【作者】 李敏；

【导师】 陈学森；

【作者基本信息】 山东农业大学 ， 果树学， 2013， 博士

【摘要】 由硬度和脆度构成的果实质地品质是苹果品质的重要构成因素之一。它不仅影响苹果的鲜食品质，也是限制苹果贮运品质的重要因素。因此探讨苹果质地品质形成的机理也具有重要的意义。本课题组育成的早熟苹果新品种‘泰山早霞’综合经济性状优良，但依然存在果实容易软化变绵这一早熟苹果的共性问题，特别是全红期采收的果实在室温条件下存放3～5d后就软化变绵甚至裂果，严重影响了苹果的品质。因此研究早熟果实质地品质的形成机理，改善早熟苹果果实质地品质是当前培育优质早熟苹果品种急需解决的问题。本文以‘泰山早霞’苹果为试材，分别测定果实生长发育期和贮藏期以及采后用乙烯抑制剂1-MCP处理果实的内源乙烯合成，PG酶活性和果实硬度的变化。克隆了五个乙烯信号转导途径的调控基因（ZMdEIL1、ZMdEIL2、ZMdEIL3、ZMdERF1和ZMdERF2）和一个功能基因ZMdPG1，并研究乙烯信号调控基因和ZMdPG1在苹果果实生长发育和成熟软化过程中的表达模式，以及对1-MCP（1.0μl·L-1）处理的应答模式，鉴别参与苹果果实成熟软化的重要基因；验证乙烯信号转导途径调控基因和功能基因ZMdPG1的作用；初步探索乙烯信号编码基因对功能基因ZMdPG1的调控作用，明确乙烯与苹果果实软化和裂果的关系。主要研究结果如下：1.‘泰山早霞’果实成熟过程中，内源乙烯大量积累，PG酶活性急剧增加，果实硬度快速下降；乙烯抑制剂1-MCP能够抑制内源乙烯合成，同时PG酶活性下降，果实硬度软化趋势被延缓，果实裂果现象也被抑制。2.在‘泰山早霞’果实整个发育过程中ZMdERF1基因持续性表达；ZMdEIL2基因的表达量在果实发育前期和后期均较高，中期比较低；而且，ZMdERF1和ZMdEIL2基因的表达丰度均在花后65d即内源乙烯开始大量积累时增高；ZMdERF2基因的表达量在花后75d升高；ZMdEIL1和ZMdEIL3基因的表达量在果实的整个发育过程中都较低。ZMdPG1基因的表达量在果实生长发育过程中较低，在软化果实和裂果果实中显著增强。用乙烯抑制剂1-MCP处理果实后，伴随着内源乙烯积累的迅速降低，ZMdPG1、ZMdERF1、ZMdEIL1和ZMdEIL2基因的转录明显被抑制，ZMdERF2的表达也降低，ZMdEIL3基因的表达未有明显的变化。ZMdPG1、ZMdERF1、ZMdERF2和ZMdEIL2基因的表达响应内源乙烯的调控，并协同调节苹果果实的成熟软化和裂果。3.利用RT-PCR技术克隆了ERFs家族的ZMdERF1和ZMdERF2，EIN3/EILs家族的ZMdEIL1、ZMdEIL2、ZMdEIL3和功能基因ZMdPG1；其中，ZMdERF1和ZMdERF2的氨基酸序列包含ERFs家族的两个保守区域YRG和RAYD，ZMdEIL1、ZMdEIL2和ZMdEIL3的氨基酸序列含有EIN3/EILs家族的特征元件AD、BDI、BDII、BDIII、BDIV、BDV和PR，并采用High-tail PCR克隆得到ZMdPG1的启动子，它包含有多个调控元件，如：A-box、AT-rich sequence、BoxI、G-Box、BoxIII、CCGTCC-box、CGTCA-motif等。亚细胞定位显示ZMdEIL1、ZMdEIL2、ZMdEIL3、ZMdERF1和ZMdERF2均定位在细胞核内，ZMdPG1定位在细胞壁和细胞质膜中。4.在拟南芥中超表达ZMdPG1基因，加速了果皮细胞壁的降解，细胞与细胞之间的结合力减弱从而引发果皮缝合处的细胞分离，导致果荚提早开裂，而反义ZMdPG1转基因拟南芥抑制了果荚正常开裂。在番茄中超表达ZMdPG1基因引起大量落花和果实不完全发育。5. ZMdPG1启动子上含有多个调控元件，其中包括蛋白结合元件BOXIII，但由ZMdPG1启动子合成的探针不与ZMdERF1蛋白结合，表明ZMdERF1不直接调控目的基因ZMdPG1。6.苹果乙烯信号编码基因之间存在相互作用，ZMdERF1/ZMdEIL2组合之间和ZMdERF2/ZMdEIL2组合之间分别存在互补关系，表明ZMdERF1蛋白和ZMdERF2蛋白特异的与ZMdEIL2蛋白互作。更多还原

