【作者】 李坤朋；

【导师】 张举仁；

【作者基本信息】 山东大学 ， 细胞生物学， 2007， 博士

【摘要】 磷作为植物生长发育所必需的大量元素之一，它不仅是核酸和生物膜的重要组分，而且在能量代谢、光合作用、呼吸作用、酶活性调节、氧化还原反应、信号传导和碳代谢等方面也扮演重要角色。因此，磷几乎参与了植物所有的生命活动过程，它的缺乏必然严重影响植物的生长发育和谷物产量。作物主要通过根系吸收土壤中的可溶性磷酸盐来满足其磷营养需求。尽管在一些土壤中总磷含量是相当丰富，然而有效磷浓度却通常在1μM左右，远不能满足植物获取充足磷营养的需求，土壤有效磷不足已成为我国乃至全世界农业生产中限制谷物产量的主要因素之一。据统计，世界30％以上耕地的谷物产量受到磷缺乏限制。为解决土壤有效磷不足的问题，在1960-2000年间磷肥投入量增加了4-5倍，但施入的磷肥80％以上被土壤固定，并且在部分地区造成水域富营养化。照此下去，未来60-80年内地球磷矿资源将被耗尽，土壤有效磷不足和潜在的磷资源匮乏将成为未来农业的潜在危机。因此，利用作物固有的生物学特性，挖掘作物自身对土壤磷素高效吸收利用的潜力，培育磷高效作物品种，是农业可持续发展和解决上述缺磷及环境污染问题的一条最有希望的途径。玉米（Zea mays L．）在我国乃至世界粮食体系中占有举足轻重的地位。近年来土壤有效磷不足在许多地区限制了玉米产量、增加了其生产成本。由于玉米植株高大，生产上利用杂交优势，采用以生物量和产量为尺度筛选磷高效基因型，不仅周期长、工作量大，而且易受环境干扰、很难准确判断。植物生物技术的发展，为获得玉米磷高效基因型开辟了新途径，然而这需要充分了解玉米低磷耐受机制。因此，深入研究玉米磷耐受机制具有重要的理论意义和应用价值。近年来，植物耐低磷机制的研究非常活跃，也取得了重大进展。低磷耐受机制已被证明是多基因或QTLs控制的数量性状，一些磷饥饿响应基因在高等植物磷胁迫反应中的作用也已被初步了解。在转录组水平上的大量工作表明植物耐低磷胁迫反应是由复杂的调控系统控制的过程。然而，蛋白是细胞功能的执行者，蛋白质组学的发展为植物基因表达分析提供了较为准确和直观的手段，但在蛋白质组水平上研究植物根系对低磷胁迫应答的工作未见报道。在以前的工作中，本实验室利用细胞工程技术获得了一系列具有不同低磷耐受性的玉米材料。经过连续6代的自交选择和耐低磷性检测，我们发现自交系99038具有稳定遗传的低磷耐受特性和优良的农艺性状，是玉米磷高效育种和低磷耐受机制研究的好材料。本论文以低磷耐受性玉米自交系99038和来源亲本齐319为材料，对足磷和缺磷处理条件下两者的根系形态和生理生化特性进行了系统的比较；检测了低磷胁迫下齐319根系蛋白质组的变化并鉴定分析了大量差异表达的蛋白；分别对足磷和缺磷条件下的99038和齐319根系进行了系统的比较蛋白质组学研究，并结合两者的根系形态和生理生化特性差异对鉴定的蛋白进行了细致的分析，旨在找出99038较齐319具有更高低磷耐受性的主导因素，为通过基因工程手段进行玉米磷高效育种提供重要参考资料。一、玉米耐低磷自交系99038和对照齐319的根系形态和生理生化特性分析连续数代的观察发现，99038植株的地上部分与齐319没有明显差别，但根系较齐319的更发达，根系形态具有明显的差异。由于磷素在土壤中低的移动性，发达的根系对于植物高效吸收土壤磷素尤为重要。与齐319相比，99038小苗的三条最长根的平均长度、根冠比、侧根平均长度和单位质量茎叶所对应的根系吸收面积、根体积、侧根数目以及总根长均显著增加，根平均直径明显变细，这一系列变化扩大了根系与土壤的接触面积和涉猎的土壤体积，提高了99038对磷素的吸收能力。通过检测不同供磷水平下99038和齐319根系与茎叶的磷含量和磷浓度，以及植株体内磷素的分配，发现低磷胁迫下99038与齐319相比将更高比例的磷滞留在植株根部，根系含磷量是齐319的1.5倍，根系磷浓度显著高于齐319，其生长发育与齐319相比受抑制程度较小（与足磷条件下相比）。酸性磷酸酶活性分析表明，低磷胁迫下两个基因型玉米老叶和根尖部位的酸性磷酸酶活性均大大提高，但足磷和缺磷条件下齐319和99038相应部位的酸性磷酸酶活性没有显著差别，说明酸性磷酸酶活性不是造成两个基因型玉米低磷耐受性差异的主要因素。根系磷吸收动力学分析表明，在足磷和缺磷条件下99038较齐319均具有更高的I_max值，更低的K_m和C_min值，具有磷高效特性。根系蛋白表达谱分析得出，在低磷条件下99038和齐319根蛋白表达谱（pH3-10）中有13.6％的蛋白存在质或量的显著差异（P＜0.01）。基于这些结果，初步认为有可能99028和齐319根蛋白的差异表达导致两者根系形态和磷吸收能力的差异，从而导致99038低磷耐受性优于齐319；同时证明了细胞工程技术在玉米磷高效育种中具有重要的价值。二、低磷胁迫下齐319根系生长和代谢改变的蛋白质组分析在对齐319和99038的根系形态和生理生化特性进行分析中，我们发现低磷胁迫下尽管齐319利99038的根系形态和磷吸收特性存在明显差异，但它们的变化趋势是一致的，即与足磷条件下相比，它们的根冠比和单位质量茎叶所对应的总根长、侧根数目、根体积和根系吸收面积均显著增加，根平均直径明显变细，根系对磷素的吸收能力显著提高。为了深入了解玉米根系对低磷胁迫的适应机制，取低磷（5μM KH₂PO₄）和足磷（1000μM KH₂PO₄）处理17天的齐319根系进行蛋白质组差异分析。双向凝胶电泳检测出约1300个蛋白点，其中254个蛋白在足磷和缺磷条件下存在量的显著差异，占检测出的蛋白总数的19.5％。通过MALDI-TOFMS鉴定出120个差异表达的蛋白，并进行了功能分类分析。这120个鉴定蛋白分布在多个功能种类范围，包括44个代谢相关蛋白、13个蛋白命运相关蛋白、6个蛋白合成相关蛋白、5个能量代谢相关蛋白、9个次级代谢相关蛋白、7个细胞结构蛋白、11个抗性或与环境互作相关蛋白、3个转运相关蛋白、7个转录/细胞周期/信号转导相关蛋白、15个未分类或未知蛋白。其中有些蛋白，如tRNA异戊烯基转移酶、GTP结合的核蛋白RAN-B1、AraC type：Arac蛋白、嘌呤丰富元件的结合蛋白B类似蛋白和蛋白激酶等，参与激素合成、细胞周期、信号传导和转录调控等过程。这些蛋白可能在连接外部磷浓度改变和植物代谢适应性调控方面扮演重要角色，以调控基因表达，维持细胞相对磷稳态。上述结果从蛋白质组水平上揭示了植物磷饥饿响应是涉及多个信号和代谢途径的复杂过程，为进一步研究一些磷饥饿响应基因在植物磷营养中的功能提供了重要启迪。三、99038和齐319根系蛋白质组差异分析为了了解99038磷高效分子机制和究竟哪些蛋白的差异表达导致99038和齐319根系形态发育和磷素吸收能力的显著差异，我们通过pH3-6和pH5-8的IPG胶条对1000μM和5μM KH₂PO₄处理17天的99038和齐319根系进行了蛋白质组比较分析。足磷条件下，双向电泳分离出1260个蛋白点，其中135个（10.7％）蛋白点在99038和齐319根中存在量的显著差异；低磷条件下检测到1637个蛋白点，其中196个（12.0％）蛋白点在两个基因型间存在量的显著差异。这些结果表明无论在足磷或缺磷条件下99038和齐319的根系蛋白质组均存在较大差异。我们通过质谱鉴定出99038和齐319根系在足磷条件下呈现显著差异的79个蛋白点和缺磷条件下呈现显著差异的108个蛋白点，这些蛋白分布于广泛的功能种类范围。差异表达蛋白涉及碳代谢、激素合成、细胞周期和信号转导等途径，如碳代谢中的柠檬酸合酶、6-磷酸葡萄糖酸脱氢酶、丙酮酸磷酸二激酶、UDP-葡萄糖焦磷酸化酶，激素合成及活化途径中的C-24固醇甲基转移酶、β-糖苷酶，具有重要调控作用的GTP结合的核蛋白RAN-B1、importinα、MCM6、PP2A、14-3-3、MAPK1、CDC48等，这些蛋白可能在调控玉米根系形态发育、细胞周期以及感知外部磷浓度变化等方面扮演重要角色。结合99038和齐319根系形态和磷吸收能力的差异，推测这些差异蛋白很可能通过参与碳代谢，激素信号途径或细胞周期运行的差异主导两基因型根系生长发育和磷吸收能力的差异。此外，在鉴定的所有差异蛋白中，有18种同功蛋白在足磷和缺磷处理的两基因型根系中均具有相同的差异表达模式，包括细胞周期调控相关蛋白和大量碳代谢相关蛋白等，它们在99038和齐319根系中的差异表达可认为主要是由基因型不同所致。据此可推测99038和齐319在碳代谢、细胞周期调控等方面存在较大的基因型差异。基于上述结果，99038具有更发达的根系和更高的低磷耐受性与其碳代谢和细胞增殖调控有利于根系细胞分裂和生长等有关。同时，这些数据也加深了对一些磷饥饿响应基因在玉米磷效率调控中的作用的认识。本论文通过对耐低磷玉米自交系99038和其来源自交系齐319的形态学、生理学和蛋白质组学的研究，初步揭示了玉米磷高效自交系的根系形态、磷吸收动力学和蛋白质组的特点，揭示了低磷胁迫对玉米根系生长发育和蛋白质组的影响，证明了采用细胞工程技术能有效地创造出玉米磷高效育种材料，加深了我们对玉米低磷耐受机制和玉米磷效率遗传控制机制的认识，为进一步研究磷饥饿响应基因在植物磷营养中的功能提供了重要资料，为拟订玉米耐低磷基因工程实验方案提供了思路和目标，有助于解决我国乃至世界磷资源匮乏和磷肥利用效率不高等问题。更多还原

