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| 沙冬青适应渗透胁迫的脯氨酸渗透调节机制的研究 | |
| 其他题名 | The study on Proline osmotic adjustment mechanism of Ammopiptanthus mongolicus under osmotic stress |
| 岳光振 | |
| 出版年 | 2015 |
| 学位类型 | 硕士 |
| 导师 | 苏彦华 |
| 学位授予单位 | 中国科学院大学 |
| 中文摘要 | 蒙古沙冬青（Ammopiptanthus mongolicus），属豆科（Legulninosae）沙冬青属，是国家二级濒危保护植物，在我国的内蒙古、宁夏、甘肃等省区有自然分布，是我国阿拉善荒漠地区特有的常绿阔叶灌木和建群植物。沙冬青的适应能力很强，在年降水量低于50mm、蒸发量2000-3000mm、极端低温为-24.8℃、极端高温为37.7℃的恶劣环境下，仍能正常生长，鉴于在这种极端胁迫条件下的生存能力，使得沙冬青成为一种研究荒漠植物抗逆性的理想材料。另外，沙冬青作为我国北方荒漠地区唯一的强旱生常绿阔叶灌木，是古老的第三纪残遗物种，具有极其重要的古生物、生态、遗传资源价值，但是由于其分布范围狭小以及人类活动的不断破坏，沙冬青的数量已逐年减少，濒临灭绝。因此，无论是为了保持物种多样性，保护稀有珍稀物种，还是为了研究沙冬青超强的抗逆性机理，本研究都迫在眉睫。 本文以沙冬青幼苗为材料，模拟干旱胁迫，观测了干旱胁迫下沙冬青根系形态与生理指标的变化，发现了渗透调节物质脯氨酸在沙冬青适应干旱胁迫过程中发挥着重要作用。在本课题组对沙冬青转录组测序的基础上，克隆获得了脯氨酸代谢过程中的未知序列基因AmP5CS1、AmP5CS2、AmProT，并进行相关的生物信息学分析，预测基因的特性；在干旱、盐、ABA胁迫下，进一步探索沙冬青脯氨酸代谢的8个相关基因在转录水平上的表达响应机制。 本文的主要研究结果如下： 1、通过模拟干旱胁迫试验，测定了沙冬青幼苗的相关生理生化指标。在强烈水分胁迫下，植株体内内活性氧出现积累，并表现出一定程度的膜脂过氧化现象。叶片相对含水量显著减少，相对电导率增加，丙二醛积累量增加。在水分胁迫处理过程中，沙冬青体内的抗氧化酶清除系统（SOD、POD、CAT、APX）酶活性的变化并不显著，显示沙冬青可能并不依赖高效的活性氧清除机制以抵抗水分胁迫。而其体内的渗透调节物质脯氨酸、可溶性糖和可溶性蛋白则有较显著的积累，其中以脯氨酸的变化尤为显著。在植株的地上部和地下部脯氨酸的含量分别增加了6.4倍和10倍。因此，以脯氨酸为代表的渗透调节物质的积累，可能是沙冬青耐受干旱胁迫的显著特征。 2、进而通过脯氨酸的合成与代谢途径，利用课题组已完成的转录组测序数据，克隆了沙冬青脯氨酸代谢过程中的8个关键基因。其中5个基因在转录组测序中获得序列，通过同源探针PCR技术克隆获得了其余3个未知序列基因：AmP5CS1、AmP5CS2、AmProT。其中AmP5CS1基因全长2366bp，编码439个氨基酸；AmP5CS2基因全长2341bp，编码757个氨基酸；AmProT，基因全长1587bp，编码442个氨基酸，其编码蛋白的二级结构含有11个跨膜区域，具备ProT转运蛋白的显著特征。 3、通过荧光定量PCR试验，明确了脯氨酸代谢相关基因在转录水平上均有相应。在干旱、盐和ABA胁迫处理下，合成相关基因AmP5CR、AmP5CS1、AmP5CS2和AmOAT 在胁迫条件下均上调，脯氨酸的主要合成路径是谷氨酸路径， AmP5CS2在合成相关基因中变化最显著；胁迫条件下，AmProDH基因受到显著抑制，预示脯氨酸的降解过程被抑制而以合成和积累为主；脯氨酸转运基因AmProT和AmLHT的表达受到胁迫处理的显著诱导，推测在此条件下该基因可能在实现脯氨酸在体内的高效转运与分配中起重要作用。 综上所述，我们的研究结果表明，沙冬青对强烈干旱胁迫的适应可能不是一个高效的抵抗机制（依赖于体内活性氧清除机制的高效诱导与运作），而很可能通过以脯氨酸为代表的渗透调节物质的积累以提升体内水分的保持从而增强其对干旱缺水的耐受能力为主要特征。进一步在分子水平对脯氨酸合成、代谢与转运关键基因表达的研究，揭示了干旱胁迫下沙冬青体内脯氨酸的代谢途径中合成与转运过程受到诱导，而降解途径则受到抑制，因而有利于这一渗透调节物质大量积累以增强沙冬青对干旱缺水的耐受能力。 |
| 英文摘要 | Ammopiptanthus mongolicus, a specific evergreen xerophyte of Leguminosae in Alashan desert and an ancient relic from the Tertiary period, can survive under the condition of the annual precipitation less than 100-200mm and the annual evaporation over 3000mm. It plays an important role in fixing moving sand and delaying further desertification. Furthermore, the species shows strong tolerance to high and low temperature, extreme salinity and drought. The ability to survive at the extreme drought condition makes A. mongolicus an ideal model for studying mechanisms of drought tolerance in plants. Due to the ecological importance and the high academic value in A. mongolicus, a deeper understanding of the mechanism in A. mongolicus adaption to desert environment should be timely and beneficially. In this study, we used four-week seedlings of Ammopiptanthus mongolicus to measure the dynamic changes of morphological and physiological parameters. And then we found that proline may play an important role in the process of adaptation to water stress. Based on the result of Ammopiptanthus transcriptome sequencing，we have cloned eight genes related to proline metabolism. Among these genes, sequencing of AmP5CS1, AmP5CS2 and AmProT