Calcium ion treatment and calcium ion fluorescence staining of yeast strain k667

To obtain the yeast strain transformed with different MdCAX genes, the four MdCAX genes (MdCAX3L-1 to MdCAX3L-4) and the truncated MdCAX3L-1 were cloned into the yeast expression vector pDR-196 and then transferred into the calcium ion-sensitive yeast mutant strain k667. After selection on SD medium (−ura) and PCR screening for the presence of the transgene, more than two monoclonal strains of each MdCAX gene were obtained and were used for subsequent Ca²⁺ treatments.

For treatments using different concentrations of calcium ions, control yeast strain (K667) or K667 strains transformed with different MdCAX3L genes were cultured in liquid YPD medium to a concentration of OD₆₀₀ = 1.0. The bacterial solutions were then diluted four times in a 10-fold gradient and placed on solid YPD or YPD medium varying in the concentration of CaCl₂. After three days, the growth of the strains was observed and photographed.

The calcium-sensitive fluorescent probe Fluo4-AM was used to observe Ca²⁺ levels inside yeast cells transformed either with pDR196 (empty vector) or with different MdCAX genes. Before fluorescence observations, yeast cells were grown on liquid SD medium (−ura) to the exponential phase and then diluted to OD₆₀₀ = 0.2 ~ 0.3 in medium supplemented with 50 mM CaCl₂. After 4 h of culture at 30 °C, yeast cells were washed with PBS solution three times to remove the medium and were then incubated with 5 μM Fluo4-AM for 20 min at 30 °C. Subsequently, the yeast cells were washed three times with PBS to remove the fluorescent dye, and the fluorescence was observed using a laser scanning confocal microscope (Leica TCS SP8 SR). The excitation and emission wavelengths for GFP fluorescence were set to 488 nm and 512–520 nm, respectively. The LAS AF software (Leica Application Suite Advanced Fluorescence, version 4.3) was used to determine the grey values of green fluorescence in vacuoles of yeast cells.