【作者】 陶园；

【导师】 郝玉金； 张宪省；

【作者基本信息】 山东农业大学 ， 植物学， 2009， 博士

【摘要】 适度低温是一些高等植物正常的生长发育所必需的,如低温层积被广泛用于提高拟南芥等种子的萌发率和一致性。赤霉素(GA)是调控种子萌发、茎和下胚轴伸长、花发育和开花等诸多生命过程的重要激素,在拟南芥、榛子和苹果中的研究表明,低温层积能提高种子内源GA含量。所以GA和低温两条信号通路是相通的,但是,人们对整合两条通路的共同节点知之甚少。ICE1是拟南芥低温信号转导通路中的一个关键转录因子,它响应低温信号,并通过调控CBFs转录因子激活COR(cold-regulated)基因的表达,从而调控植物的低温响应。本文着重研究ICE1基因在植物发育方面的功能,特别是它通过调节GA合成调控拟南芥的植株生长、开花、育性等。主要研究结果如下:1. ICE1抑制拟南芥的植株生长成年野生型拟南芥(Col-0)植株的高度为30 cm左右,而突变体ice1-t高达45 cm,在ICE1基因过表达株系中,强表型植株的高度只有10 cm左右。这说明,ICE1能够抑制拟南芥的植株生长,降低株高。2. ICE1通过调控GAMYB基因影响花粉育性在ice1-t突变体的主花序轴上,下端有10个左右的果荚不能结种子。与野生型相比,突变体对应部位的花药开裂较晚,花粉粒颜色异常。在萌发培养基上的离体萌发实验表明,野生型花粉的萌发率高达50%,而ice1-t花粉只有10%。人工授粉后的活体实验表明,ice1-t花粉在野生型和突变体的柱头都不能正常萌发,而用野生型花粉授粉可以使突变体结果荚。上述结果表明,雄性不育导致了突变体的花序基部不育。鉴于GAMYB-like基因影响花粉育性,我们分析了该类基因在野生型和突变体中的表达模式,半定量RT-PCR和Northern杂交实验结果表明,与野生型相比,突变体花中的AtMYB33、AtMYB65、AtMYB101和AtMYB120基因表达上调。3. ICE1通过调控开花相关基因影响开花时间短日照条件下,ice1-t突变体的开花时间比野生型早。进而我们分析了开花诱导相关基因的表达模式。半定量RT-PCR结果显示,突变体中促进拟南芥开花的基因LFY、CO和FT表达上调,而抑制开花的基因FLC表达下调。4. ICE1通过调控LECs基因影响种子发育ice1-t的成熟种子外表干瘪,显微观察发现,ice1-t突变体能发育出正常的球形胚、心形胚和鱼雷胚,但随后的发育受阻,不能形成正常大小和形状的弯子叶胚,导致成熟胚不能充满种皮,造成种子干瘪。鉴于LEC1、LEC2和FUS3等是胚胎发育的重要调控基因,我们对这些基因的表达模式进行了分析,实时定量RT-PCR结果显示,LEC1、LEC2和FUS3在ice1-t果荚中的表达升高,其中FUS3最明显。5. ICE1通过调控GA合成代谢相关基因影响内源GA水平鉴于突变体的各种表型,我们怀疑ICE1能够调控GA合成或代谢,因此对GA合成代谢关键酶基因的表达模式进行了分析。实时定量RT-PCR结果显示,在ice1-t突变体中,GA合成相关基因AtGA3ox1、AtGA3ox2、AtGA3ox3、AtGA3ox4和AtGA20ox1的表达水平明显升高,而GA分解代谢基因AtGA2ox2表达水平则下降。激素测定也表明,突变体的内源GA含量升高,表明ICE1基因抑制拟南芥的内源GA合成。6. ICE1蛋白与AtGA3ox4基因启动子的顺式元件结合染色质免疫共沉淀(CHIP)结果表明,在候选基因AtGA3ox1、AtGA3ox2、AtGA3ox3、AtGA3ox4、AtGA20ox1和AtGA2ox2中,ICE1蛋白能够与GA合成基因AtGA3ox4的启动子结合,特异结合的E-box元件为-206 CATTTG -200。更多还原

