【作者】 李兴亮；

【导师】 贾文锁；

【作者基本信息】 中国农业大学 ， 果树学， 2014， 博士

【摘要】 果树果实发育和成熟调控是果树学科的核心问题之一。多年来的研究表明,跃变型果实的成熟是由植物激素乙烯操纵的,但对果实中乙烯信号起源的机制却几乎没有深入了解。苹果是重要的果树品种之一,也是典型的呼吸跃变型果实。本论文以‘金冠’苹果为试材,从细胞信号转导的角度,对果实发育过程中乙烯信号起源的机制进行了研究。主要结果和结论如下：1.通过生物信息学分析,在苹果中共鉴定9个蔗糖非酵解型蛋白激酶SnRK2s亚家族成员。该亚家族成员均含有ATP结合位点及Ser/Thr激酶结构域,属于PKc类蛋白激酶。对SnRK2s亚家族全部成员的时空表达分析表明,MdSnRK2.4的表达特征与果实发育和成熟进程尤为一致,意味着MdSnRK2.4在苹果果实发育和成熟过程中可能起着重要作用。2. MdSnRK2.4在番茄’Micro Tom’中的稳定表达不仅可以促进果实发育还可以影响植物的一系列形态建成,如植株矮化、叶绿素含量上升、叶绒毛变长、叶肉细胞膨大及密度降低等。这些特征都与乙烯调控有关,说明MdSnRK2.4是操纵乙烯信号起源的上游信号蛋白。3. MdSnRK2.4在苹果果实愈伤中瞬时表达促进了乙烯合成关键酶MdACO1基因的表达及乙烯的释放,相反,MdSnRK2.4的RNAi沉默抑制了MdACO1的表达及乙烯的释放。MdSnRK2.4在番茄果实中异源过表达可显著促进乙烯合成及番茄果实成熟进程。进一步研究表明,在玉米原生质体中,MdSnRK2.4的瞬时表达可促进MdACO1启动子驱动的LUC报告基因的表达。这些数据表明,MdSnRK2.4为操纵MdACO1基因表达的上游信号蛋白,因而在乙烯合成及果实成熟调控中起着重要作用。4.通过酵母双杂交、双分子荧光互补及Pull-down检测证明,MdSnRK2.4与MdACO1转录因子MdHB存在物理互作,互作区域发生在MdSnRK2.4功能域STKC (Ser/Thr protein kinase c)与MdHB功能域HD (Homeobox domain)之间。利用原核菌株表达体系及磷酸化位点质谱检测发现,MdSnRK2.4与MdHB之间的互作导致MdHB蛋白磷酸化,磷酸化位点位于MdHB中HD结构域的三个苏氨酸位点,即T82、T95、T131。酵母双杂交、酵母单杂交检测发现,点突变该三个苏氨酸位点后MdHB与MdSnRK2.4互作强度及其对MdACO1启动子的启动活性下降。表明,MdSnRK2.4对MdHB的功能调控是通过磷酸化修饰机制。综上所述,本研究揭示了MdSnRK2.4为操纵乙烯信号产生的重要信号蛋白,其分子机制在于,MdSnRK2.4可以通过与MdACO1转录因子MdHB的直接互作,对MdHB功能域进行磷酸化修饰以调控MdHB的活性,进而调控MdACO1的表达,因而最终操纵了乙烯的产生。该研究不仅解析了苹果果实发育过程中乙烯信号产生的分子机制,对今后苹果分子改良也具有重要的指导意义。更多还原

【Abstract】 Regulation of fruit development and ripening is one of the most important questions in pomology science. It has been well established that ripening of climacteric fruit is controlled by phytohormone ethylene, but mechanisms of the signal production for ethylene production is largely unknown. Apple is well known to be one of the most important, and also, typical climacteric species of fruit tresses. Using’Golden Delicious’apple as material, this study aims to probe into the mechanisms of signal transduction for the ethylene production during apple fruit development and ripening. The major results are summarized as follows:1. Bioinformatics analysis identified nine members of sucrose non-fermenting protein kinase subfamily, SnRK2s, in ’Golden Delicious’ apple. The SnRK2s consists of nine members, all of which contain ATP-binding sites and the Ser/Thr catalytic domain of PKc. Analysis for the temporospatial characteristics of the gene expression of SnRK2s indicated that, MdSnRK2.4is closely correlated to apple fruit development and ripening, implying that MdSnRK2.4may play an important role in the regulation of apple fruit development and ripening.2. Stable transformation of MdSnRK2.4driven by CaM35S in ’Micro Tom’ not only promoted fruit ripening, but also affected plant phenotypes, e.g. plant dwarfing, increase in chlorophyll contents, long epidermal hairs and lower cell density and so on. All these phenotypes are correlated with function of ethylene, demonstrating a role of MdSnRK2.4in the regulation of ethylene production.3. Over-expression of MdSnRK2.4promoted ethylene production and the expression of MdACOl, a gene encoding the key enzyme in ethylene biosynthesis pathway, in apple fruit callus, and by contrast, inhibition of MdSnRK2.4by RNAi inhibited ethylene production and the expression of MdACO1. Additionally, transient expression of MdSnRK2.4in ’micro torn’ fruit also promoted ethylene production and fruit ripening. Maize protoplast transient expression analysis demonstrated that expression of MdSnRK2.4dramatically promoted the activity of the reporter gene GUS driven by MdACO1protmoter. All these results strongly indicated that MdSnRK2.4is a key signal controlling MdACO1expression thereby controlling ethylene production.4. Yeast two-hybrid, BiFC and full-down experiments all demonstrated that MdSnRK2.4could physically interact with MdACO1, and more over, the interaction domain took place between MdSnRK2.4catalytic domain STKC and MdHB functional Homeobox domain. Using prokaryotic expression system and mass spectrum analysis, it was demonstrated that MdSnRK2.4phosphorylated MdHB at three sites of Thr, i.e. T82, T95and T131. Mutation of those three phosphorylation sites in MdHB caused a decrease of interaction strength between MdHB and MdSnRK2.4in the yeast two-hybrid, and failed to activate transcription for MdACO1promoter detected by the yeast one-hybrid. Those showed that the expression regulation of MdSnRK2.4to MdHB is through phosphorylation mechanism.Collectively, this study demonstrated that MdSnRK2.4is a key signal controlling ethylene production. The mechanisms for the MdSnRK2.4-contrlled ethylene production is that MdSnRK2.4could phosphorylate MdHB transcription factor thereby regulating MdACO1expression and finally controlling the ethylene production. The present study is of great significance not only in the understanding of the molecular basis of ethylene signal origination but also in the molecular breeding of apple trees.更多还原