【作者】 李冬梅；

【导师】 高东升；

【作者基本信息】 山东农业大学 ， 设施农业科学与工程， 2012， 博士

【摘要】 木本植物的芽休眠是植物在生长发育过程中的一种暂停现象，是植物经过长期演化而获得的一种对不良环境及季节性变化的生物学适应性。这种生物学特性不仅对物种的生存繁衍具有特殊的生物学和生态学意义，而且对设施农业生产也有十分重要的应用价值。设施果树生产已经成为果树生产的重要分支，休眠是限制设施果树促成生产的关键因素，因此研究设施果树休眠进程有利于进行果树的熟期调控，具有重要的现实意义。蛋白质组学是以器官或组织的全部蛋白质作为研究对象，从蛋白质整体水平上来认识生命活动规律。通过研究不同光周期条件下桃树组织中蛋白质的变化，可以对其表达模式和功能模式进行分析。本研究以6年生低需冷量设施桃专用品种“春捷”[Prunus percica cv. Chunjie]为材料，通过人工设置长日照和短日照的光周期条件，研究了不同光周期对休眠的诱导效应以及休眠进程中桃树叶片和芽体的生理生化响应，同时利用高通量蛋白质组学技术平台分析了诱导期蛋白质表达特征，揭示了光周期诱导设施桃树休眠进程中差异蛋白质的表达规律及其所属功能类群。主要研究结果如下：1、短日照（SD）可以诱导桃芽进入自然休眠，较自然条件（CK）下提早1周，效果显著；长日照（LD）推迟该诱导作用的发生。休眠诱导进程呈阶段性发展。2、休眠诱导期内，叶片的可溶性糖和可溶性蛋白含量降低，淀粉积累升高。长日照有利于减小可溶性糖和可溶性蛋白的降低幅度，降低淀粉含量，短日照作用反之。休眠诱导期内，叶片总含水量、自由水含量及其所占总含水量的比例降低，束缚水含量及其所占总含水量的比例升高。LD处理提高了诱导期后期叶片超氧化物歧化酶（SOD）和过氧化氢酶（CAT）的活性、束缚水含量及其占总含水量的比值、脯氨酸含量，降低了丙二醛（MDA）含量和伤害率，表现出较强的抗逆性。SD处理下叶片抗性响应迅速，但持续时间短。3、休眠诱导期间，净光合速率、气孔导度降低，胞间CO2浓度升高，核酮糖-1,5-二磷酸羧化酶/加氧酶（Rubisco）降解、光合酶活性降低，说明休眠诱导期叶片光合速率下降的原因为非气孔因素，主要是光合酶的降解和活性下降所致。SD诱导下叶片的光合和荧光性能低于CK，主要原因在于锰稳定蛋白/光系统Ⅱ多肽超级家族类蛋白表达下调和光合酶降解。C4途径关键酶磷酸烯醇式丙酮酸羧化酶（PEPC）和C3途径关键酶核酮糖-1,5-二磷酸羧化酶（RuBPC）的活性在休眠诱导期呈降低变化。LD处理的叶片中PEPC/RuBPC比值显著或极显著低于CK，SD处理则高于CK，推测长日照条件下桃树叶片的光合途径趋向C3途径，而短日照下则趋向C4途径以提高光合速率。4、休眠诱导期内花芽底物氧化水平上磷酸戊糖途径呼吸速率和电子传递水平上交替途径呼吸速率升高，所占总呼吸速率的比例均加大，两个呼吸途径的活化是休眠诱导期重要的呼吸特征，SD处理高于CK高于LD处理。但就所占比例而言，三羧酸循环和细胞色素电子传递途径仍然是底物氧化水平和电子传递水平的主路呼吸。5、利用三氯乙酸-丙酮沉淀法提取桃树叶片和花芽总蛋白，建立了适合叶片和花芽的双向电泳技术体系。ImagemasterTM2D Platinum Software分析所得凝胶图像，每张凝胶均获得了500多个蛋白质胶点。不同光周期诱导的休眠进程中叶片和花芽的蛋白质组均有变化，两者共筛选出65个差异蛋白质点，MALDI-TOF/TOF方法鉴定出42个差异蛋白，涵盖8大功能类群：生物学进程调控；细胞周期、构建及调控；应激响应；新陈代谢过程；大分子复合体或细胞器组分等；定位、转录调控活性；催化或抗氧化活性；未知功能蛋白。初步明确了部分参与光周期休眠诱导的蛋白质种类、表达变化规律和生物学功能，为从分子水平上揭示桃树响应光周期诱导休眠机制的研究奠定了基础，为深入了解光周期诱导下休眠进程的调控机制提供了依据。更多还原

