【作者】 王慧慧；

【导师】 齐晓花； 陈学好；

【作者基本信息】 扬州大学 ， 园艺学（专业学位）， 2015， 硕士

【摘要】 黄瓜(Cucumis sativus ..2n=2x=14)起源于喜马拉雅山南麓的热带雨林区,为葫芦科甜瓜属一年蔓生草本植物。我国是黄瓜生产大国,2010年栽培总面积达98.9万公顷,年产量达4070.9万吨。随着全球气候变暖和雨涝灾害的频繁发生,涝胁迫已成为严重危害作物生长发育甚至死亡的逆境因素。我国夏季多爆发性大雨和连续性洪涝灾害,南方地区尤其明显。黄瓜根系具有浅根性、通气组织不发达、吸收能力弱、再生能力差等特点,因此极易受到涝胁迫危害。涝胁迫下黄瓜植株可以通过茎部形成不定根逐渐替代衰老的原生根系,从而缓解涝胁迫危害。有关涝胁迫诱导黄瓜不定根形成的机制尚缺乏研究。已有研究表明,过氧化氢(Hydrogen peroxide H2O2)在提高植物的抗逆能力,缓解逆境伤害等方面具有重要作用。本研究以耐涝性较强的黄瓜品系“早二N”为供试材料,首先探明H202是否参与涝胁迫下黄瓜不定根的形成,进而探讨H2O2调控黄瓜不定根形成的可能机制。主要研究结果如下：(1)黄瓜幼苗主要通过植株茎部形成不定根来缓解涝胁迫伤害。涝胁迫处理第三天,茎部产生白色根原基突起；第四天,少量根原基突起撑破表皮,形成短小的不定根；第五天时,大量的不定根形成且不断伸长。通过显微观察发现,不定根发生的进程为：12～24 h维管束中的束中形成层细胞启动脱分化、不断分裂增殖；24～48 h该细胞团继续脱分化形成不定根原基；48～72 h不定根原基细胞分裂增殖发育,并向皮层细胞穿透；72～96 h之后大量不定根突破表皮,并继续伸长。(2)通过测定涝胁迫下黄瓜幼苗茎部ROS含量发现,与对照相比,涝胁迫下黄瓜幼苗茎部超氧阴离子产生速率迅速增加且在8h时达到最大值；H2O2含量亦呈显著上升趋势,4h达到最高水平。(3)10mM的H2O2处理可以促进涝胁迫下黄瓜幼苗不定根的形成,不定根数目和长度与对照相比,分别提高了24.76%和14.87%；根系活力明显高于对照63.09%；而5μM的DPI(H2O2生成抑制剂)处理则显著抑制涝胁迫下黄瓜幼苗不定根的生成,不定根数目和长度分别比对照减少49.21%和43.08%；根系活力降低24.63%。(4)H2O2结合涝胁迫处理显著提高黄瓜幼苗茎部的H2O2含量,对超氧阴离子生成速率无显著影响；DPI结合涝胁迫处理后黄瓜幼苗茎部的H2O2含量和超氧阴离子生成速率显著增加,这可能与外源DPI对植株的损伤有关；外源H2O2结合涝胁迫处理后茎部SOD、CAT的活性增加,说明这两种酶在清除H2O2过程中起主要作用。(5)利用qRT-PCR检测9个RBOH家族基因在涝胁迫、外源H2O2和DPI结合涝胁迫处理后在黄瓜幼苗下胚轴内的表达水平变化,结果表明：CsRboh02和CsRboh06在涝胁迫下表达量显著升高,且与植株体内H2O2水平变化趋势一致,说明上述两个基因可能参与促进涝胁迫下黄瓜茎部H2O2的生成；H2O2结合涝胁迫处理后CsRboh06上调表达,变化趋势与H2O2含量变化一致,说明该基因可能参与调控外源H2O2结合涝胁迫处理诱导黄瓜茎部H2O2生成的过程。CsRboh02 和 CsRboh06可能参与调控涝胁迫下黄瓜不定根形成。(6)利用qRT-PCR技术,检测3个MT家族基因在涝胁迫、外源H2O2和DPI结合涝胁迫处理后表达水平的变化,结果表明：涝胁迫下,MT2基因的表达量显著下调；H2O2结合涝胁迫处理后MT2基因在0-8h表达量下调；MT2基因在涝胁迫下受到ROS信号抑制,导致了ROS的积累,从而促进黄瓜幼苗不定根的形成。更多还原

