【作者】 丁冬；

【导师】 郑用琏；

【作者基本信息】 华中农业大学 ， 遗传学， 2008， 博士

【摘要】 盐胁迫是一种世界范围很严重的环境胁迫,它会造成农作物减产和品质降低。作物只能在原位对抗盐胁迫,它进化出一种适应的分子机制,包括很多功能（代谢的、生理的和形态学的）基因的表达。这些基因参与非常广泛的胁迫适应生理及代谢过程,包括细胞解毒,物质转运,能量维持,激素响应等。而这些功能基因的表达又受到精致的调控。这些调控包括转录水平的,如对细胞外信号的感知,细胞内信号的生成与传递,基因转录的总体调节（染色质变化）,个别基因的特异转录调节;转录后水平的,如miRNA指导的靶基因转录本剪切或翻译抑制,蛋白质磷酸化和去磷酸化参与的信号途径,分子伴侣对胁迫相关蛋白质的保护,以及泛素化系统对蛋白转运信号的传递和蛋白质降解。这些基因在盐胁迫下的表达改变,就可重建细胞内稳态,达到对盐胁迫的适应和抵御。玉米,作为重要的粮食、饲料和生物能源作物,其产量对世界粮食安全影响巨大。玉米是一种盐敏感作物,而不同的玉米自交系之间,盐胁迫抗性相差很大。用高通量技术手段检测在盐胁迫条件下不同玉米自交系中表达量发生改变的转录本,对理解玉米盐胁迫响应的分子机制,揭示玉米自交系间盐胁迫抗性差异的分子基础意义重大。本研究利用高通量的技术手段SSH（消减杂交）和cDNA芯片技术检测了两个盐胁迫抗性差别很大的玉米自交系（盐胁迫敏感的黄早4和盐胁迫抗性的NC286）中表达量发生变化的转录本,认为这些转录本编码的蛋白质参与玉米的盐胁迫响应。许多调节因子和功能蛋白的编码基因在文库中被检测到。得到的主要结果包括:1、盐胁迫响应的蛋白激酶编码基因受到不同调节,而蛋白磷酸酯酶编码基因被诱导。说明盐胁迫条件下磷酸化/去磷酸化事件在胁迫信号传导中发挥作用。2、盐胁迫影响转录和翻译相关因子转录本的表达,进而影响转录、翻译的效率及特定转录本的转录和翻译。3、盐胁迫条件下,Ub/26S蛋白酶体系的作用加强,说明受损蛋白质增多,同时,蛋白酶体系的作用加强加快了氨基酸的代谢。4、盐胁迫抑制光合作用,从而影响能量供给。5、盐胁迫条件下,组蛋白和核糖体蛋白质编码基因倾向于被抑制表达,导致转录和翻译的全面抑制。6、盐胁迫下,细胞骨架成分蛋白质的编码基因倾向于受到抑制。7、与膜上物质转运相关的蛋白质编码基因都被盐胁迫条件诱导,加快膜上物质转运。对在SSH文库和cDNA芯片中共同检测到的8个ESTs,用RT-PCR技术构建了它们在不同胁迫（高盐、脱水和零上冷害）条件下玉米根系中的表达谱,发现盐胁迫和脱水胁迫的共同响应因子较多,而这两者与冷害的共同响应因子较少。microRNA是约21nt的非编码RNA,它在转录后水平调节基因的表达,广泛参与发育进程的调控,器官的极性,叶的生长及RNA代谢等。一些miRNA可被胁迫条件诱导,而且,一些miRNA的靶基因是胁迫响应基因,就暗示了miRNA可能在环境胁迫应答中起作用。利用一个含有10.1版本所有653个已知植物成熟miRNA探针的μParaflo^TM微流体芯片（LC science USA）,搜寻了玉米根系中盐胁迫响应的microRNAs。结果显示,在盐胁迫敏感性不同的两个玉米自交系中,存在相同响应的miRNA家族,但大多数盐胁迫响应miRNA家族在两个自交系中的表达趋势并不相同。在两自交系各个时间点表达趋势都相同的共4个,来自2个miRNA家族;在两自交系之间的表达趋势相同,但表达发生改变的时间点不同的9个,来自4个miRNA家族;在两自交系中都检测到表达改变,但表达趋势（抑制或诱导）不同的48个,来自13个miRNA家族;仅在某一个自交系中表达量发生变化的18个,来自8个miRNA家族。其中,在两个自交系中受到相同或相似调节的microRNA可能代表了共同的胁迫响应机制,而在两自交系之间受到不同调控的,尤其是仅在某一个自交系中表达量发生变化的miRNAs可用于解释两自交系不同的盐敏感性。对由高通量技术获得的盐胁迫响应ESTs和miRNA基因,分别预测了其启动子区域的主要胁迫响应cis因子。发现盐胁迫响应的编码基因和非编码基因启动子区具有共同的胁迫响应cis因子,其中,ABRE,ARE和MYBS位点的出现频率最高。这说明盐胁迫响应的蛋白编码基因与非编码基因受到相同的胁迫信号调节。更多还原

