ABSTRACT
Nanomaterials have been widely studied for their potential to become the new generation of nanocarriers in gene transfection, yet it remains still difficult to apply them efficiently and succinctly to plant cells. Poly (2-(N,N-dimethylamino) ethyl methacrylate) (PDMAEMA), which possesses temperature and pH dual-sensitivity, has largely been applied in animal cells, but it is rarely involved in plant cells. As a proof of concept, PDMAEMA as a gene carrier is incubated with plasmid GFP (pGFP) to explore its transfection ability in plants, and cationic polymer polyethylenimine (PEI) is used as a control. pGFP was efficiently condensed into the nanostructure by electrostatic interactions at an N/P (amino group from cationic polymers/phosphate group from plasmid DNA (pDNA)) ratio of 15; after complexation into nanocarriers, pGFP was protected from endonuclease degradation according to the DNase I digestion assay. After incubation with protoplasts and leaves, GFP was observed with confocal microscopy in plant cells. Western blot experiments confirmed GFP expression at the protein level. Toxicity assay showed PDMAEMA had a lower toxicity than PEI. These results showed that transient expression of pGFP was readily achieved in Arabidopsis thaliana and Nicotiana benthamiana. Notably, PDMAEMA showed lower cytotoxicity than PEI upon incubation with Nicotiana benthamiana leaves. PDMAEMA exhibited great potency for DNA delivery in plant cells. This work provides us with new ideas of more concise and more effective methods for plant transformation.
ABSTRACT
In recent years, gold nanoparticles (AuNPs) have been widely used as gene silencing agents and therapeutics for treatment of cancers due to their high transfection efficiency and lack of cytotoxicity, but their roles in gene silencing in plants have not yet been reported. Here, we report synthesis of AuNPs-branched polyethylenimine and its integration with the small interfering RNAs (siRNA) of NPR1 to form a AuNPs-siRNA NPR1 compound. Our results showed that AuNPs-siRNA NPR1 was capable of infiltrating into Arabidopsis cells. AuNPs-siRNA NPR1 silenced 80% of the NPR1 gene in Arabidopsis. Bacteriostatic and ion leakage experiments suggest that the NPR1 gene in Arabidopsis leaves was silenced by AuNPs-siRNA NPR1 . In Columbia-0 plants, compared with the control group treated with buffer solution, the AuNPs-siRNA NPR1 treatment significantly increased the number of colonies and cell death, and the leaves turned yellow, similar to the phenotype of the npr1 leaves. These results indicated this AuNPs-siRNA NPR1 silencing the NPR1 gene method is simple, effective and quick (3 days), and a powerful tool to study gene functions in plants.
