【作者】 孙扬；

【导师】 夏光敏；

【作者基本信息】 山东大学 ， 细胞生物学， 2014， 博士

【摘要】 植物作为固着生物,在漫长的进化过程中发展了复杂的响应系统以适应多变的内、外环境信号。光是植物生长发育过程中最重要的环境因子之一。光信号被植物光受体接收,经过一系列光信号元件的转导,调控光诱导基因的表达,最终引起植物的光形态建成。除了光信号,油菜素内酯(BR)作为一种重要的植物内源激素,在调节光形态建成中同样发挥关键作用。光信号转导途径和油菜素内酯信号转导途径的交叉正成为当前研究的热点。盐、旱等非生物胁迫是影响植物生长发育的另一类重要环境刺激。植物通过ABA依赖和ABA非依赖信号途径响应非生物胁迫。一些光信号元件,如CRY1,HY5也参与植物对非生物胁迫的应答。本实验室通过体细胞杂交,选育了高产、耐盐小麦新品种山融3号(SR3)。在对其耐盐机理进行抑制性消减杂交(SSH)和基因芯片分析后,克隆了参与盐胁迫应答的小麦基因TaGBF及其可能的互作蛋白基因TaCKB并进行功能鉴定。本论文发现TaGBF整合了光、油菜素内酯、非生物胁迫信号转导途径,是植物生长发育调控的关键节点；初步证明TaCKB可以与TaGBF互作,参与调控植物抗逆与发育。论文开展了以下工作：1)分析了TaGBF的组织特异性表达及光响应表达模式,研究了过表达TaGBF对拟南芥光形态建成和气孔发育的影响,确定了TaGBF在蓝光信号转导途径中的遗传位置,检测了TaGBF与光信号转导途径关键蛋白的相互作用关系。2)分析了油菜素内酯对TaGBF表达及生物学功能的影响,研究了过表达TaGBF对油菜素内酯响应的影响,确定了TaGBF与油菜素内酯信号转导途径关键作用因子BZR1的遗传关系,检测了TaGBF与油菜素内酯信号转导途径关键蛋白的相互作用关系。3)分析了TaGBF在非生物胁迫下的表达模式,构建了TaGBF小麦过表达系、RNAi干扰系,研究了TaGBF过表达小麦、过表达拟南芥在非生物胁迫下的表型,检测了非生物胁迫响应Marker基因的表达变化,探讨了TaGBF可能参与的非生物胁迫相关的信号途径。4)克隆了TaGBF潜在互作蛋白小麦酪蛋白激酶2调节亚基基因TaCKB,分析了TaCKB的组织特异性表达和非生物胁迫响应表达模式,进行了亚细胞定位,研究了TaCKB过表达拟南芥在盐处理下的表型和开花时间,探讨了TaCKB可能参与的调节种子萌发的途径,初步验证了TaCKB和TaGBF的互作关系。主要研究结果包括：1TaGBF在植物光信号转导途径中的功能研究TaGBF属于植物bZIP家族G组基因,在小麦的根、茎、叶、花中的表达量较高。小麦中TaGBF的表达受到蓝光诱导。过表达TaGBF促进了蓝光下拟南芥的光形态建成,表现为下胚轴长度显著缩短,子叶面积显著增大。同时TaGBF显著增加了蓝光下拟南芥子叶的气孔密度,促进了子叶气孔发育。遗传学实验显示,在拟南芥中TaGBF在CRY1下游发挥生物学功能。酵母双杂交的结果显示,TaGBF与光信号转导途径的关键抑制因子AtCOP1之间存在相互作用,而与bZIP转录因子AtHY5之间则不存在相互作用。以上结果表明,TaGBF是CRY1下游,蓝光信号转导的正向调控因子。2TaGBF介导拟南芥蓝光信号与油菜素内酯信号转导途径的交叉在拟南芥中,激活油菜素内酯信号转导途径可以诱导AtGBF的表达。在小麦中,施加外源油菜素内酯同样可以诱导TaGBF的表达。表明GBF响应油菜素内酯信号。蓝光下,TaGBF过表达系的下胚轴长度较野生型显著缩短,外源施加油菜素内酯后过表达系下胚轴伸长,表明过表达系中TaGBF介导的蓝光信号转导途径与油菜素内酯信号转导途径在光形态建成的调控中功能是拮抗的。过表达TaGBF导致蓝光下拟南芥对油菜素内酯及其抑制剂的响应发生改变。在黑暗中,TaGBF过表达系的下胚轴对油菜素内酯的抑制剂超敏感。TaGBF过表达系中,油菜素内酯响应基因的表达显著下调。遗传学实验显示,bzrl-1D可以部分抑制蓝光下TaGBF过表达系下胚轴的缩短,证明过表达系中TaGBF介导的蓝光信号与油菜素内酯信号转导途径在光形态建成的调控中功能是拮抗的。Bzrl-1D能够部分抑制TaGBF过表达系对油菜素内酯抑制剂的响应。通过以上实验,我们发现TaGBF作为节点整合了蓝光信号转导途径和油菜素内酯信号转导途径,提出了两条途径相互作用和动态平衡的模型。3TaGBF在非生物胁迫应答中的功能研究在小麦中,TaGBF的表达受到盐胁迫、渗透胁迫等非生物胁迫的诱导。我们构建了TaGBF的小麦过表达系、RNAi干扰系。表型分析显示,盐胁迫下,野生型小麦叶尖出现轻度萎蔫,叶片呈现绿色；TaGBF过表达小麦叶片显著萎蔫,叶片变黄,根长较野生型小麦显著缩短,表明TaGBF提高小麦对盐胁迫的敏感性。这是首次在小麦中证明蓝光信号元件参与非生物胁迫应答。在拟南芥中,过表达TaGBF导致拟南芥种子在盐胁迫、渗透胁迫下萌发率降低,萌发后子叶不能变绿。表明TaGBF提高了拟南芥对盐胁迫的敏感性。在TaGBF拟南芥过表达系中,ABA依赖途径的AtMYC2较野生型显著下调,AtAB15显著上调,表明TaGBF可能通过ABA依赖途径提高拟南芥对非生物胁迫的敏感性。4TaCKB的克隆及其在非生物胁迫应答中的功能研究在小麦中,酪蛋白激酶2的β亚基基因TaCKB的表达受到盐胁迫诱导,该蛋白是TaGBF潜在的互作蛋白,我们从小麦中首次中克隆了该基因,进行了初步的功能分析。酵母双杂交实验显示,TaCKB与TaGBF之间存在相互作用。TaCKB过表达拟南芥在种子萌发阶段表现出对盐胁迫的耐受性。TaCKB在萌发阶段表现出对GA抑制剂PAC的抗性,在ABA处理下与野生型拟南芥的萌发率没有显著性差异,所以TaCKB对拟南芥种子萌发的调控可能直接依赖于GA信号途径。种子的萌发依赖于GA和ABA的平衡,TaCKB与TaGBF如何通过互作调控胁迫下的种子萌发有待进一步研究。更多还原

【Abstract】 Being sessile organisms, plants are forced to develop complex responses to deal with endogenous and exogenous environmental stimulations.Light is an essential environmental facor regulating plant growth and development. Light signals are absorbed by photoreceptors and transducted by a series of light signaling components to modulate the expression of light-regulated genes and induce photomorphogenesis.In addition to light, brassinosteroids (BR) play a key role in photomorphogenesis. The crosstalk between light