【作者】 陈勇；

【导师】 朱睦元； 王君晖；

【作者基本信息】 浙江大学 ， 遗传学， 2012， 博士

【摘要】 Sirtuins (Sir2)是从细菌到人类都高度保守的NAD+依赖的去乙酰化酶／ADP-核糖基转移酶。Sir2最初发现于芽殖酵母并能延长其寿命。哺乳动物有七个Sirtuins (SIRT1-7)成员,都含有一个保守的NAD+结合和催化域,称作Sirtuin核心域；这些Sirtuins还有不同的N端和C端结构,定位于细胞的不同部位,并有不同的底物和生物学功能。目前,哺乳动物Sirtuins已经被越来越多的研究认为是从能量代谢、应激反应到肿瘤发生以及衰老等各种细胞过程的关键调节因子。人hSIRT4定位于线粒体基质,NAD+依赖的ADP-核糖基转移酶是它唯一的活性。有证据表明,hSIRT4能ADP核糖基化谷氨酸脱氢酶(GDH),在胰腺β细胞中对热量限制(CR)做出反应时能控制胰岛素分泌。人hSIRT6是一种核定位的染色质结合蛋白。它不仅具有ADP-核糖基转移酶活性,还有去乙酰化酶活性,并优先作用于组蛋白H3K9和H3K56. hSIRT6在DNA修复、端粒功能、基因组稳定和细胞衰老中起着非常重要的作用。hSIRT6主要是通过调节C端结合蛋白(CtBP). DNA蛋白激酶(DNA-PK)和聚腺嘌呤二核苷酸核糖聚合酶1(PARP1)等参与DNA双链断裂修复。拟南芥有两个SIRT基因家族基因,分别命名为AtSIRT1(At5g09230)和AtSIRT2(At5g55760)。过去的十年里,哺乳动物Sirtuins的研究已经取得了显著的进步,但是对植物SIRT基因研究却很少。本文以拟南芥的两个SIRT基因家族基因(AtSIRTl和AtSIRT2)为研究对象,分析它们表达部位和基因产物的亚细胞定位,并对它们的生物学功能进行了初步研究,主要结果以下：1从GenBank获得拟南芥和其他生物SIRT蛋白的相关信息,进行了同源性比较和进化树构建。分析表明,拟南芥AtSIRT1与哺乳动物SIRT4蛋白高度同源,而拟南芥AtSIRT2蛋白与哺乳动物SIRT6蛋白高度同源。2构建了AtSIRT-EGFP融合蛋白表达载体来分析AtSIRT蛋白的亚细胞定位。结果表明,AtSIRT1蛋白定位于线粒体上,而AtSIRT2则定位于细胞核。同时,构建了AtSIRT:GUS启动子报告株系。GUS的组织化学染色结果表明,AtSIRTl基因主要在拟南芥的根、茎、叶组织中表达,尤其在分生组织如叶芽和根尖表达量较高,同时发现光照条件下其表达量比黑暗条件下有明显增加。3我们购买和鉴定了针AtSIR噻因的T-DNA突变体和TILLING突变体,通过对突变体的初步研究发现,AtSIRT1突变可引起幼苗的子叶变黄和早衰。用FLAG蛋白标签过量表AtSIRT1四种转录本,表型观察时发现AtSIRT1.7-FLAG株系的种子萌发迟缓,萌发率低,幼苗个体偏小,在高糖培养基中,有停止生长、甚至致死的表型；而AtSIRT1.3-FLAG株系的种子萌发速度快,幼苗个体大,在高糖培养基中生长旺盛。SIRT蛋白的抑制剂Sirtinol不能表型模写AtSIRT1和AtSIRT2突变体,说明Sirtino1在拟南芥中的作用机制不同于其他生物。4双分子荧光互补(bimolecular fluorescence complementation, BiFC)是指两个不发光的荧光蛋白互补片段在与其融合的蛋白质的相互作用驱动下重新组装形成荧光复合物,恢复荧光特性。为了弄清AtSIRT2的底物是什么,我们构建了DNA-PK、CtIP、PARP1等多个可能与SIRT互作的蛋白的BiFC载体。结果表明,ATSIRT2与CtIP, DNA-PK没有相互作用,但与PARP1有相互作用。5DNA双链断裂(DSBs)是细胞最严重的损伤形式之一。高等动植物中主要通过同源重组(HR)和非同源末端连接(NHEJ)途径进行DNA双链断裂修复。为了研究拟南芥在DNA双链断裂修复期间AtSIRT2蛋白的工作模式,我们构建了EJ2-GFP、EJ5-GFP、HDR-GFP等一系列DSB修复方式报告载体以作进一步深入研究。我们发现AtSIRT2在两种主要的DNA双链断裂修复通路即HDR和NHEJ中有明显的作用。纵上所述,我们的初步研究表明：拟南芥AtSIRT1与人的同源蛋白hSIRT4相同,定位于线粒体,它的主要功能是通过调节线粒体活性参与子叶和叶片衰老的调节；AtSIRT2与人的同源蛋白hSIRT6相同,定位于细胞核,可能通过DSB修复途径调节细胞寿命。更多还原