【Abstract】 As one of essential macronutrients for plant growth and development, phosphorus （P） not only is an important component of nuclear acid and biomembranes, but also plays an important role in an array of processes such as energy metabolism, photosynthesis, respiration, enzyme activation/inactivation, redox reactions, signal transduction, carbon metabolism etc. P is involved in almost all life activities, so its deficiency seriously affects the growth and yield of crops. Plants mainly absorb soluble phosphate （Pi） in soil by roots to satisfy the demand for P nutrition. Although total P is quite abundant in many soils, the concentration of available Pi by roots in soil is usually about 1μM, which cannot satisfy the demand of plants to acquire the sufficient Pi. The low availability of Pi in soil has been an important constraint for crop production in the agriculture. It is reported that crop yield on above 30% of the world’s arable land is limited by Pi availability. To cope with the problem, P fertilizer use increased four- to five-fold betweenl960 and 2000, however, up to 80% of which might be fixed in the soil, and caused water eutrophication in some areas. If this situation continues, rock Pi reserves could be depleted in as little as 60-80 years. Insufficient of available Pi by roots in soil and lack of rock Pi reserves are potential crisis for agriculture in the future. Therefore, exploiting biological characteristics of crops and developing P-efficient cultivars, to acquire P in soil more efficiently, may be the most promising way of achieving sustainable agricultural development and solving the problem of P-deficit and environment pollution.Maize （Zea mays L.） occupies a pivotal position in China and even the world food system. In recent years, maize growth usually meets the problem of P-deficit, which is an important limiting factor for yield and increases its production cost. Maize plant is tall and large, and heterosis is utilized in agricultural production. Screening P-efficient maize genotypes according to biomass and yield is not only time-consuming and labor-intensive, but also easily influenced by environment. Development of plant biotechnology provides a new way to obtain P-efficient maize genotypes, while understanding the low-P tolerance mechanism of maize well is required. Therefore, the further study of low-P tolerance mechanism of maize is of important theoretical significance and practical value.In recent years, the studies of low-P tolerance mechanism in plants were active and have made great progress. It has been showed to be a quantitative trait controlled by multi-gene or QTLs, and some individual genes responsive to P-starvation in higher plants have been characterized. Some researches on transcriptome suggested that the response of plants to P-stress is a metabolic process controlled by a complicated regulation system. It is well known that protein is the bearer of cell functions, and the development of proteomics