野生老芒麦盐胁迫过程中ABI5基因作用机制研究

Wild siberian wildryegrass (Elymus sibiricus L.) is a salt tolerant forage with high quality in the grass family (Gramineae), not only has large potential in saline-alkali land management and improvement of utilization, but also is one of the important stress resistant genic source, therefore, the study on salt resistant mechanism of siberian wildryegrass is very important. Early stage of the project team had done lots of field and indoor evaluation on salt tolerance of more than 150 wild siberian wildryegrass, found ABI5 gene played an important role in the process of salt stress, in addition, the whole length of ABI5 gene in siberian wildryegrass was amplified, cloned and sequenced, and the bioinformatics analysis was carried out. On this basis, this study will use real-time fluorescent quantitative PCR technology and RNA interference technology, to focus on ABA regulation ABI5 gene expression difference analysis and ABI5 gene functional analysis in siberian wildryegrass. The relationship among ABA, ABI5 and salt tolerance of siberian wildryegrass will be revealed, and ABI5 gene function will be clearly identified. It will preliminaryily clarify that the functional mechanism of ABI5 gene in siberian wildryegrass, and it will provide excellent genetic resources for salt tolerant molecular breeding and provide a theoretical basis for the study on salt tolerant regulation and molecular mechanism of siberian wildryegrass and its related plant of wheat family. The results of this study will have important theoretical significance and application value for the development and utilization of siberian wildryegrass and its related plants.

野生老芒麦(Elymus sibiricus L.)是禾本科(Gramineae)中比较耐盐的优质牧草，不仅在盐碱地治理和改良利用中具有较大潜力，而且是小麦族作物的重要抗逆基因源，因而老芒麦耐盐机制研究非常重要。项目组前期对我国150余份野生老芒麦种质资源进行了大量田间和室内的耐盐性评价，发现了ABI5基因在老芒麦耐盐胁迫过程中起重要作用，并对老芒麦ABI5基因的全长进行了扩增、克隆和测序，进行了生物信息学分析。本研究在此基础上，将用实时荧光定量PCR技术和RNA干扰技术，重点开展老芒麦ABA调控ABI5基因表达差异分析以及ABI5基因功能分析。揭示老芒麦ABA、ABI5基因和耐盐性三者之间的关系；明确鉴定出ABI5基因的功能。初步阐明老芒麦盐胁迫过程中ABI5基因的作用机制，为老芒麦及其小麦族近缘植物的耐盐分子育种提供优良基因资源，为它们耐盐调控、 耐盐分子机制的研究提供理论基础。本研究结果将对老芒麦及其小麦族近缘植物的开发利用具有重要的理论意义和应用价值。

老芒麦（Elymus sibiricus L.）是禾本科（Gramineae）中比较耐盐的优质牧草，不仅在盐碱地治理和改良中具有较大潜力，而且是小麦族（Triticeae）作物的重要抗逆基因源。ABA途径在植物响应盐胁迫重要信号转导途径，ABI5基因是ABA信号转导过程中重要的转录因子，在植物抵御外界胁迫过程中起着重要的作用。本研究从耐盐性强的老芒麦种质中克隆ABI5基因；NaCl和ABA处理老芒麦幼苗，分析老芒麦叶片和根系ABI5基因表达的差异；转基因分析其功能。本研究旨在揭示ABI5基因的作用机制，为老芒麦及其小麦族近缘植物的耐盐调控、耐盐分子机制、耐盐育种的研究提供理论依据。主要研究结果如下：首次克隆获得老芒麦ABI5基因的cDNA全长，命名为EsABI5，GenBank登录号为MN607227.1。EsABI5全长1563bp，包括一个1170bp的开放阅读框（ORF），编码389个氨基酸，亲水性不稳定蛋白，无信号肽和跨膜区，位于细胞核，有显著卷曲区域；与大麦（Hordeum vulgare）HvABI5蛋白的同源性最高，含有与逆境表达紧密相关的bZIP46、CBLZ和bZIP1结构域；与ABA信号通路密切相关，在植物适应逆境胁迫过程中发挥着重要作用。NaCl和ABA能够调控EsABI5的表达，NaCl上调叶片和根系EsABI5表达，叶片上调的幅度大于根系上调的幅度；ABA上调叶片EsABI5表达，下调根系EsABI5表达，当ABA浓度为50μmol/L时，叶片EsABI5上调表达的幅度是对照的7倍，根系EsABI5下调表达的幅度是对照的41倍。此时生理指标分析结果表明，脯氨酸含量显著高于对照和250mmol/L的NaCl处理，根系活力最高，丙二醛含量最低；超氧化物歧化酶活性最高，因此，根系EsABI5的下调表达可能是老芒麦ABA信号转导途径的关键步骤，该基因可能起负调控的作用。盐胁迫下，转基因植株黄化萎蔫程度均较野生型植株严重，各项生理指标均表明转基因植株对盐胁迫更加敏感；EsABI5参与了ABA信号转导途径和MAPK级联反应途径抗盐反应的复杂调控网络，并且作为负调控因子发挥作用。