脱落酸介导的条斑紫菜抗氧化胁迫响应机制解析

Active oxygen radicals will be induced when the plant is subjected to environmental stresses. The burst of ROS usually leads to the up-regulation of genes related to stress responding and the increase of antioxidases activity. Our previous investigations found that the ABA content in high salinity treated Py. yezoensis showed a significant up-regulation compared with the in the control. The enzymes relating to ABA synthesis, and the kinase interacting with the ABA receptor were up-regulated. Applying of exogenous ABA, the damage caused by malondialdehyde (MDA) was alleviated, and the antioxidases activities were enhanced. All the results implied the presence of ABA-mediated antioxidative metabolism in Py. yezoensis. According to the determinations of ABA content and antioxidant enzyme activity, the samples with active stress responding will be collected to perform analysis of transcriptome and proteome. The genes which are relevant to ABA signal transduction will be obtained through blasting. For the proteins with pivotal function in ABA signal transduction will be cloned and the monoclonal antibody will be prepared. The key protein as a bait is recombined into the GAL4 DNA-binding domain and libraries of prey proteins are constructed using RNA extracted from the stressed Py. yezoensis. The yeast two-hybrid screen will be carried out and the gene information in positive clone will be sequenced. The Co-Immunoprecipitation method will be applied to test the interaction between molecules identified by the yeast two-hybrid system. The potential role of the regulatory molecule in ABA signal transduction can be analyzed through determination of the photosynthetic parameters of Py. yezoensis with or without antagonists of the target protein under salinity stress. Thus, the molecular mechanism of antioxidation mediated by ABA in Py. yezoensis could be constructed. This will provide new information for evolutionary analysis of ABA signal transduction and the theoretical principle for the breeding of Py. yezoensis.

植物在逆境下产生活性氧，诱导抗氧化酶活性升高、抗逆基因表达上调。条斑紫菜为潮间带典型物种，高盐胁迫下细胞内脱落酸（ABA）含量显著增加,ABA间接合成途径的两个关键酶及与ABA受体相互作用的激酶表达上调；外源添加ABA可诱导抗氧化酶活性升高。提示条斑紫菜中存在ABA介导的抗氧化代谢通路。本项目拟通过高盐处理，基于抗氧化酶活及ABA含量检测，获得ABA信号转导活跃的材料，开展转录组与蛋白组表达分析，通过生物信息学比对筛查ABA信号转导关键蛋白；克隆其基因并制备抗体；采用酵母双杂交系统筛选cDNA 表达文库中与诱饵蛋白相互作用的分子；免疫共沉淀验证,质谱鉴定，寻找与目标蛋白发生相互作用的分子；采用某些关键蛋白的拮抗/抑制剂，活体水平验证目标蛋白在抗氧化胁迫响应中的作用。项目的完成，可为ABA介导的逆境响应机制演化提供数据支持，为条斑紫菜抗性品系的培育提供新思路。

脱落酸（ABA）被认为是植物适应逆境胁迫的关键激素，在所有光合生物中均存在，且ABA信号通路在进化上相对保守。因此，深入研究潮间带典型藻类——条斑紫菜ABA的合成及其对下游逆境响应基因的调控，将有助于进一步了解ABA在植物中的进化历程。本项目首先对高盐胁迫下条斑紫菜抗氧化能力进行了研究，结果表明抗氧化系统在条板紫菜活性氧清除中发挥重要作用。使用ABA合成抑制剂，在高盐条件下对条斑紫菜进行胁迫与复苏处理，发现ABA间接合成途径的抑制剂可显著影响藻体光合作用，说明条斑紫菜中ABA通过类似高等植物的间接途径合成，进一步研究证明其ABA前体IPP的合成，同时存在MVA和MEP两种途径。在IPP合成抑制条件下，高盐胁迫时藻体ABA含量显著降低，抗氧化酶活性明显下调，而外源添加ABA后，该抑制作用得到缓减。对上述样本进行转录组测定与分析，发现抗氧化系统相关酶基因在抑制剂条件下表达下调，在外源添加ABA的样本中则出现上调，实时荧光定量结果与转录组分析趋势一致。基于转录组数据筛查获得了ABA信号通路关键组分，与种子植物，苔藓、丝状绿藻ABA通路的核心分子比较发现，条斑紫菜Snrk1及PP2C核心组分呈现较为原始的特点，因此，其ABA信号通路属于一种原始的通路。同时，在条斑紫菜大多数抗氧化酶基因启动子区域，鉴定到了AREB序列，因此，条斑紫菜中抗氧化酶的表达受到了ABA信号通路的调控，这种调控作用的重要性在紫菜栽培的实际生产过程中也得到了验证。然而，ABA的受体蛋白目前仍未被鉴定。为全面阐该通路的分子过程，以关键蛋白因子PP2C和SnRK1构建诱饵质粒，进行了酵母双杂交筛选，但PP2C未能在酵母中表达，SnRK1虽能表达，但未能鉴定到与其互作的蛋白因子。运用基因重组的方法，构建了条斑紫菜抗逆新品系，该品系氧自由基清除能力得到显著提升。相关成果不仅为完善ABA信号通路在植物界的进化研究提供了数据支撑，而且，在条斑紫菜抗性品系培育方面，也具有广阔的应用前景。