【作者】 樊玉杰；

【导师】 赵云龙；

【作者基本信息】 华东师范大学 ， 动物学， 2010， 博士

【摘要】 Darkener of apricot（Doa）是果蝇基因组中编码LAMMER激酶的唯一位点。因其在哺乳动物存在4个同源蛋白（CLK1-4）,LAMMER激酶也被称为CLK激酶。关于LAMMER激酶的功能了解最清楚的是它可能磷酸化SR和类SR蛋白,通过调控果蝇体细胞性别决定信号通路中关键因子doublesex的选择性剪接,从而控制果蝇的性别分化。本研究确定了eEF1Bγ和SRm160为DOA激酶的磷酸化底物,并进一步研究这两种蛋白与DOA蛋白激酶的相互作用及在其果蝇发育中的作用。扩充了LAMMER激酶参与调控的生理功能。通过酵母双杂交,发现果蝇eEF1Bγ（翻译延长因子1Bγ）与DOA激酶存在相互作用。关于eEF1Bγ基因的研究主要集中在低等生物,在果蝇及更高等的真核生物中,未见相关研究。果蝇eEF1Bγ蛋白C端的丝氨酸²⁹⁴极为保守,可能是LAMMER激酶的磷酸化位点。进一步研究发现在体外和体内实验中eEF1Bγ都能够被LAMMER激酶磷酸化,并通过点突变的方法确定了该磷酸化位点。本研究通过免疫荧光组化的方法确定该蛋白在果蝇胚胎发育中的表达及分布情况,并在蛹期的细胞核提取物中检测到该蛋白的存在。该位点的无义突变造成果蝇隐性致死,说明eEF1Bγ蛋白在果蝇中担负重要的生理功能。为了进一步揭示eEF1Bγ中DOA磷酸化的作用,我们构建了野生型载体和磷酸化位点被突变的突变载体,结果证实该磷酸化位点的缺失降低了蛋白的活性,因为诱导转化的野生型基因位点表达可以逆转内在的纯合无义等位基因的致死性,而该磷酸化位点突变后,诱导表达的蛋白只能在一定程度上逆转致死性。在mRNA前体组成型和选择性拼接过程中SRm160都是必不可少的作用因子,另外SRm160还参与mRNA前体3’端加工。SRm160蛋白含有多个可能的LAMMER激酶的作用位点。本研究通过原位杂交和免疫组化的方法,研究了SRm160 mRNA和蛋白质在胚胎中的表达及分布。SRm160基因活性的丧失及高表达对果蝇都具致死性。另外器官特异性的诱导SRm160高表达与RNAi导致多种的器官的发育异常。高表达SRm160导致果蝇复眼结构严重异常和雄性生殖器结构异常或完全缺失雄性生殖器和肛门。而DOA位点的突变抑制高表达在复眼中导致的突变表型,但增强在生殖器中高表达导致的表型,该结果显示在不同器官中,不同的信号通路参与SRm160和DOA的相互作用。此外,Doa突变可以造成雌果蝇出现不同程度的雄性化特征,而SRm160无义突变基因增强Doa突变诱导的这种性别逆转表型。该结果表明SRm160可能也是DOA激酶的底物并参与果蝇体细胞性别决定的信号通路。我们的研究结果表明Doa和SRm160基因之前存在密切的遗传上的相互作用,这说明SRm160是一个DOA激酶的作用底物。为了确切阐明二者间的相互作用机制,还需要进一步通过生物化学和分子生物学的手段进行更深入的研究。更多还原

【Abstract】 The Darkener of apricot（Doa） is the unique locus encoding LAMMER kinase in Drosophila. LAMMER kinase is also known as CLK for its 4 paralogues（CLK1-4） in mammals.The best studied role of LAMMER kinases is that it phosphorylate the SR and SR-like proteins and subsequently regulate the alternative splicing events in somatic sex-determination pathway in Drosophila.In this study,we identified eEF1Bγand SRm160 as the substrates of DOA kinase,and characterize their roles in development of Drosophila,in order to shed more light on the roles of LAMMER kinase.Drosophila eEF1Bγwas isolated as a protein interacting with DOA kinase in a yeast two-hybrid screen.It contains a predicted and conserved phosphorylation site(Serine²⁹⁴) for LAMMER kinases in its C-terminal region.The locus encoding eEF1Bγin Drosophila or any other higher eukaryote has never been analyzed,eEF1Bγprotein is phosphorylated in vitro and as well in vivo by DOA,and the phosphorylation site identified via site-directed mutagensis.The protein expression pattern in embryos is investigated via immunocytochemistry and the existence of EF-1γis detected in pupal nuclear fractions.I generated and characterized several EF-1γmutant alleles via excision of a P-element provides,including a recessive lethal null allele.To define further the function of the phosphorylation of EF-1γby DOA,we generated wildtype and mutant constructs expressing a Serine-294-Alanine substitution.The absence of this phosphorylation site reduces the activity of the protein,since the expression of the mutant construct does not rescue the lethality caused by homozygoty of the null allele as well as that of the wildtype.SRm160 functions as a splicing co-activator,which is required in both constitutive and alternative splicing.It is also involved in pre-mRNA 3’-end processing.SRm160 contains several potential phosphorylation sites for LAMMER kinases.The transcript and protein expression pattern of SRm160 during embryogenesis were performed.Loss of SRm160 activity and its ubiquitous over-expression provoke lethality.Not surprisingly,over-expression and RNA interference for SRm160 with different drivers disturbs the development of multiple organs.RNA interference of SRm160 leads to slightly rough eyes and blistered wings,respectively.Overexpression SRm160 result in extremely rough eyes and abnormal genital structure or males lacking genitalia and analia, respectively.Interestingly,Doa mutations supress the overexpression phenotype in eye,but enhance the phenotype in genitals,indicating different singal pathways are involved in the interaction.In addition,as for the interaction between Doa and SRm160,SRm160 mutant alleles enhance the female-to-male sex transformation phenotype of Doa alleles,further indicating its involvement in somatic sex determination,and its probability as a DOA substrate.Our results to date indicate the strong genetic interactions between Doa and SRm160,suggesting the idea that SRm160 is a substrate of DOA kinase.However,more biochemical and molecular investigations need to be done to elucidate and confirm the mechanism of their interactions.更多还原