signaling and BR signaling transduction pathway is becoming a hotspot nowadays.Abiotic stresses such as salinity and osmotic stresses are unfavorable environmental conditions affecting plant growth and development. Plants respond to abiotic stresses via both ABA-dependent and ABA-independent signaling pathway. Some light signaling components including CRY1and HY5are involved in abiotic stress responses.A new wheat introgression line SR3with high yield and tolerance to abiotic stresses was generated with somatic hybridization in our lab. During the study of the stress tolerance of SR3with SSH and microarray, a bZIP gene TaGBF and a potential TaGBF-interacting protein gene TaCKB were cloned and studied preliminarily.This study illustrates that TaGBF is an important modulator integrating light, BR and abiotic stress responses signaling pathway during plant growth and development Research was carried out as follows:1) The expression pattern of TaGBF in different tissues and light conditions, regulation of photomorphogenesis and stomatal development in Arabidopsis by TaGBF, genetic relationship analysis in light signaling pathway and protein interaction between TaGBF and important components in light signaling pathway.2) Expression of TaGBF in response to BR, sensitivity to BR affected by TaGBF, genetic relationship between TaGBF and BZR1, protein interaction between TaGBF and important components in BR signaling pathway.3) Expression of TaGBF in response to abiotic stresses, generation of TaGBF overexpressing lines and RNAi lines in wheat, phenotype of wheat TaGBFox lines and Arabidopsis TaGBFox lines under abiotic stresses, expression of stress responsive genes in stress signaling pathways.4) Cloning and characterization of TaCKB, a gene encoding potential TaGBF-interacting protein. The expression pattern of TaCKB in different tissues and abiotic stresses, subcellular localization of TaCKB, phenotype of Arabidopsis TaCKBox lines under abiotic stresses, flowering time, seed dormancy signaling pathway regulating TaCKBox lines, and the interaction between TaCKB and TaGBF in yeast-two-hybrid assy. The main results are summarized as follows:1. The function of TaGBF in light signaling pathway.TaGBF belongs to bZIP family group G in plant.The expression of TaGBF in wheat was relatively higher in roots, leaves, shoots and flowers, and was induced by blue light.Overexpression of TaGBF promoted photomorphogenesis under blue light, characterized by shorter hypocotyl length and larger cytoledon area. In addition to photomorphogenesis, TaGBF promoted stomatal development under blue light.Genetic analysis indicated that TaGBF functioned downstream of CRY1in light signaling pathway.Yeast-two-hybrid assay showed that TaGBF interacted with AtCOP1, and didn’t interact with AtHY5.The results showed that TaGBF functions downstream of CRY1, as a positive blue light signaling regulator.2. TaGBF mediated the crosstalk between blue light and BR signaling pathway.Expression of AtGBFs