【Abstract】 Salt stress is one of the most serious abiotic stresses threaten the crop plants worldwide. High salinity causes serious damage to crop growth, resulting in lower quality and less yield. To survive under high salinity condition in their rooted lifestyle, crop plants have evolved a considerable degree of developmental plasticity, many genes change their expression profiles, which are involved in a broad spectrum of biochemical, cellular, and physiological processes. To cope with both ironic and osmotic stresses introduced by salt, many functional and regulating genes, including those involved in the cell detoxification, membrane transporting, energy maintaining, protein processing, and phytohormone responding, changed their expression profiles. In addition, these functional genes were regulated at different levels, such as the stress transduction cascades, which regulating gene expression at transcriptional level; miRNA dependent post transcriptional gene silencing were also involved in plant salt stress response .Maize （Zea mays L.） is one of the most important cereal crops in the world, it provides not only food and feed, but also material for bio-ethanol production, it is a crop that combined food, feed and fuel in one. Maize is salt-sensitive, and the salt sensitivities were distinct between maize inbred lines.Two maize inbred lines NC286 and Huangzao4 were selected with distinct salt stress sensitivity, SSH and cDNA chip hybridizing were processed between the salt stressed and un-stressed control materials. Differently expressed ESTs were detected. These ESTs were considered to be salt stress responsive ones, which involved in a vast spectrum of cellular and physiological processes.This research aimed in clarifies the genetic mechanisms of maize salt stress responsive and elucidates the relationship between differently expressed ESTs and distinct salt stress sensitivity. The main results derived from the high throughput methods were listed as following.1. Many protein kinase transcripts were differently regulated under salt stress, and the protein phosphatase transcripts were up-regulated, hinted that the reversible protein phosphorylation may play roles in salt stress signal cascades.2. Transcriptional and translational regulations occurred under salt shock.3. UB/26S mechanism was much more active under salt stress condition than in un-stressed control, which may resulted from the increasing of destroyed proteins. At the same time, activation of the protein degradation system may also enhance the re-use of amino acids.4. Salt stress may destroy the photosynthesis system, which in turn affect the energy supplement.5. Most of the histones and ribosomal proteins transcripts were repressed under salt stress, which may influence the repression of gene transcription and protein translation.6. Most of the ESTs encoding cellular framework proteins were repressed under salt shock.7. Most of the ESTs associated with membrane trafficking were detected to be up-regulated by the salt stress, which may play roles in re-establishing of homeostasis.In all, there are 8 co-detected ESTs from the SSH and cDNA chip. Expression profiles of these 8 ESTs under salt, dehydration and chilling stress were established. For most of the ESTs, the expression profiles under salt and dehydration stress were of the same, yet they were differing from those under chilling stress.Corn responds to salt stress via changes in gene expression, metabolism, and physiology. This adaptation is achieved through the regulation of gene expression at transcriptional and post-transcriptional levels. microRNAs （miRNAs） have been found to act as key regulating factors at post-transcriptional levels. However, little is known about the role of miRNAs in plants’ responses to salt stress.A customμParaflo^TM microfluidic array （LC science USA） containing Release version 10.1 plant miRNA probes （http://microrna.sanger.ac.uk/） was used to discover salt stress responsive miRNAs using the differences in miRNAs expression between the salt-tolerant maize inbred line NC286 and the salt-sensitive maize line Huangzao4.Salt responsive miRNAs are involved in the regulation of metabolic, morphological and physiological adaptations of maize seedlings at the post-transcriptional level. The miRNA genotype-specific expression model might explain the distinct salt sensitivities between maize lines.miRNA microarray hybridization revealed that a total of 98 miRNAs, from 27 plant miRNA families, had significantly altered expression after salt treatment. These microRNAs displayed different expression profiles under salt stress, and miRNAs belonging to the same miRNA family showed the same behaviour. Interestingly, we found 18 miRNAs were only expressed in the salt tolerant maize line NC286, and 25 miRNAs showed a delayed regulating pattern in the salt sensitive line.For all the salt responsive ESTs and miRNA genes, the key stress related cis-elements in their promoter regions were predicted. The results showed that the most frequently detected stress related cis-elements ABRE, ARE and MYBs were of the same in saltresponsive ESTs and miRNA genes, suggesting that the salt stress responsive coding andnon-coding genes were regulated by the same up steam factors.更多还原