PEI介导外源基因进入植物细胞的瞬时表达

Transient Expression of Transferred Gene Induced by Polyethylenimine in Plant

【目的】医学方面的大量研究证实,聚乙烯亚胺(polyethylenimine,PEI)作为一种新型阳离子多聚物基因载体可以吸附和浓缩DNA,通过与细胞膜亲和粘附和细胞吞噬作用运载外源基因进入细胞并实现表达。本实验研究旨在探索PEI作为非病毒基因载体介导外源基因进入植物细胞并获得瞬时表达的可能性。【方法】制备PEI/DNA复合物,利用凝胶阻滞分析PEI与DNA的结合情况,采用电子显微镜观察PEI/DNA复合物的形态。以绿色荧光蛋白基因为报告基因研究不同N/P比条件下PEI/DNA复合物对拟南芥原生质体细胞的转化效率,并与PEG转化方法进行比较分析。【结果】PEI与DNA的质量比为5﹕1～1﹕4,PEI可以与DNA稳定结合,形成粒径约100～200nm的球形复合物。PEI/DNA复合物对拟南芥原生质体细胞的转化效率随其N/P比的增大而提高,N/P=5时PEI的转化效率达到最高且明显高于PEG介导的转化效率,N/P>5时转化效率反而下降,容易使细胞破碎和变形。【结论】PEI作为一种新型的外源基因运送载体对拟南芥原生质体细胞具有良好的介导基因转移效果。

[Objective] The experimental research was carried out to investigate the possibility and method ofgene delivery into plant cells by nano-vector polyethylenimine （PEI） and obtain transient expression of gene, based on some research results that PEI can condense DNA into granules of nanometer size for delivering gene into cells in the field of medical science. [Method] PEI/DNA complexes were prepared. The ability of PEI to combine and protect DNA was investigated by agarose gel electrophoresis retardation assay. The surface characteristics of PEI/DNA complexes were observed with transmission electron microscope. For Arabidopsis thaliana protoplasts, transformation efficiency of PEUDNA at different charge ratios （N/P） was analyzed and compared with PEG. [Result] When the quality rate of PEI and DNA is in range of 5 ： 1-1 ： 4, PEI could bind and condense DNA, and form stable PEI/DNA complexes with nanoparticles diameter in 100-200 nm. Transformation efficiency of PEI/DNA can be improved with the increase of N/P ratios and reached highest at N/P-5. There was higher transformation efficiency of PEUDNA complexes than PEG at N/P=5, but began to decrease at N/P 〉 5 with higher toxicity to cells. [ Conclusion ] The experimental results confirmed that PEI is a new kind of efficient gene vector for transferring genes into Arabidopsis thaliana protoplasts.