was induced by BR signaling. Exogenous brassinolide induced the expression of TaGBF. These results suggested that GBF responded to BR.The hypocotyl length of Arabidopsis TaGBFox lines was significantly shorter than wild type under blue light. Exogenous brassinolide partly suppressed the short hypocotyl phenotype of TaGBFox lines, suggesting the antagonistic function of blue light and BR signaling pathway in regulating photomorphogenesis.The responses of TaGBFox lines to both BR and BRZ were changed under blue light. In darkness, the hypocotyls of TaGBFox lines were hypersensitive to inhibitor of BR synthesis. Expression of BR responsive genes were significantly downregulated in TaGBFox lines. These results indicated overexpression of TaGBF changed BR responses.Genetic analysis indicated that bzr1-1D partly suppressed the short hypocotyl phenotype of TaGBFox lines under blue light, further supporting the antagonistic function of blue light and BR signaling pathway in regulating photomorphogenesis. Bzr1-1D partly suppressed the responses of TaGBFox lines to inhibitor of BR synthesis.These results revealed that TaGBF is a key crosstalk integrating blue light and BR signaling pathway.3. The function of TaGBF in abiotic stress responses.Expression of TaGBF was induced by abiotic stresses such as salinity and osmotic stresses in SR3.TaGBF overexpressing lines and RNAi lines were generated in wheat. Wild type wheat seedlings showed mild wilting under salt stress, wheareas their leaves were still green. TaGBFox wheat seedlings exhibited serious wilting and yellow leaves, with shorter roots than wild type. These results indicated that overexpression of TaGBF heightened the sensitivity of wheat to salt stress.Overexpression of TaGBF in Arabidopsis decreased the germination rate and inhibited cytoledon development from turning green under salt and osmotic stresses. These results indicated that overexpression of TaGBF heightened the sensitivity of Arabidopsis to abiotic stress.In TaGBFox Arabidopsis seedlings, a set of stress responsive genes were screened. Among ABA-dependent stress responsive genes, AtMYC2was down-regulated, whereas AtAB15was up-regulated. TaGBF responded to abiotic stresses via ABA-dependent signaling pathway. 4. Cloning and functional analysis of TaCKB in abiotic stress tolerance and flowering.Expression of TaCKB was induced by salt stress in wheat.TaCKB which encodes a casein kinase2(CK2) regulatory subunit was cloned from wheat.Two-yeast-hybrid assay indicated that TaCKB interacted with TaGBF.Overexpression of TaCKB in Arabidopsis increased the germination rate under salt stress. TaCKB increased PAC(inhibitor of GA synthesis) tolerance at germination stage, but didn’t affect ABA response. This indicated that TaCKB regulated seed germination via GA signaling pathway. Seed dormancy depends on the balance between ABA and GA signaling. How does TaCKB interact with TaGBF to regutate both GA and ABA signaling pathway is worth to be investigated further.更多还原