【作者】 朱骏；

【导师】 武天龙；

【作者基本信息】 上海交通大学 ， 生物医学工程， 2010， 博士

【摘要】 植物的有性生殖不仅是植物繁殖的主要途径,也是植物进化及对环境适应的基础之一。花药是植物的雄性生殖器官,是花粉发育的场所。对花药及花粉发育的研究具有重大的理论意义;植物雄性不育是作物杂种优势利用的重要途径,因此对花药及花粉发育的深入研究又有重要的应用价值。在开花植物中,花药发育是一个非常复杂的过程,包括小孢子发生及小配子发生两个不同的阶段。在拟南芥小孢子发生过程中,雄蕊原基通过细胞分化和命运决定逐渐形成花药及花丝的基本结构和形态。小配子发生过程中,花粉逐渐成熟并经过两次不均等的有丝分裂形成多核的雄配子体。花粉发育依赖于双倍体孢子体细胞层尤其是绒毡层的支持。绒毡层由花药次级壁细胞分化而成,包围着小孢子,在小孢子释放,营养供给和花粉壁的形成等过程中至关重要。在此过程中,其自身启动细胞凋亡程序,当小孢子有丝分裂完成时,绒毡层最终行使花粉含油层合成的功能并完全降解。目前在拟南芥中,已分离到一些富含亮氨酸重复的受体蛋白激酶参与到绒毡层细胞命运决定过程,此外,一些转录因子也被报道参与调控绒毡层后期的发育与功能。但从花药发育复杂性来说,应该还有许多关键因子没有被发现,而且这些关键基因的相互关系的了解还比较少。基于这些原因,我们希望用正向遗传学的方法继续挖掘未知的花药发育关键基因并对其功能进行研究。在本研究中,我们通过EMS诱变的方法获得了雄性不育拟南芥突变体ms188,采用了图位克隆的策略将MS188基因定位于第5条染色体的分子标记MDA7与K24C1之间的95.8Kb区间内,测序发现MYB家族转录因子AtMYB103的R2R3区存在一个单碱基的终止突变。不同等位突变体之间的等位杂交实验以及遗传互补实验证明了该基因的突变导致了雄性不育表型。对野生型和突变体花药组织的半薄切片观察表明突变体ms188的绒毡层外壁持续不降解,暗示其分泌功能可能受到了影响,后期大部分小孢子降解导致花粉败育;苯胺蓝染色实验表明,突变体小孢子外围的胼胝质在花药发育第7期以后的降解异常,影响了四分体的分离;对残余花粉的外壁进行扫描和透射电镜观察后发现,ms188的花粉外壁结构完全不能形成。此外,半定量和定量PCR结果显示,胼胝质酶相关基因A6以及花粉外壁形成相关基因MS2在突变体中明显下调。以上结果表明AtMYB103基因在调控拟南芥绒毡层发育,胼胝质降解以及花粉外壁形成的过程中有重要作用。我们鉴定了该基因在花药中的时空表达谱,表明其在花药绒毡层和小孢子母细胞中有较为特异的表达,暗示AtMYB103参与了绒毡层发育及后期分泌功能的调控。进一步的透射电镜发现该基因的突变导致了绒毡层的发育异常,提前降解,导致其分泌功能紊乱;此外,突变体残余花粉的外壁缺失是由于绒毡层供给功能的丧失而非小孢子初生外壁所决定。为了进一步阐明AtMYB103在花药发育的调控机理,我们利用基因芯片的方法对野生型及ms188突变体的转录谱进行了分析。基因芯片表达分析表明在ms188突变体花苞中共有821个基因的表达有明显变化(其中728个下调,93个上调)。随后我们依据‘Electronic Fluorescent Pictograph’(e-fp) Browser数据库按照不同发育时期将这些差异表达基因分为花苞表达,晚期花药表达,花粉表达等9类。对部分差异表达基因的定量RT-PCR分析表明该芯片数据较为可靠。数据的功能分类显示该基因的突变使花药发育中的一些重要的代谢和信号途径受到了严重影响:信号转导途径中许多花药发育后期表达的激酶的转录受到抑制,暗示AtMYB103可能参与了花药发育后期的信号途径;细胞壁代谢途径中多个与降解相关的果胶酶以及葡聚糖酶严重下调,这些蛋白可能参与到绒毡层壁及胼胝质壁的降解过程;大量脂肪酸合成与转运相关的蛋白也受到抑制,这可能导致了突变体中花粉外壁的缺失。然而,TUNEL实验表明,绒毡层的细胞程序性死亡过程与AtMYB103的调控并没有直接的关系。此外,我们利用原位杂交的手段鉴定了在基因芯片中差异表达的,对花药发育有潜在调控作用的转录因子。这些结果表明在AtMYB103作为一个关键基因,通过调控多个关键途径来控制拟南芥花药绒毡层的发育进程。为了弄清花药发育过程中转录调控通路,我们首先对已报道的花药发育关键转录因子突变体进行了细胞学分析,随后通过RT-PCR的策略初步确定了调控绒毡层发育与功能的转录因子之间的上下游关系。进一步的双突变分析及原位杂交实验则基本阐明了绒毡层发育过程中存在着DYT1-TDF1-AMS-AtMYB103-MS1这样一条转录调控途径。综上所述,本文的结果表明AtMYB103作为花药发育转录调控通路上的一个关键转录因子,通过调控多个关键途径来控制拟南芥绒毡层的发育及小孢子的成熟。这些分子机理的阐明加深了我们对绒毡层细胞如何调控花粉发育这个生物学问题的了解。更多还原

