GFP在表皮细胞的线粒体中表达	[73]
人工合成高分子有机物	甲基丙烯酸二甲氨基乙酯（DMAEM）聚合物	分支带正电荷,与DNA以静电作用结合	未修饰	PEG转化	烟草（N. tabacum L.）和角齿藓（Ceratodon purpureus）原生质体	YFP和GFP质粒	瞬时表达和稳定表达	[54]
	磷酸钙纳米粒子（CaPNPs）	20-50 nm,钙离子的渗透不平衡使负载DNA逃逸内含体,通过核孔。	未修饰	胚轴组培转染	芥菜（Brassica juncea L.）胚轴外植体培养物	GUS质粒	80.7%稳定转化	[74]
	阳离子荧光纳米粒子	水溶性并含有荧光发色团	未修饰	幼苗根浸染	拟南芥（A. thaliana）根和幼苗	dsRNA	SAM基因沉默表现型发生改变	[75]
	荧光共轭聚合物纳米粒子	水溶液稳定悬浮	未修饰	浸染	烟草（N. tabacum L.）原生质体	SiRNA	抑制CesA-1基因表达,丧失细胞壁再生能力	[76]