provides a more precise and intuitive way to analyze the gene expression. However, to the best of our knowledge, the analysis of roots response to P-stress at the proteome level has not been available for any plant by far.Previously, our lab obtained an array of maize inbred lines with different low-P tolerance using cellular engineering technology. During the process of low-P tolerance determination and conformity analysis for six generations, we found that the low-P tolerant trait of 99038 with good agronomic traits could be stably inherited, which is an elite material for maize P-efficiency breeding and the study of low-P tolerance mechanism. In the study, low-P tolerant maize inbred line 99038 and the original line Qi-319 were used to study the low-P tolerance metabolism. The root morphology, and physiological and biochemical characteristics in 99038 and Qi-319 under +P （1000μM KH₂PO₄） or -P （5μM KH₂PO₄） condition were studied; the proteome changes of Qi-319 roots responsive to P-starvation were analyzed systemically, and a large number of differentially expressed proteins were identified; the comparative proteome analyses for proteins isolated from roots of 99038 and Qi-319 under +P or -P condition were conducted, respectively, and differentially expressed proteins were analyzed combining with their difference of root morphology and physiological and biochemical characteristics. The aim is to investigate the main reasons for higher low-P tolerance of 99038, compared to Qi-319, and also offer the valuable information for P-efficiency breeding by genetic engineering.1. Analyses of root morphology and physiological and biochemical characteristics of low-P tolerant maize inbred line 99038 and control Qi-319During the process of observation for continuous multi-generation, we found out that plants from 99038 and Qi-319 showed no obvious difference on shoots but substantial difference on roots morphology, and roots of 99038 were more developed than Qi-319. It is well known that developed roots are especially important for P-acquisition because of its relative immobility. Total root length, the number of lateral root, root absorptive surface area and root volume corresponding to unit shoot weight, root-shoot ratio, the average length of lateral root and the three longest roots of 99038 seedlings were increased significantly, while average root diameter was decreased obviously, compared to Qi-319. These changes expanded the root surface-soil contact area and explored a larger volume of soil, which improved the capacity of P-acquisition of 99038. Under +P or -P condition, the determination of P content, concentration and distribution in 99038 and Qi-319 plants showed that under P-stress 99038 distributed the greater proportion of P to the roots, P content of roots in 99038 increased to 1.5 folds, P concentration in the roots of 99038 was higher significantly and root growth was less restrained, compared to Qi-319. APase activity assay showed that under P-stress APase activity of old leaves and root tips of both maize genotypes increased significantly. However, whether under -P or +P condition, APase activity of corresponding to the area of 99038 and Qi-319 showed no significant difference. Therefore, APase activity was not the main factor for different low-P tolerance of 99038 and Qi-319. Under +P or -P condition, the analysis of P-uptake kinetics also suggested that the I_max value of 99038 was higher and K_m, C_min