调控不同生态型拟南芥离体芽再生能力差异关键基因的分离及分子机理研究

项目名称： 调控不同生态型拟南芥离体芽再生能力差异关键基因的分离及分子机理研究

项目编号： No.31270231

项目类型： 面上项目

立项/批准年度： 2013

项目学科： 生物科学

项目作者： 李兴国

作者单位： 山东农业大学

项目金额： 75万元

中文摘要： 以172种生态型的拟南芥为试材，以根为外植体，诱导离体芽的再生，筛选再生能力差异极显著的生态型。构建高代互交-重组自交系，进而利用QTL图位克隆技术定位调控不同生态型拟南芥离体芽再生能力差异的候选基因。利用互补实验对重要候选基因进行功能验证，确定关键基因。利用mRNA原位杂交和报告基因标记技术考察关键基因在离体芽再生过程中的时空表达模式。利用高通量测序技术，结合酵母杂交、Chip-sequence、EMSA等生化技术筛选、鉴定与关键基因互作的基因。利用相关基因的突变体，研究关键基因与互作基因的上下游关系。本研究将初步揭示不同生态型拟南芥离体芽再生能力差异的分子机理。研究结果将有助于阐明干细胞离体建成的分子机理，并为深刻理解细胞全能性提供重要资料。期望本研究结果为理解不同基因型（品种）农作物和其它植物器官再生能力差异的分子机理，克服器官离体再生障碍，提高遗传转化效率提供基因资源和理论参考。

中文关键词： 芽再生；硫氧还蛋白；自然变异；活性氧；生长素

英文摘要： In order to decipher the molecular mechanism of natural variation in the in vitro shoot regeneration, 172 accessions of Arabidopsis thaliana will be used as experimental materials, and root selected as explants to induce shoot regeneration follow the shoot regeneration procedures described by the previous report. One index will be used to measure in vitro shoot regeneration capacity: "regeneration rate" calculated as the percentage of regenerating calluses. The accessions exhibiting significant differences in regenerative capacity will be identified according to the statistical results. Further, Advanced Intercross-Recombinant Inbred Lines (AI-RILs) will be established, and the AI-RIL populations used for QTL mapping and subsequent molecular identification of the candidate genes in shoot regeneration. The complementary experiments are designed to verify the function of the important candidate genes, therefore the causal gene identified and characterized. Subsequently, it will be examined that the spatiotemporal expression patterns of the causal gene during in vitro shoot regeneration process by using in situ mRNA hybridization technology and reporter genes. Furthermore, Biochemical experiments including yeast hybridization, EMSA assay and ChIP-Sequence will be performed to probe the candidate genes interacting

英文关键词： shoot regeneration；thioredoxin；natural variation；ROS；auxin