【Abstract】 Sirtuins (Sir2) are NAD-dependent deacetylases and/or ADP-ribosyltransferases that are highly conserved from bacteria to human. Sir2was originally shown to extend lifespan in budding yeast. In mammals, there are seven sirtuins (SIRT1-7). All mammalian sirtuins contain a conserved NAD-binding and catalytic domain, termed as the sirtuin core domain, but differ in their N and C-terminal domains. They have different specific substrates and biological functions, and are found in various cell compartments. Sirtuins have been increasingly recognized as crucial regulators for a variety of cellular processes, ranging from energy metabolism and stress response to tumorigenesis and aging.hSIRT4is found in the mitochondrial matrix. The only reported activity of hSIRT4is the NAD+-dependant ADP-ribosyltransferase activity. It has been shown that hSIRT4can ADP-ribosylate glutamate dehydrogenase (GDH) and control insulin secretion in pancreatic β-cells in response to CR. hSIRT6is a nuclear, chromatin-bound protein. hSIRT6is shown to have ADP-ribosyltransferase and deacetylase activity, preferentially acting on histone H3K9and H3K56. hSIRT6is playing an important role in DNA repair, telomere function, genomic stability, and cellular senescence. SIRT6is involved in DNA double-strand break repair by regulating C-terminal binding protein (CtBP), DNA protein kinase and poly[adenosine diphosphate (ADP)-ribose] polymerase1(PARP1).Arabidopsis thaliana contain two SIRT gene families, named AtSIRTl (At5g09230) and AtSIRT2(At5g55760). However, little research on SIRT in plant has been conducted. The present study focuses on the AtSIRTl and AtSIRT2of Arabidopsis. Here, we aim to reveal their expression patterns, subcellular locations and function in plants. The results are summarized as follows:1Phylogenetic trees of AtSIRTl, AtSIRT2and the homologous proteins of other model organisms is constructed by means of the MEGA method. The results show that AtSIRTl is closest homologs with mammalian SIRT4, and AtSIRT2is closest homologs with mammalian SIRT6. 2The AtSIRT-EGFP fusions are constructed to analyze the subcellular localization of AtSIRT proteins. The results suggest that AtSIRTl was targeted to the mitochondrial, and AtSIRT2was located in nucleolus. Meanwhile, PAtSIRT1:GUS report lines were generated. The histochemical localization of GUS staining indicated that AtSIRT1is expressed in roots, stems and leaves, especially in the leaf and root tips. Investigation of accumulation of AtSIRTl transcripts during a photoperiod by real-time PCR indicated that AtSIRT1gene was a diurnal-regulated gene.3We characterized the Arabidopsis T-DNA insertional mutant lines of AtSIRT. We found that mutation in AtSIRT1leaded to early senescence of cotyledons. Four transcripts of AtSIRTl were overexpressed harbouring the FLAG tag. Transgenic plants overexpressing the AtSIRT1.7-FLAG displayed low seed germination rate and delayed seedling development. When they grow on B5medium supplemented with high levels of sucrose, they fail to develop green expanded cotyledons and true leaves, and even die. Transgenic plants overexpressing AtSIRT1.3-FLAG and the T-DNA mutants exhibited vigorous growth both on B5and high levels of sucrose media. Sirtinol, a inhibitor of SIRT which did not cause the same phenotype of the mutation of AtSIRTl indicated that the mechanism of Sirtinol in Arabidopsis was different from other organism.4Bimolecular fluorescence complementation (BiFC) means two non-fluorescent complementary fragments of fluorescent protein can reassemble to form fluorescent complex and restore fluorescence when they are fused to two proteins that interact with each other. To figure out which protein AtSIRT2does to interact with, we construct a series vectors mat associate with mammalian hSIRT6, including DNA-PK-cGFP, CtIP-cGFP and PARP1-cGFP. Protein-protein interaction between AtSIRT2and substrates were studied in vivo. The results suggest that PARP1was the substrate.5The DNA double strand breaks (DSBs) is one o f the most serious form of DNA damage. Nonhomologous end joining (NHEJ) and homologous recombnation (HR) are the two major pathway to repair DNA double strand break damages in both higher plants and animals. To check the role of AtSIRT2during DSB repair of Arabidopsis, we constructed a series reporter vectors, including EJ2-GFP, EJ5-GFP, HDR-GFP. The results show that AtSIRT2is playing an important role in DNA repair.AtSIRTl was located in mitochondria as hSIRT4of human, and maybe take part in respiration and electron transformation chain to regulate cotyledon and leaf senescence. AtSIRT2was located in nucleolus as hSIRT6of human, maybe play important role in DSB repair.更多还原