values were lower, compared to Qi-319. Namely, 99038 is with P-efficiency trait. Analysis of 2-DE gel maps （pH 3-10） of proteins from roots of 99038 and Qi-319 plants grown under -P condition showed that 13.6% protein spots changed significantly in amount （P < 0.01） . Based on the results above, it could be concluded that the different expression of proteins in roots of 99038 and Qi-319 might result in the differences of root morphology and the capability of P-absorption in two genotype maize, which lead to higher low-P tolerance of 99038 than Qi-319. Simultaneously, the results also confirmed that cell engineering technology is of important value in P-efficiency breeding of maize.2. Proteomic analysis of roots growth and metabolic changes under P deficit in Qi-319 maize plantsThe analysis of root morphology and physiological and biochemical characteristics of Qi-319 and 99038 indicated that under P-stress root morphology and P-absorptive characteristics of them were significantly different, but their change tendencies were consistent. Under P-stress total root length, the number of lateral root, root absorptive surface area and root volume corresponding to unit shoot weight and root-shoot ratio of both genotypes were increased significantly, average root diameter was decreased obviously and the capacity of P-absorption were enhanced markedly etc., compared to +P condition. In order to understand the mechanism of maize roots responsive to P-deficiency, we conducted the comparative proteome analysis for proteins isolated from Qi-319 roots treated with 1000μM KH₂PO₄ （control） or 5μM KH₂PO₄ for 17 days. Approximate 1300 protein spots were detected by 2-DE, 254 （19.5 %） of which changed significantly in amount （P < 0.05; = 2 folds）. One hundred and twenty differentially expressed proteins were identified by MALDI-TOF MS and classified according to Arabidopsis MATDB. The 120 identified proteins represented a large range of functional categories, including 44 proteins in metabolism, 13 proteins in protein fate, 6 proteins in protein synthesis, 5 proteins in energy, 9 proteins in secondary metabolism, 7 proteins in cellular organization, 11 proteins in defense/interaction with environment, 3 proteins in transport, 7 proteins in transcription /cell cycle/signal transduction and 15 unclassified or unknown proteins. Several proteins, such as tRNA isopentenyl transferase, GTP-binding nuclear protein RAN-B1, AraC typr: Arac protein, purine rich element binding protein B-like and protein kinases, were involved in the biosynthesis of phytohormone, cell cycle, signal transduction and transcriptional regulation. They might play important roles in linking the change of external P concentration and the metabolic adaptation of plants, to regulate gene expression facilitating P-homeostasis. Those results above suggested that the low-P tolerance of plants is involved in a complicated process related with multiple metabolic and signal pathways at proteome level and also offered the valuable information for further characterization of function and regulation of P starvation responsive genes in plant P-nutrition.3. Difference analysis of roots proteome in 99038 and Qi-319In order to understand P-efficiency mechanism of 99038 and the different expression of which proteins resulting in the differences of root morphology and capacity of P-acquisition between both genotypes, we conducted comparative proteomic analysis for roots of Qi-319 and 99038 