【作者】 余涛；

【导师】 朱英国；

【作者基本信息】 武汉大学 ， 遗传学， 2004， 博士

【摘要】 自然界中的植物经常遭受各种各样的逆境胁迫伤害，每年由于自然灾害的原因造成大量作物减产，并严重影响各种农产品的品质。研究植物在不良环境下生命活动的规律，弄清其抗逆性的细胞及生理学基础并加以调控，不仅有助于全面和彻底地了解植物正常生理活动，而且对提高农业生产力、保护环境也有重要的指导作用，因此，它一直是植物学研究中的重要内容之一。 为了探讨植物抗逆反应机理，本文使用差异显示技术分别从单子叶植物水稻和双子叶植物烟草中克隆受逆境调控表达的基因，分析了它们在多种逆境处理下的表达模式，并使用转基因烟草研究了谷胱甘肽S转移酶在植物抗氧化胁迫中的作用，具体内容如下： 1、用改进的差异显示技术从烟草中分离受乙烯利调控表达的基因，共获得24条在对照和乙烯利处理烟草叶片中表达有差异的cDNA片段。这些差异片段经点杂交验证后表明，17条为真实的阳性片段，其中12条表达受乙烯利诱导，另外5条表达受乙烯利抑制。测序后使用GenBank搜索软件对这些差异片段进行了序列分析，结果显示，这12条受乙烯利诱导表达增强的基因主要为逆境及PCD相关基因、植物激素相关基因以及一些细胞壁相关蛋白基因。其中有5条为尚未克隆的新基因，使用RACE扩增获得其中一条基因的全长cDNA 序列。另外5条受乙烯利抑制表达的基因片段经序列分析后证实均为与植物光合作用相关的蛋白基因，这说明乙烯与植物的光合作用密切相关。为了研究这些乙烯利诱导基因与植物逆境的关系，我们采用RT-PCR的方法分析了它们在各种不同逆境下的表达模式，从而探讨了它们在植物抵抗逆境反应中可能的功能。 2、采用改进的差异显示技术从单子叶植物水稻中分离受氧化胁迫调控表达的基因，获得4条真实的差异表达片段，其中2条受氧化胁迫诱导表达增强，序列分析表明为未知功能的新基因，分别编码植物中一个可能的谷胱甘肽转运蛋白和一个推测的C末端结合蛋白。另2条受氧化胁迫抑制表达，序列分析表明它们与水稻中的两个已知基因高度同源。为了探讨它们在植物抗逆反应中的功能，我们使用Northern杂交分析了这4个基因受不同逆境及逆境信号分子(SA、ABA、乙烯)处理后的表达模式，研究了它们与植物逆境的关系。 3、用农杆菌介导的叶盘转化法将一个来自棉花的谷胱甘肽S转移酶(GST)基因转化到烟草中。转基因烟草使用Sotltllen:杂交验证表明，外源的GST基因己经整合到烟草的基因组中，同时还表明，在烟草荃因组中也存在多个内源的GST基因拷贝，证实GS厂fs是一个大从因家族。使用酶活测定和Nortllem分析鉴定了3株过表达外源(}S‘f自勺转墓因烟草，并检测了它们对氧化剂甲基紫晶(MV)诱导的氧化胁迫的抵抗能力。日一十盘离子渗漏分析表明，与对照相比，过表达外源GST基因的转基因烟草具有较强的抗氧化胁迫的能力。此外，使用酶活测定的方法检测了MV处理过程中，对照和转基因烟草中6种抗轨化酶(GS1’，谷肤卜「肤过氧化物酶，抗坏血酸过氧化物酶，过轨化城酶，超氧化物岐化醉，过氧化物酶)活力的变化，比较了氧化J])].迫卜这两种材右}中的抗氧化酶活力的变化。更多还原

【Abstract】 Plants often suffer from many kinds of stress conditions. Every year, disaster in the world made sever decrease of crop production and affect the quality of farm products. Researches on the response of plant to stress and discover the cellular and physiological mechanism of plants in defending against different stress conditions will be helpful not only to understand the normal physiological process completely but also to improve the productive potentialities of crops and protect the environment, which is always an important content in plant science.In order to discover the mechanism in plant defending against stress conditions, a differential display method was used to isolate genes regulated by stress condition in rice and tobacco, and the expression pattern of these genes in different stress conditions was investigated. Transgenic tobacco plants were used to analyze the function of plant glutathione S-transferase (GST) in defending against oxidative stress. The detailed work was as follows:1 An improved differential display method was used to identify ethephon-regulated genes from tobacco. Total 24 cDNA fragments expressing differently between control and ethephon-treated plants were isolated. In the 17 fragments that were identified by dot hybridization to be truly positive results, 12 of them were induced by ethephon and 5 were inhibited. Sequence analysis and BLAST analysis in the GenBank database indicated that the 12 fragments induced by ethephon were some stress and PCD-related genes, hormone-related genes or cell wall-related genes. Five of them represent for novel genes, and one full length cDNA were amplified by RACE method. The other 5 genes inhibited by ethephon were proved to be photosynthesis-related protein genes, which indicated that there is a tight relationship between ethylene and photosynthesis in plant. In order to explore the relationship of these ethephon-induced genes with stress. RT-PCR analysis of these genes in different stress conditions were carried out. and the putative function of these genes in stress werediscussed.2 A differential display method was used to identify oxidative stress-regulated genes from rice, and 4 truly positive fragments were obtained by this way. Two of them got enhanced expression by oxidative stress treatment, sequence analysis revealed that they present function unknown genes, encoding a glutathione transporter (GT1) protein and a C terminal binding protein (CtBP) respectively. The other two gene fragments were oxidative down-regulated, and sequence analysis indicated that they share high similarity with two known genes in rice. In order to explore the function of these genes in defending against stress conditions, Northern blot analysis were used to analyze the expression pattern of these genes in different stress conditions and the treatment of stress signal molecular (SA. ABA, ethylene), the role played by these genes in differential stress conditions were discussed.3 A glutathione S-transferase (GST) gene from cotton was introduced into tobacco by Agrobacturium-mediated leaf disk transformation method. Southern blot analysis of the transgenie plants indicated that the foreign GST gene has introduced into the tobacco genome. It’s also suggested that there are many copy of GST genes in tobacco genome. By the enzyme activity and northern blot analysis, 3 transgenie tobacco plants exhibited high expression level of foreign GST gene were selected to treat with MV inducted oxidative stress. The ion leakage results indicated that transgenie plants overexprcssing foreign GST show high resistance to oxidative stress. Enzyme activity of six antioxidate enzymes (GST, glutathione peroxidase. ascorbate peroxidase, catalase, superoxide dismutase. peroxidase) in control and transgenie plants were monitored during MV treatment. The activity change models of these enzymes were compared between control and transgenie plants.更多还原