【作者】 李凌；

【导师】 李道高； 周泽扬； 裴炎；

【作者基本信息】 西南农业大学 ， 果树学， 2002， 博士

【摘要】 碱性及钙质土壤上粮食作物和果树的缺铁现象在世界范围内普遍存在。铁在土壤中尽管含量很丰富，但通常都呈不溶状态很难被植物吸收。据统计，全球约25％～30％的土壤存在潜在的植物缺铁现象。植物缺铁，叶片脉间失绿，叶肉发黄甚至变白，叶片缩小，如果缺铁症状严重，会引起生长停滞甚至死亡，给农业生产造成严重损失。果树作为一种与人民生活密切相关的重要经济作物，由于其本身的特点，缺铁造成的产量损失远比一年生作物严重，因为果树一旦栽培在土壤中便很多年不移动，所以有明显的缺铁积累效应。可是，时至今日缺铁现象还没有完全被人们认识清楚，现行的矫治措施也收效不大，有些甚至还带来严重的环境问题。随着分子生物学和现代生物技术手段的发展，科学家们开始寻找新的方法来解决缺铁问题，创造铁吸收利用高效型的植物种类已经成为一条有希望的途径，在果树生产中，创造新的综合性状优良同时又耐缺铁的砧木便可以达到既防治果树缺铁又保护环境的目的。基于此，本研究选择了两种果树砧木，小金海棠和香橙因为它们在初步的田间鉴定中表现耐缺铁，但它们耐缺铁的生理及分子机制并不清楚。本研究通过进一步分析香橙和小金海棠的耐缺铁特性，研究它们耐缺铁的生理原因和分子基础，并通过分析三价铁螯合物还原酶基因的空间表达模式，从分子水平上去探讨植物耐缺铁的原因，为从香橙和小金海棠中克隆三价铁螯合物还原酶基因奠定基础，并为人工创造耐缺铁的果树砧木提供基础研究数据。主要的研究结果如下： 1：铁胁迫下香橙的生理反应研究 香橙在田间实验中表现耐缺铁，在pH7.8的土壤中生长正常，叶片无任何缺铁症状；但对照植物枳在同样土壤条件下却表现出明显的缺铁症状，作为缺铁程度指标的叶片叶绿素含量极显著低于香橙。枳在缺铁营养液中培养4周即可出现明显缺铁症状，而生长在同样时间内和同样缺铁培养液中的香橙观察不到任何缺铁症状，香橙的叶片叶绿素和活性铁含量均极显著高于枳；但枳的黄化叶片的总铁含量高于香橙和枳的绿色叶片。恰如以前的许多报道，铁是唯一的组织含量和缺铁程度没有相关关系的个例。 香橙的根系H”分泌迅速被缺铁激活，在缺铁4天即明显强于对照处理，在 缺铁12周依然明显强于对照处理；积的幼苗根系在缺铁4天时也有较强的H” 分泌，但随着缺铁时间的延长，在缺铁12周时H”分泌已观察不到。由于培养 液以NO厂析为N源，它会引起溶液PH的升高，但香橙的培养液在加入Fe卜， Fey和完全无 Fe 3种处理下的 pH升高的值始终低于积 3种处理的培养液的升 高值。涨醚和积的 3种处理的溶液的 eH增加的值始终是＋Fey｝＋Feh ）Fe。 很显然，缺铁激活了香橙根系净H”的分泌，使得香橙控制环境PH的能力始终 比积强，香橙根系很强的H”分泌能力与香橙耐缺铁的特性有关。 香橙根系的三价铁赘合物还原酶活性被缺铁诱导强烈增加，在缺铁4周时 酶活性增加了20倍；积在缺铁4周时酶活性仅增强了不到3倍。。很明显，香 橙根系三价铁赘合物还原酶活性的强烈增加与香橙耐缺铁的特性直接相关。香橙 比积耐缺铁的原因便是铁胁迫条件下，香橙根系三价铁螫合物还原酶恬性被缺铁 诱导增加的幅度远大于积的酶活性增加幅度。 香橙根系三价铁赘合物还原酶的还原区域与H”分泌区域重叠；而且被缺铁 激活的香橙根系H”分泌先于根系三价铁赘合物还原酶的出现。可见，增强的H“ 分泌可以降低根际土壤的PH，低的根际土壤PH不仅增加了根际土壤中铁的溶 解度，而且为根系三价铁螫合物还原酶的活动提供了适宜的PH环境，因为已经 发现该酶有PH依赖性，碱性PH会强烈地抑制该酶活性。 缺铁使香橙根系的形态发生了一定的变化。开始，香橙幼苗的根系生长比积 慢，但随着铁胁迫时间的延长，积的地上部分生长受到明显抑制，根系生长减缓， 根长而纤细，很少侧根发生；而香橙的根系则变的粗短，缺铁胁迫下的香橙的根 长和恻根的发生量虽明显低于对照处理，但侧根发生量却明显优于积的缺铁处 理，而根长又极显著低于积的缺铁处理。电子显微镜下观察到，与对照加铁处理 相比，缺铁香橙的根吸收区域表皮细胞明显稻皱隆起；而积的根系吸收区域的表 皮细胞在两种铁处理下没有明显变化。可见缺铁会使香橙的根系吸收区域发生一 定的形态变化，沼皱隆起的细胞表面增加了吸收面积，对铁吸收无疑是有帮助的， 但要肯定香橙的根系形态变化是香橙耐缺铁的原因之一尚需做进一步的较精确 的测定。 2．铁胁迫下小金海棠的生理反应研究和小金诲棠无性系的建立 小金海棠实生苗在田间土壤PH为7．8时生长正常?更多还原

