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

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 with the causal gene. Based on the results above, the mutants of relevant genes will be chosen for investigation of the relationship between the causal gene and the relevant genes in genetic pathway. This research will reveal the molecular mechanism underlying adventitious shoot formation in Arabidopsis accessions, and be helpful for elucidating the bases of stem cell establishment in vitro, and in-depth understanding the cell totipotency.

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。

芽再生是构建遗传转化系统、进行基因功能研究和转基因育种的基础。同一物种内不同基因型之间普遍存在芽再生能力的明显差异，但人们对造成这种差异的分子机理目前所知甚少。.本项目首先以拟南芥为材料，筛选出了再生能力差异极显著的两种生态型Col-0和Gu-0。利用图位克隆技术定位到可能导致这两种生态型芽再生能力差异的候选基因DCC1。Col-0生态型dcc1突变体的芽再生能力显著低于野生型。qRT-PCR和GUS报告基因的研究结果显示，DCC1在愈伤组织中表达水平较高。功能互补实验的结果表明，以Col-0生态型的DCC1的启动子驱动其基因的表达能够互补突变体的表型；该启动子驱动Gu-0生态型的DCC1基因的表达则不能互补突变体的表型，暗示DCC1基因的变异可能是导致Gu-0生态型芽再生能力低下的重要原因。DCC1蛋白含有DxxCxxC基序，属于一类新的硫氧还蛋白。亚细胞定位结果发现其定位于线粒体和叶绿体中。DCC1突变导致外植体H2O2水平显著提高，暗示DCC1通过调节外植体的活性氧（ROS）水平进而调节芽的再生。.酵母双杂交和pull-down实验的结果显示，DCC1与碳酸酐酶CARBONIC ANHYDRASE 2 (CA2)具有互作关系。利用荧光素酶进行的互补实验和免疫共沉淀（Co-IP）的研究结果进一步证实了两者的互作关系。ca2突变体的芽再生能力显著低于野生型对照，dcc1 ca2双突变的表型更加严重。CA2为线粒体呼吸复合体Ⅰ亚基的组分。DCC1和CA2突变均会导致线粒体呼吸复合体Ⅰ活性的显著降低和愈伤组织中H2O2水平的提高，而外施还原型谷胱甘肽以及过表达过氧化氢酶CAT3均能有效恢复dcc1和 ca2突变体的芽再生能力，表明活性氧对芽再生具有重要调控作用。RNA-seq分析结果显示，在dcc1突变体中，一些生长素合成基因和响应基因的转录水平显著降下调。外施H2O2明显抑制生长素报告基因DR5的信号水平，暗示DCC1通过活性氧调控生长素信号转导基因的表达进而调节芽的再生。.连锁不平衡分析和数量互补实验表明，DCC1是调控不同生态型拟南芥的芽再生能力差异的关键因子。以小麦为材料开展的初步研究结果提示活性氧水平也是决定小麦芽再生能力自然变异的重要调控因子。