核酸酶I-SceI介导的转基因斑马鱼构建及连接蛋白Connexin48．5的定位研究

I-SceI Meganuclease Mediates Transgenesis in Zebrafish and Connexin48.5 Localization Research

细胞连接蛋白是由多基因家族编码的一类结构相似、分子质量不同的蛋白质.在细胞膜上,每6个相同或不同的连接蛋白围绕中央孔排列形成一个连接子,相邻细胞膜上的连接子相互对接形成细胞间隙连接,细胞间隙连接是一种重要的通讯连接,它不仅是细胞间代谢偶联、冲动传导的结构基础,而且可以通过介导与细胞的迁移、分化、增生和器官形成有关的信号物质而在胚胎发育中起重要作用.为了进一步探讨细胞间隙连接在生物体中的作用,采用斑马鱼胚胎显微注射核酸酶I-SceI和质粒DNA的方法成功构建了绿色荧光蛋白GFP与细胞连接蛋白Connexin48.5融合表达的转基因荧光斑马鱼品系,并通过对荧光蛋白发光的观察在斑马鱼鱼体中进行连接蛋白Connexin48.5的定位.实验结果表明:核酸酶I-SceI介导下的斑马鱼转基因方法效率较高,可行性好;连接蛋白Connexin48.5在斑马鱼体内主要定位于眼球晶状体和脊索.所获得的Connexin48.5-GFP融合表达斑马鱼系,以及利用该品系进行的Connexin48.5定位研究对细胞连接蛋白在生物体内的功能研究将具有重要作用.

Connexins are a group of proteins coded by multi gene family. On the cell membrane,every 6 Connexins combine together to form a Connexon,and two Connexons of two different cells connect with each other to form a gap junction. Gap junction is a very important kind of communicational connection between cells,which is not only the constructional basic of metabolic coupling and sig- nal transduction betweens cells, but also the channel of signal substance controlling cell migration, differentiation, proliferation and or- ganogenesis. For a deeper study of gap junction function in organisms,we generated a transgenic zebrafish line that expresses Con- nexin48.5-GFP fusion protein by co-injecting meganuclease I-SceI and plasmid DNA into zebrafish embryos. I-SceI mediated trans- genesis showed high efficiency,which made it a better way to get transgenic animals than the traditional method. While the working mechanism of I-SceI remains unclear,in our opinion, I-SceI binds to its recognition site after the cleavage and promotes nuclear local- ization of the transgene could be a good explanation. Using our transgenic zebrafish line, we found that Connexin48.5 is highly ex- pressed in lens and notochord in zebrafish. In addition to its expression in lens,which has been reported preciously,our new finding suggested that Connexin48.5 could have important roles in notochord development or nervous system function.