【Abstract】 The bud dormancy in perennial plants is a temporary stagnation during growth anddevelopment, which formed during a long evolutional process, is a biological adaptability tothe changes of environmental factors and different seasons. In particular, the characteristicfeature is not only of biological and ecological interest in survive and multiplication ofspecies, but of important application value in agricultural facilities production. The produc-tion of facilities fruit tree had been an important branch of fruit tree production, dormancy isone of key factors to limit the forcing production of facilities fruit tree. So the regulationstudy on dormancy process of facilities fruit tree has an important realistic significance, whichcan regulate the mature period of fruit tree. Proteomics make all the proteins of the organ ortissue as the research objects, understand the laws of science life activities from the overalllevel. Protein expression and function mode in peach tissues could be analyzed by studying itschanges under different photoperiod conditions.Differential photoperiods (long day and short day) were set up to investigate the role ondormancy process and physiological changes in6-year-peach [Prunus persica cv. Chunjie]leaves and buds during dormancy induction. At the same time, characteristics of proteinexpression were analyzed by using high-throughput proteomics technology platform, and thedifferentially expressed proteins and their functional group were revealed.The main results were as follows:1. Short day (SD) can induce buds to natural dormancy significantly,1week earlier thannatural condition (CK), long day (LD) delayed the occurrence of induction. The dormancyinduction process was phased development.2. Soluble sugar and soluble protein were decreased, and starch content was increasedduring dormancy induction period. LD favored to reduce the starch content and the decreasingamplitude of soluble sugar and soluble protein. SD played contrary roles.Totle water content, free water content and the contribution rate of it to totle watercontent were decreased, bound water and the contribution rate of it to totle water content wasincreased during dormancy induction period. LD treatment increased SOD and CAT activities,bound water and the contribution rate of it to totle water content, proline contents in leaves in late stage of induction period, decreased MDA content and injury rate, which performance outof the strong resistance. The leaf resistance of SD treatment responsed rapidly, the durationwas short, however.3. Net photosynthetic rate, stomatal conductance and photosynthetic enzyme activityreduced, intercellular CO2concentration rised, and Rubisco degradated during dormancyinduction, which indicated that the reason of photosynthetic rate decline was due to thedeterioration and activity decrease of photosynthetic enzymes, which was non-stomatalfachtors. Photosynthetic performance in short-day leaves was lower than the natural leaves,the main reason was the manganese stabilizing protein/photosystem Ⅱ polypeptidesuperfamily-like protein is downregulated.Activities of the C4pathway key enzyme PEPC and the C3pathway key enzyme RuBPCwere reduced in dormancy induction period. The ratio of PEPC/RuBPC in LD leaf was lowerthan CK significantly or very significantly, the SD was higher than that of CK. Photosyntheticpathway was speculated tend to the C3pathway under long-day conditions and the C4pathway under short day.4. PPP respiration in substrate oxidation level and alternative pathway respiration in electrontransfer pathway level was increased during dormancy induction, and the contribu-tion rate tothe total respiration rate increased, which were the remarkable characteristics of dormancyinduction. The two different types of respiration rates of SD treatment higher than that of CK,LD treatment contrarily. According to the respective contributions of the respiration pathwaysto the total respira-tion rate, TCA cycle and cytochrome electron transport pathway were stillthe main pathways of substrate level and electron transport level respectively.5. The2-DE system was conducted to be suitable for peach leaves and flower buds byusing TCA-acetone precipitation method. The gels were analyzed by ImageMasterTM2DPlatinum software. More of500reproducible protein spots were detected, among which65protein spots were differential in both leaves and flower buds (the criterion was thedifferential protein spots changed their intensities significantly (P<0.05) by more than2.0folds),42differential protein spots were identified by MALDI-TOF/TOF MS and databasesearching. Which were classified into8groups: regulation of biological process; cellularprocess, part and regulation; response to stimulus; metabolic process; macromolecular complex and organelle part; localization, transporter activity; catalytic and/or antioxidantactivity, unknown protein. This study initially cleared that some types, expression andfunction of proteins involved in photoperiodic dormancy induction, which provided basis forrevealing peach response and process regulation to photoperiodic induction mechanism ofdormancy.更多还原