【作者】 王霞；

【导师】 袁明；

【作者基本信息】 中国农业大学 ， 植物学， 2005， 博士

【摘要】 微管在细胞分裂、分化,细胞形态决定及胞内物质运输等生理活动中起重要作用。植物有不同于动物的微管列阵,如周质微管列阵、早前期带和成膜体。在植物细胞的分裂、生长和发育过程中,微管列阵会依次转换。微管列阵的转换需要微管结合蛋白的参与。植物微管结合蛋白的研究是目前植物细胞骨架研究的前沿领域。 MAP1B是动物神经元细胞中的微管结合蛋白,用它的微管结合域的特征序列与拟南芥基因组比对,发现了一个功能未知的基因(At5g44610),其开放阅读框编码168个氨基酸,分子量为18.5 kDa,有8个类似MAP1B微管结合域的VVEKKN/EE不完全特征重复序列,因此推测其为一个微管结合蛋白。 我们通过克隆,原核表达,并纯化了该蛋白。通过共沉淀实验证明了它与微管结合。用绿色荧光染料Alex 488标记该蛋白,与红色荧光的罗丹明标记的微管蛋白在微管聚合缓冲液中共同孵育,结果显示它与体外聚合的微管共定位。证明这是一个新的微管结合蛋白,因此将它命名为AtMAP18。 激光共聚焦显微镜的观察结果表明AtMAP18虽然结合微管但是没有使微管形成微管束的效应。利用体外微管聚合系统的实验结果表明,AtMAP18对微管蛋白的聚合有影响,这种影响依赖于AtMAP18的浓度。低浓度的AtMAP18促进微管聚合,而高浓度的AtMAP18抑制微管聚合。但是,EDC交联实验表明AtMAP18不与微管蛋白单体结合。 为了研究AtMAP18的基因表达模式,构建了AtMAP18启动子-GUS质粒,并转化拟南芥植株。GUS活性检测结果显示:AtMAP18主要在根、花、果荚、子叶、下胚轴、表皮毛等器官和组织中表达。以原核表达的AtMAP18为抗原制备了抗AtMAP18抗体。Western blot实验也同样证明AtMAP18蛋白主要存在于拟南芥根和花等器官的组织中。 为了研究AtMAP18的生理功能,构建相应的质粒转化拟南芥,筛选获得AtMAP18过表达和RNAi转基因植株的纯合体株系。在RNAi转基因植株未发现明显的表型,但是在AtMAP18过表达的纯合体株系中,细胞发生了广泛的与细胞异向性生长相关的细胞形态异常:叶和子叶的铺板细胞、根的表皮细胞和根毛、下胚轴表皮细胞和皮层细胞与野生型同一部位的细胞相比,细胞形态都发生了改变;铺板细胞突起数明显减少,细胞边缘光滑;根和下胚轴表皮细胞肿胀;花粉形态异常,异常花粉没有内含物,不能正常萌发。 进一步对AtMAP18对细胞中微管骨架的作用进行了研究。免疫荧光双标记的观察结果表明拟南芥根的细胞中AtMAP18与细胞周质微管共定位。将AtMAP18连上红色荧光蛋白转化GFP-tubulin拟南芥,观察结果显示AtMAP18在子叶和下胚轴细胞中也与细胞周质微管共定位。对AtMAP18过表达植株中子叶铺板细胞微管和下胚轴表皮细胞微管观察显示,微管的排列即微管列阵与野生型的相比发生改变。AtMAP18过量表达使微管列阵的动态转换被抑制,导致细胞形态的改变。 本学位论文首先证明了AtMAP18是一个新发现的植物细胞微管结合蛋白,并对AtMAP18的生理功能进行了分析,证明了AtMAP18在对细胞微管骨架的组织和调控方面有作用,并进一步在植物细胞生长的调控和形态建成方面有重要的功能。更多还原

【Abstract】 Microtubules (MTs) play a critical role in cell expansion and division, cell morphology and intracellular transport. MT arrays in plant cells are different from that in animal cells. For example, the cortical MT array, MT preprophase band and phragmoplast are unique to plant cells, MT arrays transit in turn from one array to the another during the cell cycle. Microtubule-associated proteins (MAPs) are involved in the transformation of MT arrays. Study of MAPs is now the cutting-edge research in the field of the cytoskeleton researches.MAP1B is a known MAPs from mammal nerve cells. Blast of the gene data base of Arabidopsis gene bank, with the imperfect repetitive motif in the MT-binding domain of MAP1B, resulted in finding a function-unknown gene (At5g44610). Its open reading frame encodes 168 amino acid and has a predicted protein molecular weight of 18.5 kDa. The protein sequence contains 8 VVEKKN/EE, which assembles the repetitive motif in MAP1B.By gene cloning and prokaryotic expression, we purified the recombinant protein. Co-sedimentation assay showed that the recombinant protein bound to MTs, Furthermore, by labeling the recombinant protein with Aleax 488 and incubated with rhodamine-conjugated MTs, the observation of confocal microscopy also demonstrated that the green fluorescent recombinant protein was co-localized with red fluorescent MTs, Therefore, we concluded that it is a novel MAPs form Arabidopsis, and thus termed it as AtMAP18.Confocal microscopy observation showed that AtMAP18 had no effect on MT bundling. The data form experiments with in vitro MT polymerization system showed that AtMAPl 8 affected tubulin polymerization differently according to its concentrations. AtMAPl 8 enhanced tubulin polymerization at low concentrations, while inhibited tubulin polymerization at high concentrations. Nevertheless, EDC cross-link assay showed that AtMAP18 did not bind to tubulin dimmer.By transformation of Arabidopsis with AtMAPl 8 promoter tagged with GUS, the analysis of GUS activity showed AtMAP18 was expressed in root, flower, fruit, cotyledon, hypocotyls and trichmoe. By raised a rabbit anti-AtMAP18 polyclonal antibody, the western blot assay also showed that AtMAPl 8 mainly existed in root and flower tissues.In order to analyze the function of AtMAP18, AtMAP18 overexpression and RNAi plasmids were reconstructed, and transformed into Arabidopsis. Although the RNAi transgenic plants showed no obvious phenotypes, the the transgenic plants of AtMAPl 8 overexpression exhibited general abnormities with cells related to directional expansion, such as pavement cells of leaf and cotyledon, epidermal cells of roots, root hairs, and epidermal cells and cortex cells of hypocotyls. The pollen of AtMAPl 8 overexpression was aberrant and aborted, too.Furthermore, double-staining of anti-ATMAP18 antibody and anti-tubublin showed that AtMAPl 8 was co-localized with cortical MTs in root cells. Observation of transgenic RFP-AtMAP18 Arabidopsis with GFP-tubulin background also showed that AtMAP18 was co-localized with corticalMTs in cells of cotyledon and hypocotyls. In addition, The MT array in cotyledon pavement cells and hypocotyls epidermal cells of AtMAP18 overexpression was altered, and the transition of MT array was hindered by the overexpression of AtMAP18, as a result, leading to abnormalities of cell growth.In present study, we demonstrated that AtMAP18 is a novel microtubule associated protein form Arabidopsis, and analyzed it functions. Our investigation indicated that AtMAP18 has functions in MT organization and regulation of MT dynamics, thus, may have important roles in regulation of cell growth and morphology.更多还原