【Abstract】 Cucumber (Cucumis sativus L.2n= 2x= 14) is cucurbitaceae annual creeping herb which originates from the tropical rain forest area in the south of Himalayan mountains. Cucumber is one kind of the most important vegetable in China. The culitivation area of cucumber in China was 98.9 hectares, and with annual production of 40.709 million tons in 2010. As global wanning and flood disasters occurred frequently, waterlogging has became one of the most frequent and extensive abiotic stresses that negatively influence crop growth and productivity, and in some case resulting in plant death. Cucumber is easily affected by heavey rain and subsequent periods of soil flooding in summber, especially in South China because of its shallow-rootedness, less developed aerenchyma, weak absorption ability and poor regeneration ability. However, the adventitious root could be formed in cucumber plants under waterlogging condition which could replace some function of taproot, for lightening the damage caused by waterlogging. Research relate the mechanism of the waterlogging stress to induce the formation of adventitious roots in cucumber seedlings is still very lack. Previous research showed that hydrogen peroxide played important roles in improving plant resilience and mitigating injury. One waterlogging resistant line "Zaoer N" was applied in present study to gain insight into the physiological response of cucumber under waterlogging, and find out the possible role of H2O2 during the adventitious roots formation of cucumber. The main results were as follows:1. The most important trait for survival in waterlogging environment is adventitious roots formation of cucumber plants. After 3 d of waterlogging, adventitious root primoidia were visible on the hypocotyl surface. After 4 d, a few of adventitious root primordia were elongate to emerge from the shoot epidermis. After 5 d, numerous adventitious roots protruded from the lower part of the submerged hypocotyl. Microscopic studies revealed that root primordial had already formed 12-24 h after the start of the experiment. Primordia continued to grow and most reached the epidermis after 48 h of submergence. After 72 h, the root primordial penetrates the hypocotyl surface.2. Compared with the control plants, the superoxide anion producing rate increased quickly in waterlogged cucumber, and reached the peak level at 8 h after treatment. H2O2 content also increased significantly and maintained high level in 4 h.3. The treatment of 10 mM H2O2 could promote the adventitious roots formation of cucumber seedlings under waterlogging stress. The number and length of adventitious roots increased 24.76% and 14.87%, respectively. The root activity was also significantly higher than control about 63.09%. However,5 μM DPI treatment obviously inhibit the adventitious root formation. The number and length of adventitious roots reduced 49.21% and 43.08%, respectively. The root activity was also significantly reduced 24.63%.4. The H2O2 content of H2O2 pretreatment cucumber increased compared with the control plants. There are no significant changes of superoxide anion producing rate. However, the content of H2O2 and the superoxide anion producing rate increased in the DPI pretreated plants. It might be due to the oxidative stress caused by DPI. The activities of SOD and CAT increased in H2O2 pretreatment cucumber indicated that the previous two kind of enzyme might be play important role in eliminating the ROS.5. qRT-PCR technology was applied to analyze the expression levels of 9 RBOH genes under waterlogging, H2O2 pretreatment and DPI pretreatment. The results showed that the expression levels of CsRboh02 and CsRboh06 increased under waterlogging condition, and the change was in accordance with the change of H2O2 content. The expression level of CsRboh06 was up-regultaed by H2O2 pretreatment. It indicated that CsRboh02 and CsRboh06 might play important roles for ROS accumulation and adventitious root formation of cucumber under waterlogging stress.6. qRT-PCR analysis showed that the expression level of MT2 was down-regulated under waterlogging and H2O2 pretreatment. It indicated that the inhibition of MT2 gene might cause the ROS accumulation of cucumber under waterlogging condition.更多还原