Study On Nanoparticles As Gene Carriers In Mediating Genetic Transformation Of Plant Cells

As gene and drug carriers, nanoparticles have been applied in gene transformation and drug release in mammalian cells. This is one of the major achievements that have been gained in nano-biotechnology field. But fewer researches have been carried in plant science. At present, it was reported that Iowa State University researchers had applied silica nanoparticles to plant cells successfully. This thesis has described the systematic researches of polyethylenimine, chitosan, magnetic nanoparticles modified by PEI as plant transgenic vehicles, and a series of experiments using nanoparticles were carried out for transgenic plants. Based on these researches, a novel method of gene transformation by three nanoparticles in plant was established.1. Preparation and Characterization of PEI/DNA, CS/DNA and polyMAG/DNA gene carrier complexesThree kinds of gene carrier complexes,PEI/DNA, CS/DNA and polyMAG/DNA were constructed by mixing nanoparticles of PEI,CS and polyMAG with DNA in different amounts respectively. The nanoparticles of PEI, CS and polyMAG could bind with DNA effectively with binding efficiency more than 90%. Observed under electron microscopy, the nano-complexes were spherical, with diameter about 100～200 nm. Nano-complexes can bind DNA stably, after 24 h placement, and protect DNA against restriction endonuclease and nuclease.Gel electrophoresis illuminates that PEI/DNA and polyMAG/DNA gene carrier complexes can prevent the digest of DNA from EcoRⅠ; while CS/DNA gene carrier complexes can prevent the digest of DNA from DNAseⅠ. The three complexes can be placed stably for 24 h.2. Preparation system optimization of arabidopsis protoplast cells3～4 weeks of arabidopsis leaves were selected as experimental materials,which were used to studied on the optimal conditions and limiting factors of separating processes of arabidopsis mesophyll protoplast cells. The results indicated that most suitable conditions for enzymolysis were cellulase 1.5%, macerozyme 0.5%, pH=5.8 in enzyme solution. The enzymolysis time for the highest production of protoplast cells was.2.0h. By purification, protoplast cells production could reach 106ml-1, and protoplast vitality could keep a high level.3. Gene transformation of plant cell mediated by nanoparticle carriersPEI/DNA, CS/DNA and polyMAG/DNA were used respectively for mediating gene transformation on arabidopsis protoplast, by using Polyethylene Glycol(PEG)as a control group. The results indicated that PEI/DNA could transfer the GFP gene into the protoplast cells and express green fluorescence protein, and the transformation efficiency was about 80%, higher than PEG-mediated transformation efficiency; CS/DNA could also transfer the GFP gene into the protoplast cells expressing green fluorescence protein, but the transformation efficiency was only about 1%, much lower than that of PEI/DNA and PEG-mediated transformation; CS/DNA caused great damage to cells. polyMAG/DNA could not mediate gene expression of green fluorescence protein in the protoplast cells under driving force of magnetic field , and also caused great damage to protoplast cells.