Open AccessTackling Heterogeneity: A Leaf Disc-Based Assay for the High-Throughput Screening of Transient Gene Expression in Tobacco
Author(s) -
Natalia Piotrzkowski,
Stefan Schillberg,
Stefan Rasche
Publication year - 2012
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0045803
Subject(s) - nicotiana tabacum , recombinant dna , agrobacterium , gene , gene expression , biology , infiltration (hvac) , microbiology and biotechnology , genetics , transgene , materials science , composite material
Transient Agrobacterium -mediated gene expression assays for Nicotiana tabacum ( N. tabacum ) are frequently used because they facilitate the comparison of multiple expression constructs regarding their capacity for maximum recombinant protein production. However, for three model proteins, we found that recombinant protein accumulation (rpa) was significantly influenced by leaf age and leaf position effects. The ratio between the highest and lowest amount of protein accumulation (max/min ratio) was found to be as high as 11. Therefore, construct-based impacts on the rpa level that are less than 11-fold will be masked by background noise. To address this problem, we developed a leaf disc-based screening assay and infiltration device that allows the rpa level in a whole tobacco plant to be reliably and reproducibly determined. The prototype of the leaf disc infiltration device allows 14 Agrobacterium -mediated infiltration events to be conducted in parallel. As shown for three model proteins, the average max/min rpa ratio was reduced to 1.4 using this method, which allows for a sensitive comparison of different genetic elements affecting recombinant protein expression.