【作者】 王旭；

【导师】 傅永福；

【作者基本信息】 中国农业科学院 ， 生物化学和分子生物学， 2014， 博士

【摘要】 核孔复合物作为真核细胞中细胞质与细胞核之间物质交换的桥梁,在细胞生命活动中发挥着极其重要的作用。然而因研究手段和材料的限制,目前对于植物核孔复合物及其组分(即核孔蛋白)所参与的生物学过程及其分子机制的认识仍非常有限。本实验室前期利用生物信息学分析手段在拟南芥基因组中筛选到一批可能编码核孔蛋白的候选基因,通过大量筛选相应的T-DNA插入突变体,最终获得多个发育表型较为明显的潜在核孔蛋白基因突变体,为研究核孔蛋白在植物生长发育过程中的功能创造了条件。本论文中的Nup96基因便是其中一个。拟南芥Nup96蛋白与脊椎动物Nup96和酵母Nup145C蛋白同源,是Nup107-160亚复合体的一个组分,可以与脊椎动物SEC13在拟南芥中的同源蛋白互作。Nup96蛋白N端存在一个保守的“自酶解结构域”,研究发现该结构域在原核和植物细胞中均有活性,且自酶解作用的失活并不影响Nup96蛋白在植物中功能,这种自酶解活性的生物学意义尚有待进一步研究。Nup96基因突变会导致拟南芥提早开花,且其早花表型与光周期无关,对该基因参与开花调控机制的分子遗传学研究表明,该基因及蛋白在拟南芥体内呈组成型表达,在长日照条件下其早花表型可以被CONSTANS (CO)基因的突变所抑制,同时Nup96基因突变会导致植物的开花时间对环境低温不敏感；此外,Nup96突变还会影响开花抑制因子FLC染色质组蛋白H3Ac修饰水平,导致其表达量降低,促使拟南芥早花；在nup96突变体中miR156a,c的转录水平显著降低,推测Nup96可能通过影响miR156的转录影响miR156的累积,从而导致拟南芥早花。此外,Nup96的开花调控功能主要发生在维管组织和茎尖生长点。综上,拟南芥Nup96基因可以通过光周期、年龄、自主及环境温度等多条途径参与拟南芥开花调控。Nup96基因突变还会影响拟南芥生物钟系统的功能。通过叶片运动节律、基因表达及生物钟启动子驱动荧光素酶报告基因实验分析发现,在连续光照和连续黑暗下,nup96突变体的生物钟周期约为28小时,比野生型植株延长3小时左右,表明该基因在拟南芥生物钟周期调控中发挥重要作用。相位响应曲线显示,nup96突变会增强生物钟对光信号的敏感性,影响生物钟对光信号的门控作用(Gating)。Nup96还参与生物钟的温度补偿,nup96突变体呈现温度补偿过度表型,且低温可以恢复突变体的长周期表型。此外,nup96突变会影响几乎所有已知生物钟关键基因的表达模式,使其表达节律的周期延长。以上结果证明,Nup96参与拟南芥生物钟周期长度的调控,是一个全新的生物钟调控因子。更多还原

【Abstract】 Nuclear pore complex (NPC) embeds in the double membrane nuclear envelope (NE), the physical barrier separating the nucleus from cytosol, and serves as the sole channel for nucleocytoplasmic transport of macromolecules essential for cell survival (i.e. proteins and RNAs). Because of the limitations of research tools and materials, it was not until the last decade that the biological functions of these individual nucleoporins just began to be understood.Previously in our lab, a set of candidate nucleoporin genes in Arabidopsis genome has been identified using bioinformatical approaches. After screening the Arabidopsis mutants for those candidate genes, a few lines with discernible developmental phenotypes have been isolated for further analysis and nup96in this thesis is one of them. Nup96is a single copy gene in Arabidopsis genome and encodes a protein whose sequence shows strong similarity with vertebrates Nup96and yeast Nup145C. Nup96is one of the stable constituents of Nup107-160subcomplex in NPC, which interacts with Arabidopsis SEC13protein both in vitro and in vivo. A conserved peptidase domain is included in the N terminus of Arabidopsis Nup96, which remains autoproteolytic activities both in prokaryotic and plant cells. Failure to complete autoproteolysis would not affect the functions of Nup96in Arabidopsis, therefore, the biological function of this proteolysis still needs to be elucidated.Loss-of-function alleles of nup96exhibit a strong early-flowering phenotype regardless of photoperiod, which can be strongly suppressed by co mutation under long days. The flowering of nup96mutants is also non-responsive to different ambient temperatures. Moreover, our data shows that Nup96is required for FLC activation partially through a mechanism involving histone acetylation of FLC chromatin. Nup96mutations also reduce the expression level of miR156genes. In addition, expression of Nup96in both vascular tissue and shoot apical meristems could rescue the early-flowering phenotype of nup96mutants. Thus, we proposed that Nup96could negatively regulate flowering in Arabidopsis through multiple pathways.Mutation of Nup96also has a broad affect on circadian clock function in Arabidopsis. Given by the leaf movement assay and bioluminescence of clock gene promoter activity, we found that nup96lengthens the circadian period both in continuous light and dark, suggesting that Nup96plays an important role in determining the circadian period of circadian clock. Phase response curve shows that nup96mutants are more sensitive to light pulses in the daytime than wild type, indicating that nup96could negatively regulate clock sensitivity for gating light signals, In addition, nup96mutation also results in a temperature overcompensation phenotype and low temperatures completely rescue the long period phenotype of nup96plants. Moreover, nup96mutation also lengthens the circadian period of clock gene expressions. Taken together, we demonstrate that Nup96is a novel regulator in clock period determination.更多还原