Recombinant Calcium-sensitive Protein Yc2.1 Fission Yeast Intracellular Calcium Ion Concentration In The Distribution

YC2.1 gene is an artifical gene by Tsien et al in 1997. It's expression product —YC2.1 protein can combine with calcium reversely and change its fluorescence. It can indicate cytoplasm calcium density and distribution using confocal microscope. As we can fuse YC2.1 gene with signal-peptide sequence, the expression of YC2.1 protein can be orientated exactly. The method using YC2.1 protein to detect calcium is applied rapidly in recent years. But there is no report about the research using YC2.1 in Schizosaccharomyces pombe.In this paper, we observed the calcium distribution of cytoplasm in S.pombe. We take two steps to transform the YC2.1 gene into the S.pombe. First step, we make orientation inserting by one flat end and one sticking end ligation. And we get pESP-YC2.1" plasmid. This plasmid has promoter, start code, YC2.1 gene and terminator but no stop code. Second step, we use OLIGO software to design one Linker. This Linker has stop code no matter by reverse inserting or forward inserting. We make non-orientation inserting by both sticking end ligation. And we get pESP-YC2.1 plasmid. After sequencing and fluorescence microscope observation test, we confirm it's the right plasmid.The results show that the YC2.1 protein indicated calcium appeared mainly around the peripheral area of the cytoplasm in the fission yeasts, less in the center area of the cytoplasm .As for the fluo-3 indicated calcium, it is higher in the center of the cytoplasm not than around the peripheral area of the cytoplasm. As we know, the cytoplasm of fission yeasts is filled with organelles, such as nucleus and mitochondria. Then we use DAPI to do nucleus dyeing and its fluorescence displays in the center area of the cytoplasm.So the cytoplasm calcium distribution in fission yeasts should be in the peripheral area of the cytoplasm just as YC2.1 protein indicated and not filled in all cytoplasm center as fluo-3 indicated. This is because the fluo-3 is non-selectly sequestered by organelles as Tang et al reported. As far as fluo-3 loading method is concerned, it is not well-proportioned and can't represent the true distribution of the fission yeasts. Our results say that the YC2.1 protein indicating calcium method is better than the traditional fluo-3 loading method, and have big promising apply in the future study of fission yeasts.