are unknown. We performed 3’-RACE and 5’-RACE to obtain the full length of CDS. We predicted the function of these genes by using bioinformatics analysis. Further, we studied the expression pattern of the eight genes at the transcriptional level under ABA, salt and drought stress. The major results are as followed: 1. The physiological and biochemical indicators were measured through simulating drought stress. Our results showed that intracellular ROS was accumulated, a degree of membrane lipid peroxidation, RWC significantly reduced, the relative conductivity increased, accumulation of MDA increased. The change of antioxidant enzyme activity was not significant(SOD, POD, CAT, APX), which showed that Ammopiptanthus do not use efficient active oxygen scavenging mechanisms to resist water stress. However, the osmotic adjustment proline, soluble sugar and soluble protein was more significantly accumulation and the most significantly is proline. They increased 6.4, 10-folds in shoots and roots, respectively. Therefore, proline as the representative of osmotic adjustment substances accumulation, which may be the significant feature of Ammopiptanthus tolerance to drought stress. 2. Eight Ammopiptanthus proline metabolism key genes were cloned, include the five genes sequence were obtained in the transcriptome sequencing and by homologous probe PCR technology cloning the remaining three unknown gene sequences: AmP5CS1, AmP5CS2, AmProT. The full length of AmP5CS1 is 2366bp, encoding 439 amino acids; The full length of AmP5CS2 is 2341bp, encoding 757 amino acids; The full length of AmProT is 1587bp, encoding 442 amino acids, the secondary structure of the encoded protein contains 11 transmembrane domains, with the salient features of ProT transporter protein. 3. By quantitative PCR test, proline biosynthesis-related genes (AmP5CR, AmP5CS1, AmP5CS2, AmOAT) mainly express in the above ground, degradation related genes (AmProDH, AmP5CDH) mainly express in the under ground. We predict that proline synthesis is mainly in the shoot. Under drought, salt stress and ABA treatment, the main synthetic route is the glutamate pathway. AmP5CS2 is the key genes in the synthesis of related genes. AmProDH gene under drought stress was significantly inhibited. We suggest that the degradation process was inhibited. Under stress conditions, AmProT and AmLHT were significantly induced to achieve a reasonable distribution. In summary, we found that ammopiptanthus does not use efficient active oxygen scavenging mechanisms to resist water stress. While, proline as the representative of osmotic adjustment substances accumulation may be the significant feature of Ammopiptanthus tolerance to drought stress. Further, in the molecular level, |
| 中文关键词 | 沙冬青 ; 渗透胁迫 ; 脯氨酸积累 ; 关键基因 ; 响应 |
| 英文关键词 | Ammopiptanthus mongolicus osmotic stress proline accumulation key gene response |
| 语种 | 中文 |
| 国家 | 中国 |
| 来源学科分类 | 植物营养学 |
| 来源机构 | 中国科学院南京土壤研究所 |
| 资源类型 | 学位论文 |
| 条目标识符 | http://119.78.100.177/qdio/handle/2XILL650/287561 |
| 推荐引用方式 GB/T 7714 | 岳光振. 沙冬青适应渗透胁迫的脯氨酸渗透调节机制的研究[D]. 中国科学院大学,2015. |
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