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Studies on the N_2-laser microbeam system for efficient gene delivery and the utilization of this system for plant protection

N_2激光微束高效基因传递系统及其在植物保护中的应用研究

基本信息

批准号：
06404009
负责人：
OZAKI Takeshi
金额：
$ 12.61万
依托单位：
Osaka Prefecture University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (A)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

项目摘要

An N_2-laser with a 337.1 nm wavelength and 3 ns pulse width burned localized hole ca.0.5 mum in diameter on the cell wall and cell membrane of cultured cells of melon or tobacco under selected irradiation conditions (1.5-6 muj/spot). No visible damage due to the irradiation was detected under these conditions. Fluotescent-isothyocyanate (FITC) was used to determine the osmotic value of the medium surrounding cultured cells and 0.3 Molarity was the best to get 66.3 % of cells which showed fluorescence. Efficiency of gene delivery into plant cells was tested using this N_2 laser treatment. Mature pollen of lmpatiens balsamina, I.walleriana and Lycoris albiflora was irradiated with a laser at 3muj/spot in medium containing 0.3 M mannitol and plasmid PBI221 harboring beta-glucuronidase. The maximum frequency of transient gene expression was 24% in these treatments. It is concluded that the osmotic value of medium surrounding the pollen grains is extremely important for the GUS gene epression to use N_2-laser microbeam and it is able to introduce any gene into pollen exactly one by one. When the melon cotyledons were treated with a laser in the presence of cucumber mosaic virus RNA,virus multiplication was observed in the epidermal and around the cells 24 hr after irradiation. These results suggest that the present laser method is useful for the transformation of plant cells. Resistance in the African wild plant, Cucumis figarei Des.et.Noude to CMV was found to related to restricted virus movement, and the rates of cell-to-cell and long distance movement in C.figarei is regulated by sequences in CMV RNA 3. A cDNA clone encoding the 3a and coat protein gene of CMV (pepo strain), linked to the cauliflower mosaic virus 35S promotor, was introdeced into tobacco by N_2-laser microbeam and the transgenic tobacco plants expressing the CP gene were protected from infection with CMV.

用波长337.1 nm、脉宽3 ns的N_2激光在选定的辐照条件下(1.5-6微焦/斑)在甜瓜或烟草培养细胞的细胞壁和细胞膜上烧毁直径约0.5微米的局部小孔。在此条件下，未检测到明显的辐照损伤。用荧光异硫氰酸酯(FITC)测定细胞周围培养液的渗透值，以0.3摩尔浓度为最佳，可使66.3%的细胞呈现荧光。用这种N_2激光处理植物细胞，测试了基因导入植物细胞的效率。在含有0.3M甘露醇和含有β-葡萄糖醛酸酶的pBI221的培养基中，用3muJ/斑点的激光照射凤仙花、花叶石蒜和白花石蒜的成熟花粉。在这些处理中，瞬时基因表达的最高频率为24%。结果表明，利用N_2激光微束对GUS基因的表达，花粉周围介质的渗透值是非常重要的，可以将任何一个基因准确地导入到花粉中。用含有黄瓜花叶病毒RNA的激光照射甜瓜子叶，24小时后病毒在细胞表面和细胞周围增殖。这些结果表明，本激光方法可用于植物细胞的转化。野生型黄瓜对CMV的抗性与病毒活动受限有关，黄瓜细胞间和远距离移动的速率受CMV RNA3基因序列的调控。利用N_2激光微束将编码CMV 3a基因和外壳蛋白基因的克隆导入烟草，保护了表达CP基因的烟草植株不受CMV侵染。