【作者】 冯颖；

【导师】 林圣彩；

【作者基本信息】 厦门大学 ， 生物化学与分子生物学， 2013， 博士

【摘要】 Tankyrase,端粒聚合酶是近年发现的一个与端粒长度调节相关的酶,是人端粒复合物中一个重要组分。近十年的研究表明,tankyrase不仅参与端粒长度的调节,同时还在纺锤体组装、葡萄糖转运、中心体调控和Wnt信号通路中发挥重要的作用。到目前为止,在人类和小鼠中共发现两个tankyrases蛋白,分别命名为tankyrase1和tankyrase2,两者在蛋白结构上拥有极高的相似度。研究发现,在人细胞中,tankyrase1和tankyrase2都参与了端粒长度的调控。然而在小鼠内,研究人员发现tankyrase1或tankyrase2敲除的小鼠并没有表现出端粒缩短的现象,但同时敲除tankyrase1和tankyrase2的小鼠则表现为胚胎致死,说明小鼠中tankyrases的功能存在冗余。尽管在人类和小鼠内,tankyrase的作用已经研究得较为透彻详尽,但是tankyrase作为一个高度保守的蛋白,在果蝇发育过程中的作用至今仍未有报道。本论文以果蝇作为模式生物,通过遗传学的手段,揭示了tankyrase在果蝇发育过程中的作用。本论文研究通过果蝇P因子敲除的技术,得到tankyrase缺失的果蝇突变株,研究发现缺失tankyrase蛋白功能的果蝇能够存活,且不会影响Wnt信号通路,同时我们发现缺失tankyrase的果蝇虽然可以存活,但是在代谢和其他方面却存在一定的缺陷。在tankyrase缺失的果蝇中果蝇的产卵效率明显降低,三龄幼虫时期体内的葡萄糖含量显著升高,幼虫和成虫体内的甘油三酯(TAG)含量显著降低,且在饥饿环境压力下的生存能力也显著降低,说明在tankyrase缺陷的果蝇体内,能量的储存和代谢受到影响,但是具体的机制还有待进一步的研究探索。同时本论文还探讨了在果蝇体内过表达tankyrase所产生的效应。本论文通过果蝇显微注射获得过表达tankyrase的转基因果蝇株,通过UAS/Gal4双元表达系统,研究在果蝇体内过表达tankyrase的作用。研究发现,在果蝇全身过表达tankyrase会引起果蝇致死,在不同的组织或器官中特异性表达tankyrase也会引起组织和器官的损伤。TUNEL试验发现,在果蝇幼虫的成虫盘后部过表达tankyrase会引起细胞凋亡,这种细胞凋亡的现象可以被过表达抗凋亡蛋白p35抑制。但是,在成蝇的眼睛和翅膀中过表达p35并不能抑制因过表达tankyrase而引起翅膀和眼睛的缺陷,说明过表达tankyrase会引起细胞死亡的发生。Tankyrase作为一个多功能结构域的蛋白,通过构建tankyrase结构域缺失的转基因果蝇,我们发现其Ankyrin结构域在此过程中发挥了重要的功能。更多还原

【Abstract】 Tankyrases (TRF1-interacting ankyrin related ADP-ribose polymerase) are protein members of the poly (ADP-ribose) polymerase family bearing several ankyrin repeats domains, a SAM domain and a PARP domain, regulating the length of human telomere. They play an important role in telomere maintenance, and are associated with the sister chromatid separation and glucose uptake. Recent research has indicates that tankyrases are also involved in the Wnt signaling. In mouse, neither tankyrasel nor tankyrase2deficiency mice has any observable on telomere length maintenance. However, double knockout of tankyrasel and tankyrase2causes mouse embryonic lethality, suggesting functional redundancy of the two genes. Despite of their important roles in mouse and human, little is known about the function of tankyrase in Drosophila development. In this study, Drosophila was used as a model to examine the function of tankyrase through genetic methods. The mutant flies of tankyrase were generated through the P element-mediated knockout technique. The mutant strain was used to systematically assess possible functions of tankyrase in Drosophila. Animal lacking the activity of tankyrase appear of viable. However, some defects in metabolism and development were observed on the mutant flies These defects include low egg hatchability, high glucose concentration, low TAG concentration and decreased tolerance of starvation. These data suggest that tankyrase is involved in the regulation of energy metabolism.We also examined the function of tankyrase by overexpression approach. Overexpression of tankyrase in whole body caused lethality in larvae stage. Overexpression of tankyranse in tissues resulted in tissue damage. The TLTNEL analysis revealed that tankyrase can induce cell apoptosis in wing imagnal discs and this phenomenon can be rescued by co-expression of the Baculoviru protein p35. Nevertheless, in adult eye and wing, co-expression of p35with tankyrase only slightly blocked the defects, indicating that overexpression of tankyrase may produce other effects besides cell apoptosis. We further showed that the ankyrin-repeats domain, but not the PARP domain of tankyrase, played a unique role in fly development.更多还原