【作者】 王爱华；

【导师】 封克； 罗明； Liz Dennis；

【作者基本信息】 扬州大学 ， 植物学， 2009， 博士

【副题名】IKU1的分离和功能研究

【摘要】 种子对人类来说,代表了食物和工业原料的重要来源。种子大小是影响作物产量的重要因素之一。而胚乳又占谷类植物种子的大部分体积,决定了种子的大小。因此,在当前人口越来越膨胀、资源越来越紧缺的形势下,加强对胚乳发育的研究,对于农业生产乃至人类社会的科学可持续发展具有特别重大的战略意义。前人研究(Garcia等；Luo等)表明,拟南芥早期胚乳和种子大小至少受到三个基因IKU1、IKU2和MINI3控制。它们的突变体均表现为胚乳数量减少和种子变小。这三个基因作用于同一个遗传途径。IKU2编码LRR受体激酶；MINI3编码WRKY家族蛋白WRKY10,是调控胚乳增殖的转录因子。但对IKU1基因了解甚少。本文使用分子生物学、遗传学、细胞生物学、生物信息学、生殖生物学的方法对与胚乳发育有关的拟南芥孢子体隐性突变体——ikul (haikul)基因进行了较为深入的研究。应用了遗传标记定位技术、显微镜技术、蛋白研究技术、基因克隆、突变技术、生物信息学等技术来分离IKU1基因、研究IKU1的功能,取得了突出进展,为早期胚乳的发育和种子大小控制的机理贡献了新的知识,对农业生产中的小麦、水稻等种子发育研究有重要的参考价值。本研究利用遗传标记、PCR技术和基因测序技术把IKU1定位在At2g35230基因上。带有At2g35230全部基因信息的互补载体弥补了iku1突变体的基因缺陷,使iku1突变体表现型恢复为野生型大种子。分析其T1、T2种子的表现型和基因型,从遗传学角度证实了IKU1基因功能发生在胚乳中,验证了拟南芥的卵细胞和中央细胞可以通过花器浸泡分别转化。IKU1编码的蛋白包含了植物中特有的VQmotif。在检验IKU1蛋白功能区的遗传互补试验中,带有VQ motif突变的IKU1基因不能使iku1突变体种子恢复野生型大小,而删除了重复区域45个氨基酸的突变仍可以使突变体种子恢复野生型大小,说明VQ motif对于IKU1基因影响种子发育的功能十分重要。高表达启动子35S作用下的GFP∷IKU1蛋白在根尖细胞、茎表皮细胞、叶片绒毛细胞、气孔保卫细胞、种皮细胞的细胞核以及洋葱表皮细胞的细胞核中表达,但核仁中没有GFP信号。同时,也发现35S::GFP::IKU1在其转化株后代中,会出现基因沉默现象。而IKU1启动子作用下的GFP::IKU1仍具有IKU1在胚乳中的功能,可以使ikul突变体的种子恢复野生型性状,同时也揭示了IKU1在植株中真实表达形式。其T1植株中,GFP::IKU1表达在根尖、茎表皮、花柄、荚果隔膜、种皮和中央细胞及受精后不同阶段的胚乳细胞细胞核中,但是蛋白表面没有亮点(speckle),卵细胞和胚的细胞中也没有GFP::IKU1信号。IKU1是一个未知功能的蛋白,它与At1g32610及其它来自毛果杨、蓖麻、棉花、葡萄的VQ motif蛋白处于同一进化组群。大多数编码VQ motif蛋白的基因都没有内含子。Atlg32610与IKU1蛋白序列十分相似,它们某些功能的可能重叠也许导致我们观察不到iku1突变体在种子以外的组织器官中的表现型。双突变体种子重量的分析结果说明了IKU1和MINI3作用于同一个影响种子发育的遗传途径。酵母双杂交等试验结果也揭示,IKU1极可能与MINI3/WRKY10,一个WRKY家族的转录因子存在相互作用,形成蛋白复合体。在本试验和前人研究的基础上,提出了一个关于IKU1-MINI3/WRKY10的种子发育途径模式。在野生型植株中,IKU1、MINI3/WRKY10先形成蛋白复合体,由复合体参与调节一个控制种子大小的基因——IKU2,最终产生野生型大种子。iku1突变体造成IKU1-MINI3/WRKY10转录结合体不稳定,作为反馈,MINI3/WRKY10抑制MINI3基因的表达；同时,IKU2的表达下调,最终产生小种子。更多还原

【Abstract】 Seed represents an important source of food, feed, and industrial raw material for mankind. Seed size is one of the significant factors affecting crop production. Endosperm accounts for most of the bulk of cereal seed and determines the seed size. It will be strategically significant for the sustainable development of the agricultural production and the human society to support the research on reproductive biology, because of the resource shortage and expanding population in the world.The reproductive process in plants is an important area in biology, which still requires substantial genetic and molecular investigation to elucidate the complex process. Angiosperms, or higher plants, have a unique seed forming process, double fertilization: the egg cell is fused with a sperm nucleus and the central cell fuses to another sperm nucleus both coming from the same pollen grain. This leads to the formation of the embryo and endosperm. Three mutants ikul, iku2and mini3have been shown to give small seed size because of the reduction of endosperm growth. The expression levels of MINI3and IKU2were decreased in the ikul-1mutant. IKU2expression was reduced in a miniS-1background whereas MINI3expression levels were unaltered in the iku2-3mutant. These observation suggest the successive action of the three genes IKU1, IKU2and MINI3in the same genetic pathway of seed development. In this pathway, MINI3encodes WRKY10, a WRKY class transcription factor; IKU2encodes a LRR KINASE (At3g19700). But the identity of IKU1remains unknown.In this study, the integrated approaches involving classic genetic, molecular, cellular methodologies were used to investigate the molecular identity of IKU1. We reported here the cloning of IKU1gene which controls the seed size in Arabidopsis using map-based strategy. It was shown that IKU1is At2g35230which encodes a novel protein containing VQ motif. VQ motif contains a core sequence of unknown function and is specific in plant species. The genetic complementation of ikul phenotype suggests that IKU1gene functions in endosperm. It was also confirmed that the endosperm and the embryo of Arabidopsis thaliana are independently transformed through infiltration by Agrobacterium tumefaciens. The mutation in IKU1only causes obvious phenotype in seed, though IKU1is expressed widely. The genomic construct carrying mutations in VQ motif failed to complement the ikul seed lesion, suggesting an essential role of the VQ motif in IKU1functioning. There is evidence that the mutations in other parts of genes are not important for IKU1function. We found that IKU1is nuclear-localized, organized into speckles and led to gene silencing under35S. When driven by its native promoter, the GFP::IKU1without any speckle, able to complement the mutant phenotype, was detected in the nuclear of the early endosperm as well as other vegetative organs. The physical interaction between IKU1and MINI3/WRKY10suggests that the IKU1is a co-transcription factor of MTNI3/WRKY10. A model has been proposed to explain how the IKU1and MINI3/WRKY10regulate endosperm development and seed size. In wild type Arabidopsis, IKU1interacts with MINI3/WRKY10to form a complex which regulates the IKU2and consequently wild-type seeds will be produced. Seeds in ikul or miniS mutants become small, because IKU2is repressed. Further, in ikul mutants the IKU1-MINI3/WRKY10transcription complex becomes unstable and the un-associated MINI3/WRKY10protein starts to repress the expression of MINIS genes as a feedback mechanism. In the same time IKU2expression is down-regulated, which leads the formation of small seeds.更多还原