【Abstract】 Apple is one of the most important fruit crop in China. It is important to investigate themechanism of texture quality including firmness and fragility which influences the eatingand shipping quality.‘Taishanzaoxia’ is one of the early-ripening apple cultivars and hasexcellent quality, but it is easy softening and even dehiscence. This greatly reduce itscommercial value. Therefore, studying the mechanism of fruit texture quality is very importfor improving the fruit quality. In this study, the ethylene production, PG activity and fruitfirmness during fruit development and storage stages were analysed. The treatment with1-MCP(1.0μL·L-1) was also used during storage stage. Five ethylene signal transcriptionfactors (ZMdEIL1, ZMdEIL2, ZMdEIL3, ZMdERF1and ZMdERF2) and one functional geneZMdPG1which encodes polygalacturonase were identified. The expression patterns ofZMdEIL1, ZMdEIL2, ZMdEIL3, ZMdERF1, ZMdERF2and ZMdPG1were studied.Transcription factors and promoter interaction and functional transgenic works were alsoconducted in the study. The main results are as follows:1. PG activity and fruit softening of ‘Taishanzaoxia’ fruit rapidly increased followed byethylene burst. With treatment of1-MCP, ethylene production was sharply reduced.Furthermore, the PG activity, fruit softening and dehiscence were significantly inhibited. Itsuggested that endogenous ethylene could play important role in the regulation of fruit softingand dehiscence.2. ZMdERF1and ZMdEIL2transcription were more abundant in ‘Taishanzaoxia’softening fruit and dehiscent fruit and their expression were inhibited by an ethylene inhibitor1-MCP. Therefore, ZMdERF1and ZMdEIL2expression were response to endogenousethylene and associated with fruit softening and dehiscence. ZMdPG1expression was inducedduring fruit softening and dehiscence, but it can be blocked by1-MCP, indicating thatZMdPG1was essential for fruit softening and dehiscence and its expression was mediated bythe endogenously occurred ethylene. The expression of ZMdERF2，ZMdEIL1and ZMdEIL3maintained low level during fruit development, and there was little increase in ZMdERF2butit was inhibited by1-MCP in softening fruit. These results indicated that the transcription ofZMdERF1，ZMdERF2，ZMdEIL2and ZMdPG1were closely connected in ethylene during fruit softening and dehiscence stages.3. ZMdERF1, ZMdERF2, ZMdEIL1, ZMdEIL2, ZMdEIL3and ZMdPG1were clonedfrom ‘Taishanzaoxia’ fruit. ZMdERF1and ZMdERF2belong to ERFs family including YRGand RAYD conserved domains. ZMdEIL1, ZMdEIL2and ZMdEIL3belong to EIN3/EILsfamily shared characteristic factors AD, BDI, BDII, BDIII, BDIV, BDV and PR. TheZMdPG1promoter was cloned which contains A-box, AT-rich sequence, BoxI, G-Box,BoxIII, CCGTCC-box and CGTCA-motif regulatory factors. ZMdEIL1, ZMdEIL2andZMdEIL3proteins were located in cell nucleus, while ZMdPG1was located in cell wall andcytoplasmic membrane.4. ZMdPG1overexpression in Arabidopsis led to seed silique early dehiscence whilesuppressing ZMdPG1expression by antisense ZMdPG1prevented seed silique naturallyopening. The ectopic expression of ZMdPG1was involved in cell separation and contributedto fruit dehiscence. ZMdPG1overexpression in tomato resulted in flower and fruit abscission.5. There were multiple regulatory factors in ZMdPG1promoter including bindingprotein factor BOXIII, however, ZMdERF1didn’t bound to ZMdPG1promoter. Therefore,ZMdERF1did not directly regulate the functional gene ZMdPG1.6. BiFC(Bimolecular fluorescence complementation) experiment evidenced thatZMdERF1and ZMdERF2interacted physically with ZMdEIL2.更多还原