学科：植物学. 植物分子生物学,出版时间：2009,导师：巩志忠指导,学位授予单位：中国农业大学,论文作者：张敏著,副题名：,学科专业：,关键词：,馆藏号：,
中文
蛋白N-糖基化过程在真核生物中是非常保守的，正确的蛋白N-糖基化是保证糖蛋白正确折叠，转运以及定位到功能区域所必须的。蛋白的N-糖基化过程起始于N-连接的寡糖链的合成，这个过程在生物界中具有很高的保守性。 本文筛选得到的突变体lew3(leaf wilting 3)中，LEW3基因的突变影响了拟南芥糖蛋白的N-糖基化。LEW3编码的蛋白与酵母中的ALG11蛋白具有较高的同源性。在酵母中，ALG11具有α-1，2-甘露糖糖基转移酶活性，参与N-连接的寡糖链Man₅GlcNAc₂的合成，催化该寡糖链的组装过程中最后一个甘露糖残基的加入，此后连接在磷酸多萜醇载体上的Man₅GlcNAc₂寡糖链就从内质网膜的细胞质一侧转运到了内质网膜腔内侧，进一步进行糖链的合成和修饰。在突变体中超表达ZEW3的cDNA或者在突变体中表达ZEW3的基因组全序列，能够回复突变体叶片萎蔫等表型，确定了突变体lew3的表型确实是由于LEW3基因突变引起的。LEW3蛋白同酵母中的ALG11蛋白一样也定位在内质网上，在拟南芥的各个组织器官中都有表达。质谱结果检测显示，突变体lew3中Man₅GlcNAe₂寡糖链合成时，1-3臂的延伸受到了破坏，Man₃GlcNAc₂,和Man₄GlcNAc₂,结构的寡糖链不能有效的被利用，在内质网膜的细胞质一侧积累。这种结构的寡糖链以较低的效率转运到内质网腔内，被寡糖基转移酶OST(oligosaccharyltransferase)识别，连接到初始合成的多肽链上，继续被修饰形成复杂的糖链。此外，我们通过检测带有高甘露糖糖链修饰的PDI(Protein Disulfide Isomerase)蛋白在lew3突变体中的N-糖基化水平，证实了这种低效率的蛋白N-糖基化过程的存在，即突变体lew3蛋白N-糖基化水平低于野生型。 突变体lew3表现山发育不止常，育性低的表型，纤维素合半纤维素合成也受到了影响进而导致细胞壁萎陷。突变体lew3的细胞壁中甘露糖单糖含量与野生型没有区别，说明作为甘露糖基转移酶的LEW3并不是直接参与细胞壁的合成，推测突变体lew3中细胞壁萎陷表型是由于破坏了细胞壁合成相关蛋白的糖基化而导致的。突变体lew3在渗透胁迫和ABA处理后也表现出敏感。由于突变体lew3中糖蛋白N-糖基化的缺失，在渗透胁迫，旱胁迫以及ABA处理时，UPR途径中的marker基因BiP的表达量明显高于野生型中的表达量，说明内质网胁迫响应的UPR信号传导途径被强烈激活。这些结果都说明糖蛋白的N-糖基化过程在植物的生长发育以及逆境响应中都具有很重要的作用。 关键词：N-糖基化，α-1，2-甘露糖糖基转移酶，细胞壁，UPR信号传导途径，逆境胁迫
英文
N-linked glycosylation is an essential protein modification that helps protein folding, trafficking, and translocation in eukaryotic systems. The initial process for N-linked glycosylation shares a common pathway with the assembly of a dolichol-linked core oligosaccharide. Here we characterized a new Arabidopsis mutant lew3 (leaf wilting 3); lew3 has a defect in an alpha-1,2-mannosyltransferase, a homologue of ALG11 in yeast, that transfers mannose to the dolichol-linked core oligosaccharide in the last step on the cytosolic face of the endoplasmic reticulum in N-glycan precursor synthesis. We confirmed the mutation by over-expression of the LEW3 cDNA and by genomic complementation of the lew3 mutant. LEW3 is localized to the ER membrane and expressed throughout the plant. The lew3 mutant contains low levels of aberrant Man₃GlcNAc₂ and Man₄GlcNAc₂, has only low levels of normal high mannose-type structures, and has increased levels of complex-type glycans. Extension of the 1-3 arm of the glycan with α-1,2-linked mannoses residues is partly impaired. Apparently, the truncated N-glycans are nevertheless flipped over the membrane and transferred to protein by the OST complex, resulting in proteins with abnormal N-glycans. In addition, the efficiency of this transfer is affected as evidenced by the underglycosylation of PDI. The lew3 mutant showed abnormal developmental phenotypes, reduced fertility, impaired cellulose synthesis, abnormal primary cell walls, and xylem collapse due to disturbance of the secondary cell walls. Mannose content in cell walls did not differ in the wild type and lew3, indicating that LEW3, a inannosyltransferase, did not directly add mannose to the cell walls. It is therefore likely that other proteins involved in cell wall biosynthesis are impaired by the lew3 mutation. lew3 mutants were more sensitive to osmotic stress and abscisic acid (ABA) treatment, and transpired less water than the wild type. The protein N-glycosylations were reduced and the unfolded protein response was more activated by osmotic stress and ABA treatment in the lew3 mutant than in the wild type. The expression of BiP in lew3, one of the UPR pathway maker genes, was more induced than in the wild type under abiotic stress, including drought as well as osmotic stress and ABA treatment. These results demonstrate that protein N-glycosylation plays crucial roles in plant development and response to abiotic stresses. Key words: N-glycosylation, alpha-1,2-mannosyltransferase, cell wall, unfolded protein response, abiotic stress
