【作者】 冯海龙；

【导师】 孟庆伟；

【作者基本信息】 山东农业大学 ， 植物学， 2013， 博士

【摘要】 在自然界中，植物不可避免地遭受各种环境因子的刺激，例如低温，干旱和盐渍胁迫。在长期的进化过程中植物形成了响应胁迫信号和提高自身抗性的应变机制，它们通过调节抗性基因的表达来引起生理生化反应，在体内建立新的物质和能量代谢平衡，从而在环境改变后得以存活。低温冷害作为一种常见的非生物胁迫，它影响植物的生长，生存和时空分布。经常会对作物生长和农业生产带来负面的影响。低温冷害对于植物细胞的直接影响是使之代谢缓慢，间接影响是引起渗透胁迫和氧化胁迫等。温带植物在遭受严寒时，自身会主动引起生理生化反应，这一过程称为冷驯化或冷锻炼，进而提高其耐冷性。ICE转录因子是植物冷驯化过程中的一个关键“开关”，它响应低温信号，并通过调控CBF/DREB1转录因子进而激活下游COR（cold-regulated）基因的表达，从而调节植物的耐低温能力。目前关于ICE基因的研究多集中在拟南芥，小麦等耐冷植物中，对于冷敏感植物番茄的研究较少。番茄是重要的蔬菜作物，分布广泛，具有重要的经济和社会效益。本研究从番茄叶片中克隆到一个新的ICE1基因，对其进行了表达和功能分析，具体结果如下：（1）利用同源克隆的方法分离了番茄中一个新的MYC类的bHLH转录因子，并命名为SlICE1a，GenBank注册号为JX625139。该基因ORF为1596bp，编码一条531个氨基酸的多肽，预测分子量为58.3kDa。蛋白质结构中包含了保守的bHLH和ACT-Like结构域，以及丝氨酸富集区和潜在的SUMO结合位点。（2）将SlICE1a与GFP融合后在洋葱表皮细胞瞬时表达发现，SlICE1a蛋白定位于细胞核中；通过与GAL4-BD融合后在酵母中表达发现，SlICE1a具有转录激活能力，且激活结构域位于蛋白的N末端。（3）利用荧光定量PCR的方法对SlICE1a基因在番茄中的表达特性进行了研究。结果表明，SlICE1a基因在叶片中表达量高于其它器官；转录水平受到低温、氧化、干旱以及高盐等非生物胁迫和ABA等信号分子的诱导表达，所以我们认为SlICE1a可能在植物响应胁迫信号过程中起重要作用。（4）构建由35S启动子驱动SlICE1a的植物表达载体，利用农杆菌介导的方法转化烟草，转基因烟草经卡那霉素筛选后进一步RT-qPCR分析，选取了表达量不同的三个株系（L8，10，26）T3代植株进行后续试验。在正常生长条件下，转基因植株表型没有发生改变。（5）与野生型植株相比，过表达SlICE1a一定程度上提高了烟草的冷驯化能力，并且显著提高了抵御渗透胁迫，盐胁迫和氧化胁迫的能力。过表达SlICE1a基因烟草在胁迫处理时可以激活并提高下游CBF/DREB以及多个逆境响应基因的表达，尤其是与渗透调节物质合成相关的基因。转基因植株积累了更多的游离型脯氨酸和可溶性糖，来维持细胞渗透平衡和内环境稳态，清除了更多的活性氧物质，有利于维持细胞膜的稳定性，进而降低了环境剧变引起的渗透和氧化伤害，提高了其抗逆性。（6）分析发现SlICE1a可以结合下游SlCBF1和SlCBF3的启动子上的MYC特异序列，说明在冷敏感作物番茄中，同样存在重要的ICE-CBF低温信号通路。由于过表达番茄ICE1和CBF转录因子都能够提高转基因植物的耐低温能力，我们认为位于番茄CBF下游的调控网络中的基因可能是导致番茄对低温敏感的决定性因素。这些研究结果，不仅使我们进一步的了解了ICE转录因子在植物应答非生物胁迫中的重要作用，并且对于通过基因工程手段改良植物的抗逆性具有一定的理论和实践意义。更多还原

