Pax6基因和Pax6基因突变体在非洲爪蟾眼睛发育过程中的相互作用及分子机制

Among all the amphibian model animals which are commonly introduced in developmental biology lab, Xenopus laevis is probably the most well studied species. As model species，it offers studies of the development and function of in gene level, as well as their related protein in vertebrates. In our previous studies, we studied temporal and special expression of pax6 and pax6 varient gene in the process of Xenopus laevis tadpole by gene cloning and PT-PCR for yeast 2-hybrid systenm and Co-immunoprecipitation, the aim of this study was to explore the relationship between Pax6 variant and Pax6.TALENs was also used to establish the model of the relationship between Pax6 variant and Pax6. Revealing the main role both in eye development processas well as exploring the molecular mechanism of related theory. By conducting in-depth studies of the dominant gene Pax6 , in particularly the determination and Analysis on the expression of specific variant gene in eye development, would be of great beneficial for not only the molecular process of in-depth study of amphibious animal development pathway and lay a solid foundation, but also for better understand the molecular regulation of vertebrates eye development system in the process of related; research results will provide the basis for technology theory and application of disease and treatment of human eyes.

非洲爪蟾(Xenopus laevis)作为两栖类模式动物，在研究脊椎动物的发育以及相关基因调控、蛋白质间的相互作用等方面发挥着重要作用。本项研究以非洲爪蟾为实验材料，采用国际上先进的研究方法，通过RT-PCR、建立酵母表达系统以及免疫共沉淀实验（Co-IP），证实眼睛发育过程中Pax6 与Pax6突变基因之间的关系；并且利用TALENs技术，在无尾两栖类动物中建立Pax6基因和Pax6突变基因模型，力争揭示两者在眼睛发育过程中的相互作用，从理论上探讨相关的分子机制。 通过对眼睛发育的主导基因Pax6的深入研究，特别是对其特异性突变体基因表达的分析测定，不仅为深入研究两栖动物眼睛发育过程中分子信号通路打下坚实的基础，也有助于我们更好的了解脊椎动物发育的分子调控体系；相关研究成果也将为人类眼睛疾病及治疗的技术理论和应用提供依据。

非洲爪蟾(Xenopus laevis)作为两栖类模式动物，在研究脊椎动物的发育以及相关基因调控、蛋白质间的相互作用等方面发挥着重要作用。本项研究以非洲爪蟾为实验材料， 研究通过RT-PCR获得两者的开放阅读框，构建载体Pax6-pGAD-T7和Pax6-variant-pGBK-T7，Pax6-pGBK-T7 和Pax6-variantpGAD-T7，建立酵母表达系统，进一步提取胚胎发育20期总蛋白，进行免疫共沉淀实验（Co-IP）。序列比对发现Pax6 variant与正常Pax6基因比较，两者同源性很高；与正常Pax6蛋白氨基酸序列相比较，二者的差别在PST结构域；酵母双杂交实验表明：当两种基因同时存在时，酵母菌能够在营养缺陷型培养基生长，并同时有阳性反应，证明Pax-6和Pax6 variant在酵母体内存在相互作用；免疫共沉淀实验发现正常Pax6蛋白能够和Pax6 variant蛋白形成复合体，证明在其体内依然存在相互作用；用3D-PSSM程序对这两种蛋白的PST结构域进行分析预测，发现Pax6 variant比正常Pax6蛋白少两个α-螺旋。. Pax6是眼睛发育的重要的调控因子，Pax6与Pax6 variant之间的相互作用对眼睛的发育起到重要的作用；Pax6 variant蛋白通过负显性调控来调节Pax6，间接的调控眼睛发育。前期有文献表明Pax6的突变发生在3′端并引起眼发育病变。本项研究结果表明Pax6 variant参与了非洲爪蟾眼的正常发育，由此推测Pax6基因的进化从PST蛋白结合区域开始，进化速度很快，形成很多功能复杂的突变体，而在其他两个DNA结合区域存在高度保守，进化速度相对很慢。由于蛋白结构的差别使得Pax6 variant 与Pax6蛋白相互作用形成复合体，改变正常Pax6蛋白的构象进而通过剂量效应来调节Pax6的转录功能，促进眼睛的正常发育。