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拟南芥花药发育相关基因MPS1和AMS功能的初步研究
【作者】 江华；
【导师】 曹凯鸣；
【作者基本信息】 复旦大学 ， 生物化学与分子生物学， 2009， 博士
【摘要】 纺锤体是细胞分裂过程中形成的由微管、摩托蛋白以及其他一系列复合物所组成的一个纺锤形的结构,它均等地将染色体分配到子代细胞中,从而保证染色体倍数的稳定性。植物细胞分裂中的纺锤体缺乏动物和酵母中的中心体类似物,因此植物中纺锤体形成的机制可能有所不同,但是目前对于植物中纺锤体形成的机制特别是分子机制仍然所知甚少。我们筛选到一株雌雄育性剧烈下降的拟南芥突变体,等位分析和遗传互补证明了At5G57880敲除导致突变表型。细胞学观察发现突变体的花药中形成大量异常的四分体,特别是六分体、五分体和不规则的四分体,这表明突变体的减数分裂可能出现问题。进一步的细胞学分析发现突变体减数分裂中前期与野生型相似,但是在分裂后期染色体分离出现异常,染色体向多个方向分离,或者向两极不均等分离。由于突变体的雌性育性同样剧烈下降,我们又分析了雌性减数分裂的过程,结果在雌性减数分裂的后期也观察到了这种染色体向多个方向分离的现象,说明该基因影响拟南芥雌雄减数分裂的染色体分离。免疫荧光的结果显示减数分裂的纺锤体形状不规则,出现多个聚集的极点,形成多极纺锤体,这个结果说明减数分裂中纺锤体错误的极性建成是突变体染色体不均等分离的根本原因。因此,我们将这一突变体命名为mps1(MultipolarSpindle 1)。MPS1编码一个高等植物特有的未知功能的蛋白,软件预测MPS1含有Coiled-coil结构域,并且在N端有核定位信号区。原位杂交的结果显示MPS1在雄性和雌性减数分裂细胞中都有很强的表达,这一结果与MPS1参与减数分裂的功能相吻合。RT-PCR的结果表明MPS1在各个器官中都有表达,特别在幼苗中表达最高,提示MPS1在营养生长,特别是有丝分裂中可能发挥作用。由于MPS1是高等植物中首次发现的一个特异性的参与细胞减数分裂纺锤体极性建成的基因,该基因的深入功能研究将有助于植物减数分裂中纺锤体极性建成机制的了解。绒毡层作为一种独特的分泌细胞,在植物的花粉发育过程中起着非常重要的作用。绒毡层的整个发育过程复杂而有序,牵涉到细胞的分化、物质的分泌以及细胞的凋亡等,通过绒毡层发育的研究可以使我们对植物花药发育有更为深入的认识。通过对T-DNA插入突变体ams-2的研究和分析,进一步地阐明了AMS在花药发育,特别是绒毡层发育中的功能。对ams-2组织切片后发现突变体的绒毡层细胞比已报道的ams突变体空泡化时间提早。从第6期开始绒毡层细胞就开始不断肥大和空泡化,小孢子被挤在药室中央,最后小孢子提前降解。进一步的透射电镜观察发现绒毡层细胞在减数分裂时期并没有转变为分泌型的细胞,小孢子和花粉发育所需要的物质在绒毡层中没有形成。TUNEL assay的结果显示绒毡层细胞自身降解的细胞凋亡机制也没有启动,使得绒毡层细胞在花药发育后期无法按时降解。通过细胞学观察、芯片数据的分析以及Real-time PCR的验证,四分体释放所需要的胼胝质酶和果胶酶的表达水平在突变体花序中剧烈下调,暗示了AMS对这一生理活动的转录调控的功能。原位杂交的结果显示AMS在花药发育的第6、7期的减数分裂细胞和绒毡层细胞中强烈表达。酵母实验和芯片分析的结果说明AMS是一个转录激活因子,在绒毡层发育的过程中调控了大量基因的表达。综上所述,我们在AMS已有研究的基础上进一步地阐述了该基因在绒毡层分化和发育中的功能,加深了我们对这一过程的理解。
【Abstract】 The spindle is an array of microtubules, microtubule motor proteins, and a series of other complex, which is essential for segregating chromosomes to generated cells during cell division. The spindle in higher plants is lack of centrosomal-like structures as in animals and yeasts, so the mechanism of spindle organization in higher plants should be different form others. Now the mechanism, especially the molecular mechanism of spindle organization in higher plants is still not well understood. We isolated an Arabidopsis T-DNA mutant with severely reduced male and female fertility, and cloned the mutated gene At5g57880. Cytological analysis showed a large number of abnormal tetrads in mutant anther, especially hexads, pentads and irregular tetrads, which indicated abnormalities in meiosis. Further analysis showed that chromosome segregation was abnormal in both male and female mps1 meiocytes. Most chromosomes separated to three or more directions at anaphase, or showed an uneven separation to two directions. Immunolocalization showed unequal bipolar or multipolar spindles in mps1 meiocytes, which indicated that aberrant spindles resulted in disordered chromosome segregation. The MPS1 protein encodes a 377 amino acid functional unknown protein with putative coiled-coil motifs, and a nuclear localization signal in N terminus. RT-PCR showed MPS1 is expressed in almost all tissues, especially in young seedling. In situ hybridization analysis showed that MPS1 is strongly expressed in both male and female meiocytes. The possible role of MPS1 in meiotic spindle organization is also discussed. In Arabidopsis, the tapetum plays important roles in anther development, providing enzymes for callose dissolution and materials for pollen wall formation, and supplying nutrients for pollen development. Tapetum development is a complicated and precise process contains cell differentiation, material secretary, programmed cell death. Here we report the identification and characterization of a T-DNA mutant ams-2, and further elucidate novel functions of AMS in tapetum development. Cytological analysis showed that tapetum vaculation in ams-2 was earlier than the ams mutant had been published. The tapetal cell became hypertrophy and vaculation before tetrad releasing. Transmission Electron Microscopy (TEM) examination further revealed tapetum didn’t differentiate into secretary- type cell at stage 6. There is no material required for microspore and pollen development in tapetal cell. TUNEL assay showed that there was no signal of PCD in tapetum, which results in aborted tapetum degradation at later stages. Callose staining and gene expression analysis suggested that AMS may be a crucial component in controlling callose dissolution. In situ hybridization analysis revealed that AMS is highly expressed in tapetum and meiocytes during anther development. AMS encodes a bHLH transcription factor, it showed transactivating ability in yeast experiment, and the activation domain was in N terminal. In conclusion, our results showed that AMS plays a vital role in tapetal differentiation and development.