【Abstract】 In the natural environment, plants are unavoidable subjected to the environmentalstresses, such as drought, high salinity and low temperature. Plants adapt to these stressesthrough the physiological and biochemical changes, including the expression of stressresponsively functional and regulatory genes, which reprogram the biological activities andestablish a new metabolism balance.Low temperature is an important environmental factor limiting the geographicaldistribution and growing season of plants. Cold stress often affects plant growth and cropproductivity, which causes significant crop losses. Cold stress limits the normal growth anddevelopment of plants directly by the inhibition of metabolic reactions and indirectly throughcold-induced osmotic and oxidative stresses. Temperate plants are capable of developingchilling tolerance when they are exposed to low non-freezing temperatures, a process knownas cold acclimation, which is associated with biochemical and physiological changes. ICE(inducer of CBF expression) is a key transcription factor in cold signaling pathway as well asa master regulator of cold acclimation. It regulates the expression of CBF/DREB1by bindingto MYC recognition elements and activate COR (cold-regulated) genes in turn.So far, researches about ICEs are mainly focus on the LT-tolerant plants, such asArabidopsis and wheat, and the information of ICE in tomato is relatively limited and hasemerged only recently. Tomato is an important vegetable around the world, while it ischilling-sensitive and incapable of cold acclimation. Thus, the research of chilling tolerancemechanism is an important theoretical and practical value. A series of studies have beenconducted on the isolation, sequence and expression analysis, function identification ofSlICE1a. The main results are as follows:(1) We isolated a novel MYC-type bHLH transcription factor, designated SlICE1a(JX625139), from tomato. It consists of a1596bp open reading frame (ORF) that encodes apolypeptide of531amino acids with a predicted molecular weight of58.3kDa. SlICE1acontains all conserved bHLH domain, S-rich region and ACT-domain, and a putative SUMO conjugation motif.(2) To observe the subcellular localization of SlICE1a, an expression cassette wasconstruct by fusing SlICE1a with the GFP reporter gene, and expressed transiently in onionepidermal cells. For transcriptional activation analysis, different coding regions of SlICE1awere constructed with the sequence of GAL4DNA-binding domain, respectively. The fusionconstructs were introduced into yeast strain Y2HGold. It is confirmed that SlICE1a localizesto the nucleus and harbors transcription-activating activity in the N-terminal.(3) RT-qPCR analysis showed that the expression of SlICE1a was stronger in leaves thanin other tissues. SlICE1a transcript was slightly up-regulated by cold, high salt, osmotic andoxidative stresses, and ABA can also induce the expression of SlICE1a. These results indicatethat SlICE1a may be involved in the adaptive responses to abiotic stresses.(4) The expression vector pBI-SlICE1a was constructed and transformed into tobaccoplant (Nicotiana benthamiana) with Agrobacterium tumefaciens-mediated leaf disc method.Transformants were evaluated by RT-qPCR after the filtering with kanamycin. Base on thevarious advanced levels of SlICE1a over-expression in transgenic lines, L8, L10and L26(T3)were chosen for further experiments. There were no obvious morphological differencesbetween WT and transgenic plants during all lifecycles under normal growth conditions.(5) SlICE1a conferred transgenic tobacco enhanced tolerance against cold, osmotic,oxidative and high salinity. Over-expression of SlICE1a in tobacco enhanced the induction ofCBF/DREB1and their target gene, consequently elevated the levels of proline, soluble sugarsand LEA proteins, which play important roles in stabilizing membranes of plant cells and inalleviating osmotic and oxidative injuries induced by many abiotic stresses.(6) SlICE1a could bind to two MYC DNA fragment in SlCBF1and SlCBF3promoter,indicating that tomato has complete ICE-CBF cold response pathways. Since both SlICE1aand SlICE1can transactive the expression of cold-responsive genes and regulate coldtolerance, suggesting that some downstream factors may be involved in chilling-sensitivity oftomato.These results broaden our knowledge about ICE genes in stress tolerance and provide a new clue for the application of genetic engineering in crop improvement.更多还原