【作者】 汪道涌；

【导师】 谢维；

【作者基本信息】 东南大学 ， 遗传学， 2006， 博士

【摘要】 我们从野生型黑腹果蝇胚胎cDNA文库中筛选并克隆得到ecp全长cDNA序列。生物信息学分析表明:ECP在进化过程中是十分保守的,从果蝇、斑马鱼到哺乳动物中均有其同源蛋白存在。从结构方面来看,ECP在其氨基端带有一个亮氨酸拉链结构基序,羧基端带有一个eIF5C结构域。这一结构域是eIF5与eIF2β的结合位点,因而ECP可能与eIF2β相互作用进而参与翻译过程。RNA原位杂交及胚胎抗体染色的结果都表明:在果蝇胚胎中,ecp无论在RNA水平上,还是在蛋白水平上都呈现广泛表达和分布,没有明显的组织特异性。Western Blot结果表明,ECP在果蝇的胚胎、幼虫、蛹及成虫中都有持续的高表达。这些结果提示ecp可能是一个管家基因。GFP标记分析及S2细胞免疫荧光结果显示,ECP主要分布在细胞质中,推测其可能通过与eIF2β相互作用而参与翻译过程。Western Blot分析发现, ECP蛋白在果蝇中存在两种迁移率不同的形式,ECP-S和ECP-F。从这两种形式在果蝇不同发育阶段所呈现的切换规律,即:胚胎、蛹和成虫都主要表达电泳迁移率较慢的形式(ECP-S),伴随着果蝇胚胎孵化成幼虫时即开始表达迁移率较快的形式(ECP-F),并持续到三龄幼虫开始蛹化时又逐渐被ECP-S替代,这些结果表明ECP-F可能与幼虫的生长发育有关。通过免疫共沉淀分析发现多种核糖体大小亚基蛋白能被抗ECP单抗所沉淀。与此同时,蔗糖密度梯度离心的结果也表明:有部分ECP能与40S、60S核糖体亚基结合。通过酵母双杂交,我们找到了两个可能与ECP相互作用的蛋白质, eIF2β和RPL5。这些结果表明ECP可能是一种核糖体相关蛋白,又因其可以和翻译起始因子eIF2β相互作用,因而ECP可能在翻译起始过程中起某种作用。S35-Methionine掺入实验表明,通过RNAi抑制S2细胞中ECP的表达后,蛋白质合成速率下降约45%。另一方面,在ecp的第一个内含子带有P因子插入突变的果蝇l(3)03022表现为短刚毛,生长发育迟缓,翅膀变小,隐性致死等典型的minute表型。这些结果进一步支持ECP可能参与蛋白质生物合成的推论。更多还原

【Abstract】 The cDNA [GenBank accession No.AF325529] of ecp was cloned from Drosophila embryonic brain cDNA library. Little is known about the function of ecp till Josh Dubnau reported that P-element insertion mutant line C0150 of Drosophila defected in long term memory which indicate it may be involved in learning and memory. ecp mRNA is 1572 bp in length and contains a complete ORF encoding a protein of 422 amino acids. Homologs of Ecp exist in various organisms from Danio rerio, xenopus laevis to mammalians. Prediction by using PROSITE shows that Ecp contains an N-terminal leucine zipper motif (amino acids 39-60) and a C-terminal eIF5C domain (amino acids 327-406), which was responsible for the binding of eIF5 with the K domain of eIF2β. The high similarity of the eIF5C domain between ECP and eIF5 suggests ECP might also interact with eIF2β.In order to analyze function of ECP, monoclonal and polyclonal antibodies against ECP were generated, and both could specifically detect recombinant ECP from bacterial and endogenous ECP. The results of Drosophila embryo immunostaining using these antibodies show that ecp expressed ubiquitously in Drosophila embryos, which was consistent with that of embryo whole mount RNA in situ hybridization. Western blotting results also indicated that ecp expressed continuously throughout Drosophila development from the embryo to adult. Both GFP tagged ECP in cell line HEK293 and endogenous ECP in Drosophila S2 cells and embryos were predominantly distributed in the cytoplasm. These results are consistent with the predicted function of ECP to be involved in protein synthesis through interaction with eIF2β.Two isoforms of ECP could be detected in Drosophila by Western Blotting. In embryo, pupa and adult stage, the slow mobility isoform (ECP-S) is the predominant one while the fast mobility isoform (ECP-F) is the main one expressed in the first and second instar larva. Moreover, ECP-F substitutes the slow one in embryos immediately after the hatching of the eggs. On the other hand, ECP-F gradually shifts back to ECP-S when the third instar larvae starts to form pupae. The periodical shifts of the 2 isoforms during Drosophila development indicate that ecp might be related to the larva growth.The results from mass spectrum show that many ribosomal proteins including Drosophila RPL5 exist in the immunoprecipitated complex formed by monoclonal antibody against ECP. In addition, Ecp cosediments with both 40S and 60S ribosomal subunits on sucrose gradient. These results indicate that Ecp might be a ribosome-associated protein. eIF2βand RPL5 were found to interact with ECP in a Y2h screening. The interaction of ECP and RPL5 was confirmed by GST pulldown and coIP. Moreover, it was showed that the fragment (1–150) which contained the predicted leucine zipper motif was sufficient for its binding with RPL5.The above results support ecp might play some role in protein synthesis. Actually, incorporation of S35-Methionine decreased by 45% compared to control after knockdown of ECP by RNAi in S2 cells. Besides, typical minute phenotypes of short bristles, slow development and更多还原