【作者】 赵强；

【导师】 郝玉金；

【作者基本信息】 山东农业大学 ， 果树学， 2015， 博士

【摘要】 铁是果树生长和发育过程重要的微量元素,是影响果实产量和品质的重要因素之一。影响铁离子吸收的环境因素很多,如pH、营养元素、激素等。其中pH是影响果树吸收铁离子的重要因素。为了维持果树体内铁离子的动态平衡,果树根系通过质膜H+-ATPase酶外泌质子使得根系周围酸化,将难溶态铁转化为可溶态,供根系直接吸收。质膜H+-ATPase酶除了外泌质子和产生跨膜电势梯度外,还能为离子的转运提供能量。因此,质膜型(PM)H+-ATPase酶在铁离子的吸收和动态平衡过程中起着重要的作用。植物缺铁响应的分子机理在模式植物中已经被研究清楚,但是对于多年生木本植物缺铁响应的分子机理尚不清楚。植物的Ib类和IVc类bHLH转录因子在铁离子的吸收过程中扮演者重要的角色。本研究从苹果中克隆到一个IVc类bHLH转录因子基因,命名为MdbHLH104, RT-PCR分析发现其表达受缺铁诱导。为了验证其功能,将35S::MdbHLH104过表达载体遗传转化并获得了苹果的转基因植株和愈伤组织；表型分析发现,在缺铁条件下,MdbHLH104转基因株系表现出明显抗缺铁能力,并且根际质子外泌和三价铁离子还原酶活性都增加,植物体内铁离子含量也比野生型对照高。进一步研究发现,MdbHLH104蛋白能够直接结合到MdAHA8基因(编码PMH+-ATPases)的和Ib类bHLH转录因子基因的启动子上,激活这些基因的表达,从而正调控PM H+-ATPases的酶活性和Ib类bHLH转录因子的转录能力。以MdbHLH104为诱饵蛋白进行酵母双杂交筛选cDNA文库,得到一些与MdbHLH104互作的蛋白。酵母双杂交和Pull-Down分析的结果表明,MdbHLH104蛋白分别与MdPYE、MdILR3 (MdbHLH105)、MdbHLH115、MdbHLH11和MdbHLH121等其他IVc类bHLH转录因子相互作用,形成异源二聚体共同正调控下游基因MdAHA8的表达,分别共表达MdbHLH104与MdPYE、MdILR3 (MdbHLH105)、MdbHLH115、 MdbHLHll或MdbHLH121明显增强转基因苹果愈伤组织中的H+-ATPase活性和铁含量。此外,还筛选到了2个BTB/POZ-TAZ蛋白MdBTl和MdBT2,序列搜索比对发现,苹果基因组还编码另外三个BTB-TAZ蛋白,分别为MdBT3.1、MdBT3.2和MdBT4,结构域分析表明,5个MdBTs蛋白都含有三个保守的结构域,即N端的BTB/POZ结构域,中间位置的TAZ结构域和C端的钙调素结合结构域。酵母双杂交、Pull-Down和Co-IP(免疫共沉淀)分析试验表明,5个MdBTs蛋白中只有MdBTl和MdBT2能够与MdbHLH104相互作用,具体互作区域是MdBT1和MdBT2的TAZ结构域和MdbHLH104的C末端区域。Western杂交分析的结果显示,MdbHLH104蛋白在35S::MdBT1/2-Anti(反义沉默表达载体)转基因苹果愈伤组织中的积累水平高于,而在35S::MdBT2(正义过量表达载体)转基因苹果愈伤组织中的积累水平低于对照,表明MdBTs蛋白负调控MdbHLH104蛋白的丰度。深入研究表明,MdBT2蛋白能够与MdCUL3互作形成CRL3BT2E3泛素化连接酶复合体,离体和活体泛素化以及体外降解试验表明,CRL3BT2 E3泛素化连接酶复合体介导MdbHLH104蛋白的泛素化修饰,进而通过26S蛋白酶体途径降解,因此,MdBT2蛋白通过MdbHLH104调控植物体内质子外泌和铁离子的吸收及动态平衡。最后,我们还发现MdBT1和MdBT2蛋白的稳定性受铁离子的直接调控,即铁存在条件下促进MdBT1和MdBT2蛋白的积累,缺铁则抑制两个蛋白的积累。总之,本文阐述了一个缺铁逆境应答和调控途径：铁离子浓度H+-ATPase活性一逆境应答。更多还原

