【作者】 王敏；

【导师】 寿惠霞；

【作者基本信息】 浙江大学 ， 植物学， 2016， 硕士

【摘要】 铁是植物生长和发育所必需的微量元素之一,它参与植物的叶绿素合成,并且是很多酶的重要组成部分。在面对复杂的生存环境时,植物维持体内的铁稳态非常重要,所以植物进化出一系列的Fe吸收和转运的调控机制。解析参与铁吸收、代谢和体内平衡的重要调控基因,研究植物缺铁的响应机制,培育富铁植物品种具有重要意义。本实验室在前期通过分析缺铁水稻的基因芯片,发现一个缺铁诱导的碱性螺旋-环-螺旋(bHLH)家族的转录因子-OsIRO3。OsIRO3,该转录因子负调控水稻的缺铁信号响应(Zheng et al.,2010)。通过进化分析,我们还发现水稻中有一个OsIRO3的高度同源基因OsbHLH62.本研究主要目的是发展OsIRO3和OsbHLH062的超表达和突变体遗传材料,并初步分析这两个基因的功能。本研究中,利用一系列分子生物学与植物生理学方法,我们分析了OsIRO3和OsbHLH062的蛋白结构、表达模式、亚细胞定位和蛋白互作等。通过分析Genevestigator中的基因芯片数据发现,OsIRO3在水稻组织中广泛表达,而OsbHLH062在种子,根和花中广泛表达,在叶片、茎和节等地上部分表达相对较低。进一步利用启动子接GUS报告基因,发现OsIRO3和OsbHLH062主要在维管组织中表达,同时OsIRO3受缺铁诱导表达。基因枪轰击洋葱表皮细胞及原生质体转化,证明OsIRO3和OsbHLH062融合的GFP蛋白定位在细胞核中,符合预期。但是酵母实验发现,OsIRO3具有转录因子的自激活功能而OsbHLH062不具备自激活功能。进一步的酵母双杂交分析发现,OsbHLH062能够与其自身和OsIRO3相互作用。利用BiFC实验,我们进一步确认了OsbHLH062和OsIRO3在水稻原生质体中的相互作用。最后,我们利用水稻转基因和CRISPR/Cas9等技术,培育了OsIRO3和OsbHLH062的超表达和突变体材料。本研究中,我们明确了OsIRO3和OsbHLH062的表达,亚细胞定位与相互作用方式,利用发展的超表达和突变体等遗传材料,下一步将对OsIRO3和OsbHLH062在水稻铁信号调控网络中的功能进行更详细的研究。更多还原

【Abstract】 Iron is one of the essential micronutrients for the survival and proliferation of plants. It is an important component in chlorophyll biosynthesis and various enzymes. To maintain iron homeostasis under different environments, plants have evolved a complicated signaling network in regulation Fe uptake and mobilization. Analyzing the genes that are involved in Fe uptake, metabolism and homeostasis at molecular level is pivotal for breeding crops with increased iron content.In a previous research, we have characterized an iron deficiency-induced bHLH (basic Helix-loop-Helix) gene in rice, OsIRO3, which negatively regulates Fe deficiency signaling in rice (Zheng et al.,2010). OsbHLH062, a highly homologue of OsIRO3, has been identified by phylogenetic analysis here. This study have analyzed the expression patterns and constructed the overexpression and mutant genetic materials for these two genes.Microarray data for Genevestigator showed that OsIRO3 is widely expressed in all rice tissues and OsbHLHO62 is mainly expressed in seeds, roots and flowers, and relatively low in leaves, stems and internodes. Promoter::GUS lines of OsIRO3 and OsbHLH62 demonstrated that the two genes were selectively expressed in the vascular tissues. As expected, OsIRO3 and OsbHLH62 fused GFP proteins localized in nuclear in the onion epidermal cells and rice protoplast. However, only the OsIRO3 showed transcription activation activity in yeast cells but not OsbHLH62. A further Y2H assay indicated that OsbHLH62 could interact with itself and OsIRO3. The protein-protein interaction between OsIRO3 and OsbHLH62 was further proved by BiFC in rice protoplasts. In order to analyze the function of OsIRO3 and OsbHLH062 in iron signaling network, overexpression and mutant lines were generated by transformation and CRISPR/Cas9, respectively.In summary, this study confirmed the tissue expression patterns, subcellular localization and protein interactions of OsIRO3 and OsbHLHO62. In the future research, the detailed molecular and physiological function of OsIRO3 and OsbHLHO62 will be analyzed by using the generated overexpression and mutant lines.更多还原