treated with 5μM or 1000μM KH₂PO₄ using pH 3-6 and pH 5-8 strips. Under +P condition, 1260 proteins were detected on 2-DE gels, 135 （10.7 %） of which were significantly different in amount （P < 0.05; = 2 folds）; under -P condition, 1637 proteins were detected, 196 （12.0 %） of which were significantly different in amount. Therefore, whether under -P or +P condition, the roots proteome of 99038 and Qi-319 were significantly different. Seventy nine （+P） and 108 （-P） differently expressed proteins were identified by MALDI-TOF MS. They represented a large range of functional categories, respectively. Differently expressed proteins were involved in carbon metabolism, phytohormone synthesis, cell cycle, signal transduction etc., such as citrate synthase, 6-phosphogluconate dehydrogenase, pyruvate phosphate dikinase, UDP-glucose pyrophosphorylase, C-24 sterol methyltransferase,β-glucosidase, GTP-binding nuclear protein RAN-B1, importin a, MCM6, PP2A, 14-3-3, MAPK1, CDC48 and so on, which might play important roles in regulating root morphology and cell cycle, sensing the change of external Pi concentration etc.. Based on functional analyses of differently expressed proteins identified, combining the differences of root morphology and P-absorption ability between 99038 and Qi-319, we presumed that these differently expressed proteins were very likely to result in the difference of root morphology and P-absorption ability by participating in the regulation of carbon metabolism, phytohormone signal pathways or cell cycle. Moreover, in all differently expressed proteins identified, 18 protein species with the same or similar function changed in the same pattern under +P or -P condition, including cell cycle regulation related proteins, several carbon metabolism related proteins etc., different expressions of which in roots of both genotypes were thought to be caused by genotypic differences. Therefore, it is presumed that there were great genotypic differences in carbon metabolism and cell cycle regulation between 99038 and Qi-319. Based on the results above, the more developed roots and higher low-P tolerance of 99038 than those of Qi-319 were related with its carbon metabolism and cell proliferation regulation, which were more advantageous for root cell division and growth. Simultaneously, these data also promoted our understanding on the role of some P-starvation responsive genes in maize P-efficiency control.In this study, the analyses of maize low-P tolerance mechanism were conducted on root morphology, physiology and proteome with low P tolerant inbred line 99038 and original inbred line Qi-319. The results showed the characteristics of P-efficient maize inbred lines in root morphology, P uptake kinetics and proteome, reflected the effects of low-P stress on the growth and development, and proteome of roots, confirmed that P-efficient maize breeding materials could be obtained by cellular engineering technology, promoted our understanding on mechanisms of maize low-P tolerance and P-efficiency genetic control. All those offered the valuable information for further study in functions of P starvation responsive genes in plant P-nutrition, and also provided ideas and objects for establishing the experiment designs for improving low-P tolerance of crops by genetic engineering, thereby contributing to resolve the status of P-deficit and low utilization of P-fertilizer in the agricultural production of China and even the world.更多还原