【Abstract】 Iron is definitely essential for fruit trees due to its various roles in life processes, fruit trees are one of the most affected crops by iron deficiency, which significantly decreases fruit yield and quality. Iron uptaking is regulated by various factors such as pH, mineral elements and hormones and so on. The pH is an important factor affecting iron uptaking. In order to maintain the balance in the tree-soil system during iron uptake, the root of plant excretes amount of protons mediated by PM H+-ATPase to lead to the acidification of rhizosphere, which in turn makes iron soluble and available in soil for uptake. Besides proton extrusion and the associated electrochemical gradient, PM H+-ATPase also supplies energy for iron uptake and transportation. Therefore, PM H+-ATPase plays a crucial role in iron acquisition and homeostasis in plant. The molecular mechanisms of iron deficiency response have been increasingly studied in model plants, the underlying molecular and genetic bases of this process remain unclear in apple trees.In Arabidopsis, Ib and IVc subgroups bHLH TFs, play a central role in modulating the expressions of the major Fe acquisition genes。In this study, we described an IVc subgroup bHLH TF gene MdbHLH104, which is up-regulated under iron deficiency conditions. 35S::MdbHLH104 transgenic apple plantlets exhibited an improved tolerance to Fe deficiency. The transgenic apple plantlets and calli showed an enhanced proton excretion, ferric oxidoreductase activities and Fe contents. Furthermore, we demonstrate that MdbHLH104 directly activates the expression of the MdAHA8 and Ib subgroup bHLH TFs by binding to the E-box element in its promoter.In order to identify the interaction genes MdbHLH104, yeast two-hybrid library screening was used with a full-length MdbHLH104 as bait. In our work, yeast two-hybrid and pull-down showed that MdbHLH104 interacts with other IVc subgroup members including MdbHLH105 (MdILR3), MdbHLH115, MdPYE, MdbHLH11 and MdbHLH121. The heterodimers also activated the expression of MdAHA8. Co-overexpression MdbHLH104 and MdbHLH105 (MdILR3), MdbHLH115, MdPYE, MdbHLH11 and MdbHLH 121, respectively, enhanced proton excretion, H+-ATPase activity and Fe contents in the transgenic apple calli.In addition, two BTB-TAZ proteins were found named MdBT1 and MdBT2 from apple. MdBT subfamily genes were identified from the apple genome database following a BLASTN homology search using each of the AtBT protein sequences, and the other three MdBT genes were found, named MdBT3.1, MdBT3.2 and MdBT4. Conserved domain analysis showed that All of them share similar structures, i.e. a BTB domain in the N-terminal part, a TAZ domain in the center and CaM domain in C-terminal. Yeast two-hybrid, pull-down and Co-IP showed that MdBTland MdBT2 interacted with MdbHLH104, and the TAZ domains of MdBT1 and MdBT2 interact with the C terminus of MdbHLH104. Western Blot analysis showed that more MdbHLH104 protein in 35S::MdBT1/2-Anti transgenic calli and less MdbHLH104 protein in 35S::MdBT2 transgenic calli, indicating that MdBTl and MdBT2 negative the MdbHLH104 protein accumulation. Furthermore, the BTB domain of MdBT2 protein is responsible for the interaction with MdCUL3 and the associated partners such as RBX1 to form CRL3BT2 complex. The cell-free degradation assays and ubiquitination modification in vivo and vitro were used, and the results showed that the BTB domain of MdBT2 recruits MdCUL3-containing CRL3 complex to regulate MdbHLH104 stability by ubiquitination and degradation via the 26S proteasome. Besides, the accumulation of MdBTl and MdBT2 protein are induced by Fe sufficiency, and reduced under Fe deficiency conditions. Taken together, our study provides evidence for that Iorn-MdBT2-MdbHLH104-MdAHA is an ubiquitin-dependent degradation pathway for development and growth for responding iron dificient in apple.更多还原