应用重组钙离子敏感蛋白YC2.1研究裂殖酵母胞内钙离子浓度的分布

【作者】 王少慧；

【导师】 袁生；

【作者基本信息】 南京师范大学 ， 微生物学， 2006， 硕士

【摘要】 YC2.1基因是1997年Tsien等人工构建的一个基因，它的表达蛋白——YC2.1蛋白可以与钙离子进行可逆的结合反应，反应前后有荧光的变化。使用激光共聚焦仪器能够测量活体细胞内钙离子的浓度和分布。同时可以将YC2.1基因和定位于不同细胞器的引导序列融合进行细胞器内的定位表达，在最近几年里得到了迅速的推广应用。但是，用YC2.1蛋白来进行裂殖酵母细胞内钙离子分布的研究，目前还没有文献报道。 本文首先构建了含YC2.1基因的粟酒裂殖酵母表达质粒pESP-YC2.1。第一步，从p35S-YC2.1-Kan质粒上(适用于在植物细胞中表达)把YC2.1基因切下，通过一端平末端，一端粘性末端的定向插入将YC2.1基因导入到pESP-3质粒中。该质粒含有启动子，起始密码子，YC2.1基因以及终止子序列。但是不含终止密码子。第二步使用OLIGO软件设计一个Linker，该Linker含有终止密码子，而且不论正向插入还是反向插入最终都会引入终止密码子。通过单酶切粘性末端不定向插入实验得到pESP-YC2.1质粒。将该质粒转化裂殖酵母菌株SP-Q01，经过测序和荧光显微镜观察证实该质粒在胞质中可正常表达YC2.1蛋白。 利用YC2.1蛋白研究和观察了裂殖酵母细胞内钙离子的分布情况。激光共聚焦显微镜下可见，表达的YC2.1蛋白指示的钙离子呈细胞周缘胞质较高浓度分布，而在细胞中部钙离子浓度相对低一些。与此相比较，用常规的钙离子荧光指示剂fluo-3染色的粟酒裂殖酵母细胞的胞内钙离子呈内部区域高的钙离子荧光信号，而在周缘的胞质区相对较弱。众所周知，在裂殖酵母的细胞质内填充着大量的细胞器，如细胞核，线粒体等。用DAPI对裂殖酵母的细胞核进行活体染色，发现细胞核确实占据了胞质的中间区域，因而使得YC2.1蛋白所指示的细胞胞质内的钙离子主要集中在细胞质的周缘区域，而不是整个细胞质全着色。因此Fluo-3荧光指示的裂殖酵母胞内钙离子的分布不能真实反映裂殖酵母胞内钙离子的分布，Fluo-3荧光给出的是细胞质和细胞器游离钙离子浓度总的分布信息。利用YC2.1蛋白测定裂殖酵母胞内钙离子的方法优于以Fluo-3位代表的钙离子荧光探针的方法，能够比较真实的反映细胞内游离钙离子的分布情况。另外，本研究表明，当细胞横隔出现时，在横隔附近显示的钙离子浓度较高。这提示钙离子可能与裂殖酵母细胞分裂有关，但是需要进一步的实验证明。更多还原

【Abstract】 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.更多还原