【Abstract】 Appropriate low temperature favorably contributes to plant growth and development, for example, cold stratification is widely applied to promote germination both in frequency and synchronization. Phytohormone gibberellins (GAs) play crucial roles in seed germination, stem and hypocotyl elongation, floral development and flowering time. Cold stratification can increase the endogenous GA contents in Arabidopsis, hazel and apple seeds. Therefore, cold and GA signal pathway should be integrated to regulate plant development in a crosspoint which is unclear as yet. ICE1 is a key transcription factor in cold signaling pathway of Arabidopsis. It controls the response of plant to cold signal by initiating CBFs transcription factors and COR (cold-regulated) genes in turn. In this study, the functions of ICE1 in plant growth and development were observed and investigated with emphasis on the regulation of ICE1 to plant growth, flowering and fertility by tuning GA synthesis. The main results are shown as follows:1. ICE1 represses plant growth in ArabidopsisMature WT (Col-0) plant was about 30 cm in height, while ice1-t mutant up to 45 cm. Arabidopsis plants overexpressing ICE1 gene exhibited a dwarf phenotype. The plant height was only 10 cm. So, ICE1 could suppress plant growth in Arabidopsis.2. ICE1 controls male sterility by tuning GAMYB-like genesThe first ten siliques failed to set seed in the base of florescence of ice1-t mutant. The anthers of corresponding flowers dehisced later in mutant than WT. In addition, mutant pollens exhibited abnormal in color relative to WT. Germination in vitro on medium demonstrated that 50% pollens were able to germinate in WT while only 10% in mutant. Furthmore, germination in vivo on pistils artificially pollinated showed that mutant pollens failed to germinate on both WT and mutant pistils, while artificial pollination with WT pollen lead to success in setting seeds in mutant siliques. Those findings indicate that male sterility causes the failure to set seeds in the basal flowers of mutant.Considering the regulation of GAMYB-like genes to pollen development, gene expression patterns were investigated in mutant and WT with RT-PCR and RNA-blot assay. The results showed that the transcript level of four GAMYB-like genes, i.e. AtMYB33, AtMYB65, AtMYB101 and AtMYB120, were up-regulated in ice1-t mutant relative to WT.3. ICE1 controls flowering time by modulating the relative genesice1-t mutant flowered earlier than WT under short day (SD) conditions. Subsequently, the expression patterns of key genes involved in floral initiation were examined with semi-quantitative RT-PCR. The result indicated that the inducing genes LFY, CO and FT for floral initiation were up-regulated in ice1-t mutant, while the suppressor gene FLC was down-regulated.4. ICE1 controls seeds development by tuning LECs genesThe mature seeds of ice1-t were scrawny and shriveled in seed skin. Microsopic observation found that ice1-t produced normal globular, heart and torpedo embryo. However, the embryo development of mutant was inhibited in the following stages and failed to form embryo with curled cotyledon. As a result, the embryos were smaller in mutant than WT, which made the mutant seed looking scrawny.Because LEAFY COTYLEDON (LEC) genes, i.e. LEC1, LEC2 and FUS3, play key roles in controlling embryo development, we detected their expression patterns. Real-time RT-PCR showed that all three genes, especially FUS3, were up-regulated in the siliques of ice1-t mutant relative to WT.5. ICE1 controls endogenous GA level by regulating the expression of genes involved in GA synthesis and metabolism Based on those phenotypes of ice1-t mutant, it was predicted that ICE1 can regulate GA synthesis or metabolism. Real-time RT-PCRs were carried out to verify the hypothesis. It was found that GA synthesis-related genes AtGA3ox1, AtGA3ox2, AtGA3ox3, AtGA3ox4 and AtGA20ox1 were up-regulated in ice1-t mutant relative to WT, while GA degradation-related gene AtGA2ox2 was down-regulated. In fact, it was also found that GA level was much higher in mutant than WT. So, ICE1 repress the synthesis of endogenous GA in Arabidopsis.6. ICE1 protein binds to the promoter of AtGA3ox4Chromatin immunoprecitation (CHIP) showed that ICE1 protein could bind to the promoter of GA biosynthetic gene AtGA3ox4 among 6 candidate genes, i.e. AtGA3ox1, AtGA3ox2, AtGA3ox3, AtGA3ox4, AtGA20ox1 and AtGA2ox2. The binding E-box element is -206 CATTTG -200.更多还原