【作者】 殷丽丽；

【导师】 韩振海； 张新忠； 王忆；

【作者基本信息】 中国农业大学 ， 果树学， 2014， 博士

【摘要】 缺铁是限制果树特别是苹果树生长和果实品质的重要因素之一。多年来,人们围绕植物抗缺铁机理开展了许多研究,然而苹果抗缺铁的分子机理仍然不清楚。在实验室筛选到苹果铁高效基因型砧木小金海棠的基础上,本研究以小金海棠为试材,从中克隆到了两个受缺铁诱导表达的转录因子,命名为MxFTT和MxIRO2,分析及验证了其特性和功能,并研究了其与小金海棠铁吸收相关的两个重要功能基因MxIRT1和MxFRO2启动子之间的关系。研究结果为小金海棠铁吸收机制的深入挖掘奠定了基础。根据苹果金冠基因组和已报道的与铁吸收相关转录因子的信息,克隆到了小金海棠MxFIT基因,其开放阅读框为966bp,编码321个氨基酸,属于螺旋-环-螺旋(bHLH)结构的转录因子,亚细胞定位分析表明MxFIT定位在细胞核,酵母单杂交表明MxFTT在酵母细胞中具有较强的激活能力。通过将MxFIT纯化蛋白注射新西兰大白兔获得了MxFIT抗体,利用实时荧光PCR和Western Blot分析了其在转录和翻译水平的表达,MxFIT主要在小金海棠根系中表达,在叶片中几乎不表达。在根中,缺铁不仅可以强烈诱导MxFIT基因表达,而且可以诱导MxFIT蛋白积累,说明MxFTT在转录和翻译水平上均受缺铁调控。在拟南芥中过表达MxFIT可使转基因植株在缺铁条件下增强铁的吸收应答反应,提高了植株的抗缺铁能力。因此,MxFIT在缺铁应答反应中起着重要的作用。同时克隆到了另一个bHLH家族转录因子MxIRO2,该转录因子基因的开放阅读框为762bp,编码253个氨基酸。亚细胞定位显示MxIRO2定位在细胞核,MxIRO2和GAL4DNA结合域的融合蛋白在酵母中能激活LacZ报告基因的表达,其激活能力较弱,可被1mM3-AT抑制。通过将纯化的MxIRO2蛋白注射新西兰大白兔获得了MxIR02的抗体,利用实时荧光PCR和Western Blot分析了其在转录和翻译水平的表达,结果表明MxIRO2在小金海棠的根部和叶部均受缺铁诱导表达,在转录和翻译水平的表达均先升高后降低。酵母双杂交表明MxFIT和MxIRO2之间存在互作关系,利用烟草注射瞬时表达方法发现MxFIT和MxIR02同时注射烟草时可激活MxIRT1和MxFRO2启动子；将含启动子删除片段的农杆菌注射烟草表明MxIRTl启动子在-485～-236bp处存在与MxFIT和MxIRO2转录因子结合的序列,MxFRO2启动子在-813～-564bp处存在与MxFIT和MxIR02转录因子结合的序列。通过转化原生质体方法检测了MxIRT1和MxFRO2启动子的活性, MxIRT1启动子在-764～-485bp处存在增强启动子活性的元件,MxFRO2启动子在-1704～-1228bp和-564--329bp处存在增强启动子活性的元件。本研究从转录因子与功能基因启动子的关系入手证明了MxFIT和MxIRO2互作后可调控MxIRTl和MxFRO2,同时利用转基因拟南芥也证明了IRT1和FRO2是FIT调控的下游功能基因。因此,MxFTT和MxIRO2是小金海棠铁吸收调控过程中的重要转录因子。更多还原

【Abstract】 Iron deficiency is one of the important factors that affect the yield and quality in fruit trees, especially for apple tree. People have conducted a lot of researches about the mechanism of resistance to iron deficiency for years. However, the molecular mechanism of resistance to iron deficiency in apple tree is still not very clear. Malus xiaojinensis (Cheng et Jiang) is the first apple genotype identified as being resistant to iron deficiency by our research team. In this study, two iron-deficiency-induced transcription factors, MxFIT and MxIRO2, were cloned in M. xiaojinensis and the characteristics and functions were also analysed. MxIRTl and MxFRO2had been proved as two major genes related to iron uptake. The relationship of the promoters of MxIRTl and MxFR02with MxFIT and MxIR02transcription factors was studied in this paper. This work offers an important basis for further investigating the mechanism of fruit tree adaptation to iron-deficiency stress.According to the genome of Golden Delicious and the transcriptome sequencing of M. xiaojinensis, an iron deficiency response transcription factor gene, MxFIT, was cloned from M. xiaojinensis. MxFIT encoded a basic helix-loop-helix protein and contained a966bp open reading frame. MxFIT protein was targeted to the nucleus in onion epidermal cells and showed strong transcriptional activation in yeast cells. The antibody was obtained by injecting a New Zealand rabbit with the purified MxPIT-His fusion protein. Real-time PCR and Western blot analysis revealed that MxFIT was up-regulated in roots under iron deficiency at both mRNA and protein levels, while almost no expression was detected in leaves irrespective of iron supply. Ectopic expression of MxFIT resulted in enhanced iron deficiency responses in Arabidopsis under iron deficiency and stronger resistance to iron deficiency. Thus, MxFIT might be involved in iron uptake and plays an important role in iron deficiency response.In this study, another bHLH iron-related transcription factor IRO2, containing a762bp open reading frame and encoding253amino acids, had been cloned from M. xiaojinensis. The MxIRO2protein was targeted to nucleus in onion epidermal cells and had weaker transcription activation ability in yeast cells. The activation degree was restrained by1mM3-AT. The antibody was obtained by injecting a New Zealand rabbit with the purified MxIR02-His fusion protein. The spatiotemporal expression of MxIR02under iron deficiency was then assayed. The results indicated that MxIR02was induced in both roots and leaves when subject to iron deficiency. The expression model in transcription and translation level was increased at first and then decreased with iron deficiency stress.Yeast two-hybrid analysis proved that MxFIT could interact with MxIRO2. The promoters of MxIRTl and MxFRO2were activated in the tobacco leaves by coinfiltrated with agrobacterium containing MxFIT and MxIRO2. The binding sequences with MxFIT and MxIRO2transcription factor might locate-485～-236bp in MxIRT1promoter and-813～-564bp in MxFR02promoter. There might be reinforcing elements in-764～-485bp of MxIRT1promoter and-1704～-1228bp and-564～-329bp of MxFOR2promoter. Therefore, based on the relationship between transcription factors and functional gene promoters, we proved that MxFIT and MxIRO2could regulate MxIRTl and MxFRO2after interactions. Meanwhile, we found IRT1and FRO2were regulated by FIT in transgenic Arabidopsis. MxFTT and MxDRO2were two important transcription factors in regulation of iron absorption in Malus xiaojinensis.更多还原