【作者】 吕宏君；

【导师】 夏光敏；

【作者基本信息】 山东大学 ， 遗传学， 2014， 博士

【摘要】 小麦是全球最重要的农作物之一,其高产得益于绿色革命,其中对光周期不敏感的特性赋予了小麦更广泛的适应性。盐早等非生物胁迫严重影响小麦产量,因而认识耐逆机制、分离耐逆相关基因并通过分子辅助育种培育耐逆小麦新品种尤为迫切。在提高耐逆能力的同时,保持小麦高产和广泛适应性的性状也非常重要。因此,有必要深入揭示小麦和其他植物中光周期(生物钟)和非生物胁迫应答之间的协作关系。本文基于小麦渐渗系山融三号(SR3)转录组数据,分离了一个在非生物胁迫下表达明显上调的光周期响应基因TaCOLl,并初步分析了它在调控植物发育和非生物胁迫应答中的功能和作用机制。1、TaCOL1是一个参与调节光周期的时钟类蛋白新基因通过筛选小麦耐盐渐渗系SR3离子和渗透胁迫基因芯片中光周期相关基因,克隆了一个B-box类锌指蛋白的胁迫诱导型基因TaCOLl。TaCOL1是一个定位在细胞核的转录激活因子,含有两个B-box结构域和一个CCT (CO, COL, TOC1)结构域,与拟南芥生物钟蛋白CO及其他CO-like (COL)等B-box家族成员结构类似。TaCOL1在拟南芥中没有同源基因,而与大麦、水稻和玉米等禾本科植物中同源基因相似性很高,这些基因的功能均不清楚。结果显示,TaCOL1及其同源基因是单子叶和双子叶植物在进化分离后形成的。TaCOL1主要在叶中表达,而其他组织中表达量较低；叶片中,TaCOL1在拔节期表达量最高,其他发育时期较低。TaCOL1是一个光周期响应基因,不同光照条件下均表现出近似24h的节律性表达,但表达模式存在差异。长日照条件下,TaCOL1在白天表达,并在光照后14小时达到最大值,明显高于其他光照条件下波峰表达量。短日照条件下,TaCOL1在光照开始前2小时表达量达到最大值。全日照条件下,TaCOL1表达的节律性降低,表达模式与短日照类似。全黑暗条件下,TaCOL1表达不具有节律性,表明TaCOL1表达的节律反应依赖于光照条件。长日照下TaCOL1的高表达暗示它在小麦春季的旺盛生长中发挥重要作用。为了分析TaCOL1在生物钟系统中的作用,我们将TaCOL1在拟南芥中进行异源表达。TaCOL1过表达株系中,生物钟系统三个核心基因AtTOC1\AtCCA1和AtLHY的表达模式均发生了改变,其中AtTOC1的波峰发生了迁移,导致AtCCA1和AtLHY的表达量降低,并且AtLHY的表达基本失去了节律性。TaCOL1过表达还改变了其他生物钟相关基因的表达模式,包括输出通路相关基因AtELF3和AtGI。试验结果证明TaCOL1蛋白能够结合到AtTOC1基因的启动子上,使其波峰发生了迁移,并最终导致了一系列生物钟相关基因的表达模式发生了改变。因此,TaCOL1是一个能够调控生物钟系统的时钟类蛋白基因。2、TaCOL1影响植物的发育不同光照周期下,TaCOL1过表达拟南芥表现出发育受阻的生长素过量表型,包括根长变短、植株矮小、叶片变小并上卷、分支增多、角果变短。TaCOL1过表达株系对外源IAA的敏感性较低,相对生长量高于野生型,高浓度的IAA抑制叶片上卷。TaCOL1过表达株系中多个生长素合成和代谢、信号转导途径相关基因的表达明显上调,包括促进叶片上卷的生长素甲酯转移酶基因AtIAMT1等。结果表明,TaCOL1通过促进生长素合成和信号转导途径调控发育。长日照和短日照条件下,TaCOL1过表达植株开花延迟。进一步研究发现,TaCOL1过表达株系中促进开花基因AtFT表达均明显降低,而抑制AtFT表达的ATFLC也明显降低。根据生物钟系统通过输出通路的CO调控AtFT的表达,可以推断TaCOL1通过调节生物钟系统的GI-CO通路使CO表达量降低,进而抑制AtFT的表达来延迟开花。与野生型相比,白光下TaCOL1过表达植株的下胚轴更长,蓝光或红光下则更短,黑暗下长度相近,表明TaCOL1调控了光形态建成过程。与野生型在蓝光或红光下下胚轴长度比在白光下更长不同,TaCOL1过表达植株在各种光照条件下长度无显著差异,暗示TaCOL1可能通过调控蓝光和红光信号通路下游COP1、 HY5等共同组分影响光形态建成。但是酵母双杂交和单杂交分析显示,TaCOL1不能与COP1、HY5结合,也不能与其编码基因的启动子结合。这表明TaCOL1可能通过通过间接影响COP1和HY5的活性或调控光信号通路的其他因子参与光形态建成过程。TaCOL1过表达株系叶片气孔密度增加,而且部分气孔发生凝集现象。Real-time PCR分析显示,过表达植株中调控气孔发育的YDA通路相关基因的表达量均出现不同程度的增加。研究表明,YDA通路受油菜素内酯(BR)的调控,而且过表达植株的气孔表型与BR合成突变体det2-1一样,暗示TaCOL1调控气孔发育与BR相关。我们发现,TaCOL1过表达系中BR合成通路关键基因DWARF4的表达量明显降低,表明TaCOL1通过抑制BR合成调控气孔发育。3、TaCOL1通过调控氧化还原平衡提高耐逆能力NaCl和PEG处理后,TaCOL1表达量在0.5小时显著提高,而在24小时恢复到正常水平,推测TaCOL1可能作为转录因子参与胁迫早期响应过程。NaCl和PEG处理下,TaCOL1过表达植株根长和茎鲜重的相对生长量均明显提高。控水试验显示,过表达植株的抗旱能力明显提高。研究发现,胁迫条件下ABA大量合成,并激活下游信号通路,产生胁迫应答反应。但是,ABA处理后TaCOL1表达无明显变化,TaCOL1过表达不改变拟南芥对ABA的敏感性,而且TaCOL1过表达株系离体叶片的失水率也明显高于野生型,表明TaCOL1在胁迫应答中的作用与ABA无密切相关性。相反,H202处理后TaCOL1表达明显上调,TaCOL1过表达系对H2O2的抗性提高,并且与野生型相比,H2O2处理后不仅相对生长量更大,而且主根更长。进一步分析显示,TaCOL1过表达系中ROS合成相关基因RBOHC的表达增强,ROS清除相关酶SOD活性提高,ROS含量也明显增加,表明TaCOL1通过调控氧化还原平衡增强耐盐抗旱能力。TaCOL1过表达株系中SOD活性与光照条件相关。与野生型相比,短日照下转基因植株中SOD活性没有明显区别,但是长日照下明显较高。这说明TaCOL1对SOD酶活性的调控与光周期相关,暗示TaCOL1可能通过调节氧化还原平衡建立生物钟和下游激素和胁迫响应通路,实现对生长发育和胁迫应答的协同调控。更多还原

【Abstract】 Common wheat (Triticum aestivum L.) is one of the most important staple food crops in the world and is grown in very different environments. The high yield of wheat mast due to the "green revolution", and its wide adaptability to a broad range of agricultural environments is conferred by insensitivity to photoperiod (day length neutrality). Abiotic stresses such as salt and drought seriously restrict the yielding of wheat, so it appears to be urgent to unravel the mechanisms of abiotic stress tolerance, identify stress tolerance associated genes, and finally breed stress tolerant cultivars via the molecular assistant breeding strategy. Along with improving abiotic stress tolerance, it is important to reverse the excellent traits of high yield and wide adaptability. Therefore, it is necessary to uncover shield insight into the scenario of the interacting mechanisms between photoperiod and abiotic stress response in wheat and other plants. In this study, we cloned a clock-associated gene TaCOL1that was induced by abiotic stresses, confirmed its role in regulating development and abiotic stress response, and primarily analyzed how its role is performed.1. TaCOL1is a circadian clock gene