Nano-bio-material In Gene-transfer Optimizing And In Fluorescence Labelling

In this study, firstly, according to the special requirements of gene transformation, bio-nanomaterials were synthesized including chitson nanopartciles and starch nanoparticles as well as magnetic nanopaticles, and then bio-nanopaticle gene vectors were constructed. Based on traditional transgene methods, the nanoparticle delivery systems were optimized, and novel transgene techniques were established. The results indictated that the efficiency of transformation was improved. Secondly, based on the method of synthesizing one-kind-material nanoparticles, two-kind-material compound nanoparticles were constructed, in which soluble starch and iron oxide nanoparticles were used as raw materials to synthesize magnetic starch nanoparticles. Those particles have bifunction including fluorescence labeling and magnetic separatation for real-time cell imaging. The results of this disertation were listed as follows:1. Synthesis of bio-nanoparticles and their propertiesChitosan nanoparticles(CSNPs) and statch nanopaticles(StNPs) as well as magnetic nanopaticles(MNPs) were synthesized with cocervation process and water-in-oil and chemical precipitation microemulsion correspondingly. The particles were characterized with scan electron microscope and Zeta-Sizer measurement, and then agarose gel electrophoresis was used to detect the bioconjugation of nanopatciles and DNA molecules. Chitosan nanoparticles were shown to be spherical structure distributed evenly, the average sizes of the particles were about 50 nm in diameter, and the particles were 11.1 mv charges. PLL-StNPs were prepared by linking poly-L-lysine (PLL) on the surface of StNPs with the size about 50 nm, and they distributed evenly without congregation. The needle-shape magnetic nanoparticles have uniformity diameter about 2～10 nm, with performance of stability and equality dispersion. The agarose gel electrophoresis showed that all of those nanoparticles could conjugate DNA effectively; furthermore, the complexes could protect the absorbed DNA from DNase I damage.2. Chitosan nanoparticle as gene vector in mammal cell transformationPlasmid DMA was absorbed to chitosan nanoparticles by electrostaficforces, and gene vetor was constructed with chitson loading plasmid pEGFP DNA, which comprised the reporter gene of green fluorescence protein. Exogenous gene could be transfected into COS-7 cells, and green fluorescence protein was expressed efficiently in cells. As no-virus gene vector, chitson nanoparticle had favourable biocompatibility with mammal cells and would be widely used in bio-medical field.3. Chitosan nanoparticle as plant gene vector mediated by particle bombardment Based on the traditional particle bombardment method, chitosan nanoparticle was used as gene vector instead of traditional powder of Au and W. Chitosan nanoparticle-DNA mixture were transformed into the onion cell via particle bombardment, and onion cells were observed under invert-fluorescence-microscope. The results showed that GFP gene was expressed in the onion cells. It indicated that chitson nanoparicle gene vector could break through the plant cell wall and deliver gene into the cell successfully mediated by particle bombardment, and this technique woud enlarge the application of chitson nanoparticle in plant gene expression.4. Magnetic nanoparticle as plant gene vector mediated by electric shockIn order to avoid the disadvantage of DNA damage in gene transfer by electric shock, magnetic nanoparticle was synthesized and was used to protect banded DNA from electric shock damage. Magnetic nanoparticles absorbed plasmid DNA to form gene vectors, and were mixed into electrode cup and the cup was shocked. Fluorescence could be observed in transformed suspended cells obviously under inverted fluorescence microscope. The result showed that GFP gene could be transformed into the rice cells by magnetic nanoparticles and expressed green fluorescent protein in the cells. Therefore, nanoparticle gene vector pierced the plant cell wall, and the GFP gene expressed in the cells successfully via electric shock.5. Starch nanoparticle as plant gene vector mediated by floral dipBased on the traditional floral dip gene-transformation method, starch nanoparticle was used as plant gene vector instead of Agrobacterium. The nanoparticle vectors were tranformed into arabidopsis seedlings by floral dip. The seeds of tranformed plants (T0) were collected, and were cultured in greenhouse. The seedlings were screened by bastar. The seeds of anti-bastar plants (T1) were collected, and were cultured in greenhouse together with the wild type plant. The results showed that the transgenic plants flowered earlier and showed varying degrees of dwarfing compared with wild-type ones. Moreover, the transgenic plants had phenotype of defection in stem elongation and leaf development. Besides, GFP fused protein expressed in the cells of hypocotyl of transgenic plants. It indicated that exterior gene was delivered to plant cell successfully and expressed stably. This method had the advantage of avoiding complex culture of microbe, and thus, a new transgene technique would be established with nanoparticle mediated by floral dip. 6. Synthesis of compound-material fluorescent/magnetic nanoparticle and its use for real-time cell imagingIn order to avoid the disadvantage of transgene detection in traditional method, fluorescent/magnetic nanoparticle was used to label and separate cells in one step. Magnetic starch nanoparticles (MSNPs) were synthesized in water-in-oil microe-mulsion under room temperature.Soluble starch and iron oxide nanoparticles were used as raw materials with POCl3 as crosslinking agent.MSNPs were characterized via TEM, FTIR, Zeta potential system, TGA and VSM. The average diameter of MSNPs was about of 220 nm, with uniform in size, well dispersed and magnetic resonance. MSNPs were coated with poly-L-lysine (PLL). Then the surface of PLL-MSNP was combined with fluorescein isothiocynate (FITC) and tetramethyl rhodamine isothiocyanate(TRITC). The results showed that fluorescent/magnetic starch nanoparticles(FMSNPs) had more stable capability against photobleaching compared with free FITC and TRITC, and the particles had low bio-toxicity and certain function of magnetic separatation. A novel bio-nano-technique of multiple fluorescence labeling and magnetic response would be established. The technique would be used for ultrasensitive detection and real-time cell imaging.