Apoplast Calmodulin Localization In Transgenic Tobacco Cells Using GFP As Reporter

Calmodulin, as a versatile Ca²⁺ sensor, plays many important biological functions. Meanwhile, our previous experiments suggested that calmodulin exists in the apoplast of plant cells and has many physiological functions. Further more, we put forward a view that apoplast CaM maybe a multiple functional polypeptide signal in plants. In recent years, multiple genes of CaM isoforms have been found in many plants, which have some differences in expression and functions. In order to understand the apoplast CaM gene and the relationship between the apoplast CaM and CaM isoforms, we intended to get transgenic plants harboring soybean calmodulin isoform genes (SCaMs) fused to gfp as a reporter and study the subcellular localization of SCaMs.First, SCaM5, one of the soybean calmodulin isoforms, was amplified by PCR and was inserted into pUC-GFP. Then the chimeric genes (SCaM2-GFP, SCaM5-GFP) were inserted into the binary vector pGTV.The binary vectors which respectively contain the chimeric genes (SCaMl-GFP, SCaM2-GFP, SCaM3-GFP, SCaM4-GFP, SCaM5-GFP) were used to transform tobacco, and pGTV-GFP was used as control. The chimeric genes were transformed into tobacco plants via Agrobacterium tumefaciens. The transgenic tobacco harboring SCaMs genes were obtained and verified by reporter gene examination, PCR, Southern blot detection. Then we establish the transgenic callus. The plasmolyzed cells of transgenic callus were treated with 0.45M mannitol (PH 7.0) and observed using LSCM(laser scanning confocal microscope).Green fluorescence was found in the cell wall of transgenic SCaM1, SCaM2, SCaM3 callus. However there was no green fluorescence in the cell wall of transgenic SCaM4, SCaM5 callus. These results indicated that SCaMl , SCaM2 , SCaM3 can be secreted into the apoplast of plant cells, while SCaM4, SCaM5 don't exit in the apoplast of plant cells. This work laid a foundation for elucidating the in vivo functions of apoplast CaM by means of molecular biology.