RESUMO
Tobacco mild green mosaic virus (TMGMV)-like nanocarriers were designed for gene delivery to plant cells. High aspect ratio TMGMVs were coated with a polycationic biopolymer, poly(allylamine) hydrochloride (PAH), to generate highly charged nanomaterials (TMGMV-PAH; 56.20 ± 4.7 mV) that efficiently load (1:6 TMGMV:DNA mass ratio) and deliver single-stranded and plasmid DNA to plant cells. The TMGMV-PAH were taken up through energy-independent mechanisms in Arabidopsis protoplasts. TMGMV-PAH delivered a plasmid DNA encoding a green fluorescent protein (GFP) to the protoplast nucleus (70% viability), as evidenced by GFP expression using confocal microscopy and Western blot analysis. TMGMV-PAH were inactivated (iTMGMV-PAH) using UV cross-linking to prevent systemic infection in intact plants. Inactivated iTMGMV-PAH-mediated pDNA delivery and gene expression of GFP in vivo was determined using confocal microscopy and RT-qPCR. Virus-like nanocarrier-mediated gene delivery can act as a facile and biocompatible tool for advancing genetic engineering in plants.
Assuntos
Arabidopsis , Proteínas de Fluorescência Verde , Arabidopsis/virologia , Arabidopsis/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Técnicas de Transferência de Genes , Plasmídeos/genética , Poliaminas/química , Protoplastos/metabolismo , Nanoestruturas/química , DNA/química , DNA/administração & dosagemRESUMO
A laboratory exercise for undergraduate advanced students of enzymology and biocatalysis is presented. Since enzyme assays can be quenched or continuous, this experiment compares the performance of two quenching agents for lactase, in a continuous setup. Enzymatic activity of ß-galactosidase (Aspergillus oryzae) was determined based on the release of 4-nitrophenol from 4-nitrophenyl ß-D-galactopyranoside using a microplate reader. Sodium carbonate and borate buffer were tested as quenching agents, and experimental control was the unstopped assay. Based on released 4-nitrophenol, enzyme activity, and rate constant k, the students could assess the performance of each termination agent. The experiment promotes disciplinary and transversal competencies, including research-based learning, critical thinking, and introduce the students to high-throughput techniques that are common in the research and development environment.