【作者】 万磊；

【导师】 朱英国；

【作者基本信息】 武汉大学 ， 遗传学， 2014， 博士

【摘要】 微管作为细胞骨架的一种,对于细胞的形态建成、细胞内的物质运输、细胞分裂、一些特殊结构的组成(如鞭毛、纤毛)和植物细胞壁的合成等重要的生命活动是必不可少的。微管具有高度动态的特性,原因之一是受到微管相关蛋白的调控。Katanin是一类可以切割微管的微管相关蛋白,由两个亚基组成,P60和P80,P60具有ATPase的活性和微管切割的活性；P80不具有微管切割的活性,其主要负责将P60导向中心体和调控P60的微管切割活性。P60本身参与了许多重要的生命活动,而P80亦在线虫减数分裂、人神经细胞发育、小鼠雄配子发生、低等生物的cytokinesis以及鞭毛发生中扮演重要角色。在拟南芥细胞中,KATANIN P60参与了细胞各向异性的延伸和根尖细胞的有丝分裂,并且作为生长素信号传导末端的效应子参与皮层微管排列。但是高等植物中P80的功能仍然不甚明了。在本研究中,我们利用水稻中的KATANIN P60(KTN60)作为诱饵蛋白淘洗水稻幼穗的细菌双杂交cDNA文库,获得了OsKTN80b,通过BLAST水稻蛋白数据库获得了另外两个同源蛋白(OsKTN80a和OsKTN80c),这3个同源蛋白均与人和小鼠的KATANIN P80具有极高的序列相似性。通过酵母双杂交验证、结构域分析和进化关联分析,证实这3个基因即为katanin p80在水稻中的同源基因。我们在水稻中过表达OsKTN80a (KTN80a-OX),导致水稻幼苗根的生长受阻。分析发现是由两个原因造成的：一是KTN80a-OX根尖细胞的延长障碍,主要是由于KTN80a过表达后导致伸长区细胞皮层微管的排列异常；二是KTN80a-OX根尖细胞的分裂滞留于有丝分裂后的细胞质分裂(cytokinesis)时期,可能是由于遗传物质的分离异常以及成膜体微管的形态和功能异常。上述两个原因可以解释为KTN80a抑制了KTN60的微管切割活性,从而导致微管的动态受到破坏。证据如下：1)KTN80a和KTN60相互作用；2)KTN80a-OX的根尖细胞变短和伸长区细胞皮层微管变得紊乱的表型均和KTN60的突变体(fra2)极为相似。3)当KTN80a过表达后,和野生型相比,其根尖的生长和伸长区的皮层微管更加耐受微管特异性的去稳定药物oryzalin的处理,表明KTN80a促使微管更稳定。我们讨论了KTN80a参与细胞延伸和细胞分裂的分子机制。从上述KTN80a的作用机制可以看出,控制KATANIN的活性对于水稻根尖细胞的伸长和细胞的分裂是必须的。更多还原

【Abstract】 As a kind of cytoskeletons, microtubules are essential for cell morphogenesis, the intracellular transportation, cell division, composition of some novel cellular apparatus such as flagellum and cilia and the plant cell wall biosynthesis and so on. Microtubules are highly dynamic and one of the reasons is that it was regulated by microtubule associated proteins (MAPs). Katanin, a kind of MAP involved in microtubule severing, is composed of two subunits, one is P60which has ATPase and microtubule severing activity and the other is P80which has no microtubule severing activity but plays roles in targeting P60to the centrosome and regulating P60’s microtubule severing activity. P60itself are involved in many significant life activities while P80also functions essentially in C. elegans meiosis, human neuronal development, the male gamete genesis in mouse and in cytokinesis and flagella biogenesis in lower order species. KATANIN P60is necessary for the anisotropic cell expansion and cell division in root tip and also acts as the effector in cortical microtubule orientation in auxin signaling pathway in Arabidopsis cell. When it comes to the other KATANIN subunit P80in higher plants, its function remain largely unknown.In this study, we used rice KATANIN P60(KTN60) as the bait to screen the bacteria two hybrid cDNA library of rice immature panicle and obtained a KTN80b. Through BLAST the protein database of Oryza sativa using this protein, we found the other two homologous proteins (KTN80a and KTN80c) in rice. There are high sequence similarity between the3homologous proteins mentioned above and the KATANIN P80in human and mouse. Later the3homologs are demonstrated as katanin p80orthologs in rice after yeast two hybrid assay, the domain analysis and the phylogenic analysis. The overexpression of OsKTN80a in rice (KTN80a-OX) caused the delayed primary root growth of rice seedling and two reasons results in it:the first is that cell elongation dysfunction in root tip in KTN80a-OX which is thought as the disordered cortical microtubule arrangement in elongation zone after Ktn80a overexpression; the second is that cell division are stalled at cytokinesis which are mainly caused by the incomplete separation of genetic materials into the daughter cells and the abnormal phragmoplast morphology and function. The above two reasons could be explained that the KTN60microtubule severing activity are repressed by KTN80a and the event caused the broken microtubule dynamics. There are3evidence supporting the idea:1) KTN80a interacts with KTN60;2) The phenotype of the shorter cell in root tip and the disordered cortical microtubule array in elongation zone when KTN80a is overexpressed could perfectly mimic the case in KTN60mutant (fra2);3) Compared with the wild type, the root tip growth and cortical microtubules in elongation zone are more resistant to treatment of oryzalin, a kind of microtubule-special destabilizing drug, which implies that KTN80a stabilizes microtubules.We discuss the molecular mechanism of the role of KTN80a plays in cell expansion and in cell division. From the above mechanism of KTN80a function in, we can get the conclusion that controlling the KATANIN activity is essential for cell expansion and cell division in rice root tip.更多还原