【作者】 李珂；

【导师】 王宝山；

【作者基本信息】 山东师范大学 ， 植物学， 2011， 硕士

【摘要】 环境胁迫是目前影响世界农作物产量的主要因素之一,它严重地影响到植物的生长、发育和作物的产量与品质。当环境如温度、土壤含水量、土壤盐度等的变化幅度超过植物的耐受范围时,都会使植物体内产生大量的有毒害作用的活性氧。它们可导致植物体内蛋白质变性和膜质过氧化,使植物体遭受氧化胁迫。因此,植物在长期的进化过程中产生了一系列保护机制来清除过多的活性氧,参与这些机制的包括一些小分子物质如抗坏血酸、谷胱甘肽及酚类物质等和抗氧化酶类如SOD、APX和CAT等。叶绿体作为植物特有的能量捕捉和转换器官,也是活性氧产生和攻击的主要部位,其中H2O2是叶绿体电子传递链产生的一种主要的活性氧,而APX被认为是叶绿体中清除H2O2的关键酶。定位在叶绿体上的抗坏血酸过氧化物酶包括类囊体膜APX和基质APX。该酶在抗氧化胁迫中的重要性已在某些转基因植物中得到初步证实。但是,到目前为止对APX的研究结果都是针对非盐生植物中的基因进行的,例如番茄,豌豆、水稻及拟南芥等,对盐生植物中叶绿体APX分子水平的研究还比较少,而且结果也是值得讨论的。因此研究盐生植物叶绿体APX与植物抗逆性之间的关系具有重要意义,同时也有待更进一步的深入探讨,APX在抗逆胁迫中的作用可能因植物材料的种类、发育阶段及处理条件的不同而有所差异。盐地碱蓬作为一种叶肉质化稀盐盐生植物,在进化过程中可能形成了特殊的耐盐机制,盐生植物和甜土植物在活性氧解毒机制上可能存在显著的差异。此外,目前关于盐分对拟南芥种子萌发效应的研究已有报道,但多集中于植物在基因表达及生理水平对盐胁迫的响应,极少将离子效应和渗透效应分开来研究盐胁迫机理。异源表达盐地碱蓬叶绿体APX能否提高甜土植物的耐盐能力,鉴于拟南芥良好的遗传背景,本研究以哥伦比亚野生型拟南芥和过量表达Ss.sAPX纯合体植株为材料,结合盐胁迫条件下二者的各项生理指标,探讨了二者在盐胁迫下的抗氧化机理,分析了该基因在盐胁迫诱导的氧化胁迫中的作用,希望获得抗逆性提高的植物材料。主要研究结果如下:正常生长条件下,野生型和转基因拟南芥的生长状况良好且没有区别。而在NaCl(≥120 mmol/L)处理下,野生型和转基因拟南芥的生长状况均受到一定程度的抑制。与野生型相比,转基因植株在种子萌发阶段受盐胁迫的抑制程度较轻,其种子萌发率、子叶长出率、存活率、主根长度及叶绿素含量均显著高于野生型,表现出较强的耐盐性。通过采用NaCl、LiCl和等渗甘露醇处理野生型和过量表达Ss.sAPX拟南芥种子,并测定二者萌发指标,以及与离子胁迫、渗透胁迫及氧化胁迫相关生理指标和抗氧化酶活性,得出盐胁迫下植物的萌发生长受抑制与渗透胁迫和离子毒害两种效应有关,其中NaCl对野生型和过量表达Ss.sAPX拟南芥种子萌发的抑制作用主要是由渗透胁迫造成的,而离子毒害处于次要地位。同时在NaCl处理下,过量表达Ss.sAPX拟南芥的钠钾离子含量、脯氨酸含量、SOD及CAT活性在NaCl处理前后与野生型并无显著差异,而其子叶中的H2O2及MDA含量随着NaCl处理后APX总酶活性的显著升高呈现显著性差异,维持在一个较低的状态。因此,这些结果表明,转基因植株在种子萌发阶段受盐胁迫的抑制程度较小并不是因为其离子毒害和渗透胁迫得到缓解而是由于Ss.sAPX及时清除了叶绿体中的H2O2,从而缓解了盐胁迫导致的氧化胁迫。更多还原

