Figure 1.
Overview of the cloning strategy for the creation of expression vectors targeting YA to the thylakoid lumen and thylakoid membrane, and analysis of aequorin expression in Arabidopsis transgenic lines. A and B, The targeting sequences for the thylakoid lumen (A) and for the thylakoid membrane (B) were cloned into an expression cassette in front of YA using the restriction enzymes ApaI and NotI. The complete expression cassettes were transferred subsequently into the binary vector pBIN19 carrying Basta resistance. C and D, Analysis of aequorin expression by RT-PCR (C) and immunoblotting (D) in Arabidopsis transgenic lines stably transformed with the constructs encoding TL-YA and TM-YA. For RT-PCR analyses, actin was used as a housekeeping gene. For immunoblot analyses, total protein extracts (50 μg) were separated by 10% SDS-PAGE, transferred to PVDF, and incubated with an anti-aequorin antibody (diluted 1:10,000). Black and white arrowheads indicate the YA chimeras targeted to the thylakoid lumen and the thylakoid membrane, respectively. The black arrow indicates the YA targeted to the cytosol, excluding the nucleus, in the Arabidopsis line CPK17G2A-NES-YA (Cyt-YA), used here as a positive control.