转录抑制子IDS1调控水稻耐盐性的分子基础

Soil salinization, which has been becoming environmental resource constraint of the sustainable development of the world irrigated agriculture, affects the yield of the crop. Mining of more salt tolerance genes and recovering the regulation network of salt response, are the prerequisite and basis of the breeding of new rice salt tolerance cultivated varieties. AP2 transcription factors are plant-specific transcription factors which are composed by many family members. Studies in recent years recover that this transcription factor family play important roles in regulation of plant growth and development, as well as response to phytohormones and biotic/abiotic stress. EAR (ethylene-responsive element binding factor-associated amphiphilic repression) motif is a conserved amphipathic leucine-rich sequence which endows the transcription repression activity to a transcription factor. EAR motif plays important roles in stress response and stress-related genes expression. We analysized 374 EAR-motif-containing ERF transcription factors and found 57 of them are differentially expressed under salt stress. By using amiRNA interferance knock-down technique and land-field screening, several AP2 transcription factors were identified as regulators of rice salt tolerance. The knock-down lines of IDS1, one of the transcriptional repressor of AP2 transcription factor family, showed significant salt tolerance. All the physiological and biochemical data indicated that IDS1 negatively regulates the accumulation of osmotic adjustment substances. Gene expression analysis demonstrated that expression of many stress-related genes were suppressed by IDS1. We also found that IDS1 can directly bind the promoter region of LEA3, which encodes an osmotic regulation protein, indicating that IDS1 regulates rice salt tolerance maybe by adjusting the accumulation of osmotic adjustment substances. Based on these experiments, by confirming the function of IDS1 in rice salt tolerance by using null mutant and overexpression lines, analyzing the transcriptional repressor activity of IDS1, finding the downstream target genes of salt response and mapping the active amino acids residue sites and cis-element of LEA3; this project will analysis the molecular pathway of the IDS1 on regulation of the rice salt tolerance, reveal relationship of IDS1 and salt stress response to elucidate the molecular basis of the IDS1 on regulation of the rice salt tolerance, and provide the useful genes and new thoughts to the molecular breeding of the crop stress tolerance.

剖析耐盐基因及其调控的分子途径是培育水稻耐盐材料的前提和基础。鉴于AP2转录因子是调控植物生长发育的重要因子，采用人工小RNA干扰技术及田间筛选方法，我们鉴定到多个AP2转录因子在水稻耐盐性中的调控功能。其中转录抑制子IDS1的干扰株系表现明显的耐盐性；生理生化检测表明IDS1负调控渗透调节物质的积累；基因表达分析表明IDS1抑制了大量胁迫相关基因的表达，且IDS1可结合渗透调节蛋白LEA3基因启动子，表明IDS1可能通过负影响渗透调节物质的积累调控水稻的耐盐性。本项目拟在以上研究基础上，采用功能缺失突变体及过表达材料剖析IDS1调控水稻的耐盐性功能、转录抑制活性；进一步结合转录组表达谱分析，剖析IDS1调控水稻耐盐性的靶基因；通过剖析IDS1与靶基因启动子结合的作用位点及靶基因启动子的顺式元件，解析IDS1调控水稻耐盐性的生化和遗传基础，以期为作物抗逆性分子育种提供新的思路和有用的基因。

土壤盐碱化问题，已经成为世界灌溉农业可持续发展的资源制约因素。水稻是我国重要的粮食作物，对水稻耐盐基因的充分挖掘并通过遗传改良提高水稻耐盐性，是保证粮食安全和改善生态环境的重要途径之一。我们前期发现，转录因子IDS1在水稻耐盐性中的调控功能。其干扰株系表现明显的耐盐性；生理生化检测表明IDS1负调控渗透调节物质的积累；基因表达分析表明IDS1抑制了大量胁迫相关基因的表达。本项目我们集中在鉴定IDS1在水稻耐盐性中的调控作用，分析IDS1的转录抑制活性，鉴定IDS1的靶标基因，鉴定 IDS1 和靶标基因启动子结合的作用位点，挖掘IDS1相互作用蛋白并阐明IDS1调控下游靶基因的分子基础。.通过项目实施，我们鉴定了IDS1在水稻耐盐应答中的作用，分析了IDS1的时空表达特性以及盐胁迫条件下IDS1的表达，同时证明了IDS1是一个转录抑制子，能抑制靶标基因的转录；我们通过EMSA、双荧光素酶系统、酵母单杂交系统等综合手段鉴定了盐应答重要基因LEA1、LEA3、SOS1是IDS1的靶标基因，并鉴定出IDS1的相互作用蛋白TPL，以及IDS1与TPL相互作用的结构域，我们还鉴定出另一个相互作用蛋白HDA1，并证明了IDS1是通过与转录抑制协同因子TPL相互作用并招募组蛋白去乙酰化酶HDA1到靶标基因的启动子上，通过改变靶标基因的乙酰化水平来抑制基因表达。.我们的部分研究结果发表在国际著名植物学杂志《植物生理学（Plant Physiology）》上。鉴于此成果是世界上首次直接揭示了植物组蛋白的乙酰化水平与植物抗逆性的直接关系，该杂志特别邀请了国际同行对我们的研究结果发表了评论，在同一期杂志上发表。IDS1是一个既参与水稻成熟又参与水稻耐盐的重要基因，因此我们找到了一个重要的、平衡产量与抗逆性的关键基因IDS1。后期我们将利用该基因挖掘水稻的耐盐潜力、利用耐盐基因资源培育水稻耐盐新品种。