【Abstract】 Abiotic stress is one of the major enviromental stresses for crop yield and quality worldwide, which severely affects the plant growth and development. Biotic and abiotic stress conditions (such as temperature, soil water content, and soil salinity) produce excessive concentrations of reactive oxygen species (ROS), causing protein denaturation and lipid peroxidation. Because plants are frequently subjected to abiotic stress, they have developed several strategies to avoid and alleviate injury by ROS. Antioxidative defense systems include non-enzymatic and enzymatic components. Non-enzymatic components include ascorbate, reduced glutathione, phenolic compounds, and several other compounds. Enzymatic components include superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT) and so on.The chloroplast, where light harvesting and tranformation occur, is the organelle in which most ROS are generated under light condition. The electron transfer chain of the chloroplasts is the best-documented source of H2O2, and APXs are the key enzymes responsible for removing H2O2. Chloroplastic APXs include stromal APX (sAPX) and the thylakoid membranes (tAPX). Although researchers recently have examined preliminarily the role of the APX isoforms in protecting against oxidative stress induced by abiotic stress by transgenic plants, so far, all of which focused on the APX of nonhalophytes such as tobacco, rice and Arabidopsis, little study has been done on the role of chloroplastic APXs in the halophytes. Moreover, the results were controversial. Therefore, the research of the relationship between halophyte chloroplate APX and stress tolerance is important, which needs to be further studied. The role of APXs in protecting against oxidative stress tolerance may differ depending on plant species, plant developmental age and stress intensity.Suaeda salsa L. is a leaf succulent euhalophyte that may have evolved the unique salt-tolerant mechanism and may have an effective antioxidant system to protect against oxidative stress induced by salt treatment. In addition, the effect of NaCl on seed germination of Arabidopsis has been studied, but all focused on the reponse to salt tolerance at gene expression and physiological level. Little is known about the ionic effect and osmotic effect. To clarify the contribution of Ss.sAPX in the regulation of ROS levels and plant protection against oxidative stress induced by salt tolerance, we produced Arabidopsis lines overexpressing Ss.sAPX and investigated the performance of wild type (WT) and two of these transgenic lines of Arabidopsis under different NaCl conditions. The main results are showed as follows:The transgenic plants grew as well as WT plants under normal conditions (no NaCl added) in MS medium. Although the growth of both WT and transgenic lines was inhibited when NaCl (≥120 mmol/L) was added, germination, cotyledon growth, survival rate, root length and total chlorophyll content were all greater in the transgenic lines than in the WT. Overexpressing Ss.sAPX improves salt tolerance of Arabidopsis.The seed germination of WT plants and Arobidopsis lines overexpressing Ss.sAPX were examined under NaCl, LiCl and iso-osmotic mannitol treatments, and Na+, K+, proline content, antioxidant enzymes activities, H2O2 and MDA contents of their cotyledons under salt stress were also determined. Iso-mannitol and NaCl had the similar effect, which supports the hypothesis that ion toxicity was not the main cause of germination inhibition, and the osmotic component of salt stress was the main factor. Level of MDA and H2O2 was significantly lower in the transgenic Arabidopsis than in the wild type. Correspondingly, the transgenic lines had higher total APX activity than the WT. Interestingly, transgenic Arabidopsis plants and wild type did not differ in both Na+, K+, proline contents and SOD, CAT activities under salt stress. In contrast, the overexpression of Ss.sAPX in transgenic Arabidopsis markedly enhanced salt tolerance by maintaining a low level of H2O2, which consequently protected the transgenic plants from damage caused by oxidative stress, not by the alleviation of ionic effect and osmotic effect.更多还原