that participates in regulating photoperiodThrough screening photoperiod associated gene from the transcriptomic data of wheat responding to ionic and osmotic stresses, we cloned an abiotic stress responsive gene TaCOLl, encoding a B-box zinc finger protein. TaCOL1localizes in the nucleus, and exhibits transcriptional activation activity. This protein possesses two B-box domains in the N-terminus, and one CCT (CO, COL, TOC1) domain in the C-terminus, and the domains are characterized in CO and other COLs of Arabidopsis. TaCOL1has orthologues in Gramineaes such as barley, rice and maize, but not in Arabidopsis; the function of these orthologues in Gramineaes has not been investigated. These results indicate that TaCOL1and its homologues were produced after the divergence between monocots and dicots.TaCOL1transcribed higher in leaves than in other tissues; in leaves, the transcript abundance of TaCOL1at elongation stage was higher than that at other stages during the whole life course. TaCOLl was photoperiod responsive, and exhibited a24h circadian rhythmic expression pattern under various photoperiod conditions. Under the long-day (LD) condition, TaCOL1was expressed during from early daytime until dusk, and its transcript peaked at14h after light onset and was significantly higher than those under other photoperiod conditions. In contrast, under the short-day (SD) condition, TaCOLl transcribed earlier, with a peak at2h before light onset. Under the continuous light (CL) conditions, TaCOL1expression was similar to that under the short-day condition, but the rhythmic pattern was weakened. Under the continuous dark (CD) conditions, the circadian rhythm of TaCOL1was disturbed, resulting in no apparent rhythmicity. According to this, the circadian rhythm of TaCOL1may depend on light. Note that the high expression level of TaCOL1under the LD condition suggests that it play important role in the vigorous growth of wheat in spring.To know the role of TaCOL1in circadian clock regulation, it was ectopically expressed in Arabidopsis Col-0. In TaCOLl overexpression lines, the expression patterns of AtTOC1, AtCCA1and AtLHY, three key genes of circadian clock central regulatory machinery, were all influenced. Of them, the peak of AtTOCl transcript was shifted, and appeared occurred in the dark, nearly4h later than that of the Col-0, which reduced the expression levels of AtCCA1and AtLHY, and led to the loss of circadian rhythm of AtLHY. TaCOL1overexpression also altered the expression profiles of other circadian clock associated genes, AtELF3and AtGI. These findings demonstrate that TaCOL1is a circadian clock gene.2. TaCOLl impacted the developmentUnder either LD or SD condition, TaCOLl overexpression resulted in development-blocked phenotypes, including short primary roots, dwarf plants, small and upward-curled leaves, more branching, short siliques, which phenocopied the alteration by auxin over-production. Consistently, TaCOLl overexpression lines had lower sensitivity to exogenous IAA and higher relative growth rate, and their leaf upward-curling was erased by high concentration of exogenous IAA. A set of genes involved in auxin synthesis and metabolism as well as auxin signaling pathway were upregulated, including AtIAMTl, encoding an IAA methyltransferase that has proved to contribute leaf up-curling. These results indicate that TaCOLl modulates plant development via promoting auxin synthesis and signaling pathway.Under both SD and LD conditions, the flowering-time was delayed by TaCOLl overexpression. Further studies revealed that in TaCOLl overexpression lines, the expression of AtFT, a flowering-accelerated gene, was restricted, while that of AtFLC, a flowering-blocked gene by inhibiting AtFT transcription, was also down-regulated. Based on the previous conclusion that AtFT was positively regulated by CO, whose encoding gene had lower expression level in TaCOL1overexpression lines, we