A Fluorescence Enhancement Method for Real-Time Observation of Ca2+ Signaling Transduction in BY2 Cells

Abstract: Calcium ion as the second messenger is an important member of cell signal transduction and is also a focused object of cell signal transduction research. It is meaningful to develop a method more sensitive and dynamical for real-time observation of calcium ion signal in plant cells to study the cellular signal transduction. In this paper， the gene of a calcium ion responsible aequorin (AEQ) original from jellyfish was transformed into tobacco BY2 (bright yellow 2) cell that was SCMP2 (secretive carrier membrane proteins 2) labeled with green fluorescent protein (SCAMP2-GFP). The bivalent transgenic BY2 cell lines were screened out and verified. The calcium ions response real-time fluorescence signal in the cells was clearly observed when the transformed cells were treated with coelenterazine and subjected to light microscopic observation. It indicated that the weak fluorescence， released by coelenterazine of AEQ response the Ca2+， could further stimulate GFP fluorescence and make the weak fluorescence converted into enhanced GFP green fluorescence. It maked the real-time observation of calcium in plant cell more activity. The fluorescence strength had been able to satisfy the direct observation and acquisition signal under microscope although the green fluorescence is a bit weak comparing with the GFP direct excitation. A clear calcium response fluorescence image was obtained at three times the exposure time of external excitation with the same cell. The bivalent enhance methods possess its advantages that it enable research on calcium signal in identical subcellular localization if label some identical protein markers with GFP.

CSTR: 32200.14.cjcb.2018.11.0008