【Abstract】 Plant sexual reproduction is not only a major approach of plant propagation, but also one of the foundations for evolution and adaptation to the environment. Anther is the male reproductive organs of plants which is the place for pollen development. Therefore, the study of anther and pollen development has important theoretical significance. As the male-sterile varieties are valuable resources that greatly facilitate the production of hybrids via cross-pollination, in-depth study of anther and pollen development has important application value. In flowering plants, anther development is a very complex process which consists of two sequential phases: microsporogenesis and microgametogenesis. In Arabidopsis microsporogenesis, stamen primordia gradually formed the primarily morphologic structure of anther and filament through cell differentiation and fate decision. In microgametogenesis, pollen matured with two asymmetric mitotic divisions to form the multi-nulcei male gametophyte. Pollen development depends on the diploid sporophytic cell layer, especially the tapetal cells. The tapetum, which arises from secondary parietal cells, surrounds developing reproductive cells. It plays an important role in pollen development by contributing to microspore release, nutrition, pollen-wall synthesis and sporopollenin deposition. With the pollen development, it initiates the programmed cell death (PCD) process. With the completion of mitosis of pollen, tapetal cells undergo lysis and provide the materials for pollen coat. So far, in Arabidopsis, several leucine-rich repeat receptor-like protein kinases were reported for cell fate determination of tapetum. Besides, several transcription factors were also identified which involved in regulating tapetum development and function. However, for the complexity of anther development, it should be there are many key factors not characterized, and the relationship of these reported factors is still not clear. Based on these reasons, we use forward genetics to identify novel key genes which important of anther development, and illustrate the function of them.In this study, male sterile mutant ms188 was generated by ethyl-methane sulfonate (EMS) mutagenesis. A map-based cloning approach was used, and ms188 was mapped to a 95.8-kb region on chromosome 5 between molecular marker MDA7 and K24C1. Sequence analysis revealed that ms188-1 had a pre-mature stop codon in the R2R3 region of AtMYB103 which belongs to MYB transcription factor family. Allelism tests and genetic complementation indicated that AtMYB103 corresponded to MS188. The observations semi-thin section of wild-type and ms188 anthers revealed that mutant tapetal cell walls remained intact in late stages, suggested that the sceretory function of tapetum was altered. Most of the mutant microspores underwent degradation during late phases. Aniline blue staining showed the degradation of callose wall was altered, resulted in separation of tetrad. The scanning and transmission electron microscopy observation showed the surviving microspores lacked exine in ms188 locules. Semi-quantitive and quantitive RT-PCR analysis indicated that the callase-related gene A6 and the exine formation gene MS2 were obviously downregulated in mutant anthers. These results implicate that AtMYB103 plays an important role in tapetum development, callose dissolution and exine formation in A. thaliana anthers.We also characterized the detail expression pattern of AtMYB103. it was specifically expressed in tapetum and microsporocyte, indicated that it regulated tapetum development and secretory function. In addition, disrupted development and secretory function of tapetal cells in ms188 mutant were analyzed by TEM. Furthermore, the aberrant exine formation of ms188 is due to the aborted tapetal functions rather than the primexine formation. In order to illustrate the regulatory mechanism of AtMYB103, we performed global expression profiling analysis of wild-type and ms188 mutant. A total of 821 genes (728 downregulated and 93 upregulated) in ms188 flower buds were identified. Based on ‘Electronic Fluorescent Pictograph’(e-fp) Browser dataset, we assigned these differentially expressed genes to nine groups as diverse stages of anther development, such as bud expressed, late anther expressed, pollen expressed, and so on. Selected differentially expressed genes for quantitative RT-PCR analysis showed the microarray experiments are reliable. Functional classification of microarray data showed that loss-of-function of AtMYB103 impairs several key metabolic and signaling pathways throughout anther development: expression of many late-stage-expressed kinases is affected, suggested that AtMYB103 might act as a regulator in signal transduction of anther development at the late stage; several pectinase and callase related genes were suppressed, indicated that they probable involved in degradation of tapetal cell wall and callose wall; many genes involved in lipid synthesis and transport were also significantly downregulated, resulted in absent of exine of ms188 pollen. However, TUNEL assay illustrated the PCD process of tapetum was not regulated by AtMYB103. In addition, we applied RNA in situ hybridization to characterize several novel regulators of microsporogenesis which identified in the microarray experiments. These results indicated that AtMYB103 acts as a key regulator for several pathways during Arabidopsis anther development.To elucidate the transcriptional regulatory pathway in anther development, we analyze the mutants of reported transcription factors through cytological observation. Besides, we preliminarily identify the relationship of several key regulators in anther development though RT-PCR strategy. Further double mutant strategy and in situ analysis confirm the DYT1-TDF1-AMS-AtMYB103-MS1 transcriptional regulatory pathway in anther development.Summary, the results indicated that AtMYB103 acts as a key transcription factor of anther transcriptional regulatory pathway, and affects several pathways to regulate tapetum development and pollen maturation. Investigation of these mechanisms deepenes our understanding of the roles of tapetal cells in pollen development.更多还原