propose that TaCOL1delays flowering time via modulating the GI-CO pathway of the circadian clock system to lower the expression of CO, and consequently the expression of AtFT.In comparison with Col-0, the hypocotyl length of TaCOLl overexpression lines was longer under white light (both SD or LD photoperiod), was shorter under either blue or red light, but was comparable under the CD condition, indicating TaCOL1participate in photomorphogenesis. Unlike Col-0who had shorter hypocotyl under white light than under red or blue light, TaCOL1overexpression lines had similar hypocotyls under white, red and blue light, suggesting that TaCOLl participate in photomorphogenesis via modulating the downstream components such as COP1and HY5shared by blue and red light signaling pathways. However, yeast two hybridization and yeast one hybridization assays showed that TaCOL1did not interact with either COP1or HY5, and it also did not bind with the promoters of the two genes. This implies that TaCOL1may contribute to photomorphogenesis through modulating other component(s) of light signaling pathways or indirectly influencing COP1and HY5.TaCOL1overexpression lines had higher stomatal density, and some of stoma were agglutinated. Real-time quantitative PCR analysis showed that, in TaCOL1overexpression lines, the expression of genes in YDA pathway that governing stomatal development was increased by various extent. It has proved that YDA pathway is regulated by by BR, and the increased stomatal density by TaCOL1overexpression was similar to the phenotype of some BR mutants. This suggests that the role of TaCOL1in stomatal development is associated with BR. Exactly, we found that TaCOL1overexpression lines had lower sensitivity to BR, and produced less transcript of DWF, encoding a key enzyme for BR synthesis pathway, indicating that TaCOLl modulates stomatal development via inhibiting BR synthesis.3. TaCOLl enhanced abiotic stress tolerance via modulating redox homeostasisWhen exposed to either NaCl or PEG, the expression level of TaCOL1was drastically increased after0.5h, while descended to the normal after24h, suggesting that TaCOL1sever as a transcription factor to play roles in the response to early stage of abiotic stress. Under either NaCl or PEG treatment, TaCOLl overexpression lines had higher relative growth rates of both primary roots and shoot fresh weight. Water-withholding assay indicates that TaCOL1overexpression lines had superior drought tolerance. It has been concluded that, abiotic stress induced a mass of ABA, which triggers the downstream signaling pathway to produce abiotic stress response. However, TaCOL1was not ABA-responsive, and TaCOLl overexpression did not change the sensitivity to ABA, and TaCOL1overexpression lines had higher water loss rate that Col-0, indicating the lack of association between ABA and the role of TaCOL1in abiotic stress response. By contrast, H2O2induced the expression of TaCOL1, and TaCOLl overexpression lines had pronounced tolerance to H2O2in comparison with Col-0. Further analysis showed that in TaCOL1overexpression lines, both the expression level of RBOHC for ROS production and the activity of SOD for ROS scavenging were elevated, and ROS level was increased, indicating the enhancement of abiotic stress tolerance by TaCOL1is partially achieved via regulating redox homeostasis. Interestingly, the elevation of SOD activity by TaCOLl overexpression is accociated with photoperiod. In comparison with Col-0, the SOD activity in TaCOL1overexpression lines had no obvious difference under the SD condition, but was significantly higher under the LD condition. This demonstrates that TaCOL1modulates SOD activity in a photoperiod-dependent manner, which speculates that TaCOLl links circadian clock and downstream phytohormone and abiotic stress responsive pathways via regulating redox homeostasis to orchestrate development and abiotic stress response.更多还原