【Abstract】 IntroductionIron chlorosis of food crop plants and fruit trees grown on alkaline, or calcareous soils is a widespread agricultural problem in the world. Although abundant in these soils, iron is often insoluble and therefore is unavailable for the plants roots. Statistics show that potential iron-deficiency exists in plants growing in 25%~30% soils of the globe. Iron deficiency causes interveinal chlorosis, mesophyll yellowing or paling and leaf size reduction and, if severe enough, retards the growth of the plants and may even cause their death, thus resulting in great losses to agricultural production. Fruit trees are very important economic crops in people’s life, the losses of fruit production caused by iron chlorosis are usually higher than those of other annual economic crops because fruit tress, as perennial plants, will remain there for many years after they are planted in a field and an evident accumulation effect may take place. Unfortunately, the widespread problem of iron chlorosis of fruit trees remains poorly understood and the results of the methods for its correction are not satisfactory, and they sometimes cause serious environmental problems as well. With the advances in molecular biology and modern biological engineering technology, scientists begin to look for new ways to tackle this problem. It has become possible to create new iron efficient plant materials or to avoid soil environmental pollution by creating new rootstocks with high iron efficiency as well as excellent complex characters. C. junos and M. xiaojinensis were found to be tolerant to iron chlorosis and were able to acquire iron from soils of low iron availability in previous field experiments, but the physiologicaland molecular mechanisms for their iron efficiency have remained unclear. The purposes of this project were to further analyze the characteristics of their iron efficiency under iron stress, to study the physiological and molecular mechanisms of iron efficiency under iron deficiency in C. junos and M. xiaojinensis , and to analyze the spatial expression model of FCR (ferric chelate reductase) gene under iron stress with the hope to cast a new light on iron stress tolerance on the molecular level, to lay solid foundations for cloning FCR gene in C. junos and M. xiaojinensis, and to provide some basic data for creating new rootstocks with excellent complex characters and iron efficiency. The main results are presented as follows: 1. The physiological reaction of C. junos under iron stressIn field experiments, C. junos manifested itself as tolerant to iron stress. No chlorosis symptom was not found in its leaves when it was grown in a soil with pH 7.8. In contrast, severe chlorosis was found in the control plant P. trifoliata grown under the same soil conditions, and leaf chlorophyll content as an indicator for the degree of Fe deficiency in P. trifoliata was much lower than in C. junos. In solution culture, evident chlorosis symptoms were observed in the leaves of P. trifoliata after 4 weeks of iron deficiency treatment, while no chlorosis symptoms were observed in the leaves of "iron efficient" C. junos under the same culture conditions. The content of leaf chlorophyll and active iron in C. junos was much higher than that of P. trifoliata. However, iron content of chlorotic leaves of P. trifoliata was found to be even higher than that of the green leaves of P. trifoliata and C. junos. Just as reported before, Fe is only case where its content in plant tissues is not correlated with the degree of its deficiency.Net H excretion in C. junos was rapidly activated by iron deficiency and its rate was enhanced 4 days after the start of the treatment as compared with the non-deficiency control, and this trend lasted over 12 weeks under iron deficiency. In P. trifoliate, net H+ excretion rate was also enhanced under 4 days’ iron deficiency, but was no longer observed under iron deficiency for 12 weeks. NOs-N was used as the N source of the culture solution and it increased pH of solution. The extent of the increa更多还原