【作者】 安秀红；

【导师】 郝玉金；

【作者基本信息】 山东农业大学 ， 果树学， 2013， 博士

【摘要】 果皮色泽是苹果果实最为重要的品质性状之一，着色好坏直接影响其商品价值。苹果果皮的颜色主要是花青苷积累的结果。花青苷是类黄酮途径的次生代谢物之一,其合成受温度、光照、营养物质和植物激素等许多因素的影响。但这些外界因素影响花青苷积累的分子机理却仍不清楚。到目前为止，已经陆续从苹果中鉴定出多个调控果实着色的调节因子，然而，各调节因子之间的关系尚不明确，调控的分子机制有待阐明。本文以红星（Malus×DomesticaBorkhcv.Starkrimson）果实为材料，克隆了三个与花青苷代谢相关的调节因子基因，分别为MdTTG1、MdMYB9和MdMYB11。为了研究它们在苹果花青苷代谢途径中所发挥的作用，将这三个基因分别转化拟南芥和王林愈伤组织鉴定其功能。主要结果如下：1.MdTTG1转基因拟南芥促进花青苷积累为了检测MdTTG1基因在植物体中的功能，构建了35s::MdTTG1过量表达载体，并转化拟南芥。MdTTG1异位表达促进转基因拟南芥幼苗积累花青苷，在持续光照条件下尤为明显，转基因拟南芥中类黄酮代谢途径结构基因表达上调。这说明MdTTG1参与植物花青苷代谢调控。2.MdTTG1是WBM蛋白复合体成员酵母双杂交及双分子荧光互补实验证明，MdTTG1与MdbHLH3、MdbHLH33互作，但与MdMYB1不互作，而MdMYB1、MdbHLH3和MdbHLH33都参与到苹果花青苷合成的调控。MdTTG1表达与果实颜色积累成正相关模式，转基因拟南芥促进幼苗花青苷积累，暗示，苹果中也存在由WD40、MYB及bHLH形成的WBM蛋白复合体。3.MdMYB9和MdMYB11参与类黄酮代谢调控本文鉴定了两个新的参与苹果类黄酮代谢调控的MYB转录因子：MdMYB9和MdMYB11。转基因愈伤中积累大量的花青苷与原花青苷，过表达愈伤组织中类黄酮代谢途径结构基因表达明显上调。ChIP-PCR实验发现MdMYB9和MdMYB11都可以结合MdANR等结构基因启动子，说明MdMYB9和MdMYB11可能直接结合到这些结构基因的启动子上，进而调控苹果花青苷与原花青苷的代谢。4.MdMYB9和MdMYB11参与JA诱导的花青苷与原花青苷代谢调控MdMYB9和MdMYB11基因表达受JA与创伤强烈诱导，JA处理转基因愈伤组织无论是花青苷还是原花青苷积累都明显增多。ChIP-PCR实验发现，JA处理可以增强MdMYB9和MdMYB11蛋白对花青苷代谢途径下游结构基因启动子的结合。由此说明，JA可能通过MdMYB9和MdMYB11来调控苹果中类黄酮代谢途径的。更多还原

【Abstract】 Fruit color is one of the most important quality traits. It determines the diet and commercial values of apple fruit to a great extent. Apple fruit coloration results from the accumulation of anthocyanins which synthesized through flavonoid secondary metabolic pathway. The biosynthesis of anthocyanins is greatly influenced by various stimuli, however, the molecular mechanism through which those stimuli regulate anthocyanins biosynthesis is largely unknown.In these study, three genes, i.e. MdTTGl, MdMYB9and MdMYBll, were isolated from apple fruit(Malus×Domestica Borkh cv. Starkrimson). To characterize their functions, they were genetically transformed into Arabidopsis and apple callus, respectively. Main results are shown as follows:1. MdTTGl promotes anthocyanins accumulation in transgenic ArabidopsisTo characterize the function of MdTTGl in planta, we constracted35S::MdTTGl construct, and then transformed it into Arabidopsis. The results showed that ectopic expression of MdTTGl in Arabidopsis promoted anthocyanins accumulation, especially with continuous light treatment. The structural genes associated with anthocyanins biosynthesis were remarkably up-regulation compared to WT. These results indicated that MdTTGl involved in anthocyanins accumulation in planta.2. MdTTGl is a member of the WBM complexY2H and BiFC assay showed that MdTTGl interacts with MdbHLH3and MdbHLH33, respectively, but not with MdMYB1protein. In apple, MdMYB1, MdbHLH3and MdbHLH33were proved to be involved in the regulation of anthocyanins biosythesis. The transcription level of MdTTG1was associated with fruit pigment, and transgenic Arabidopsis overexpressing MdTTGl produced more anthocyanins than the WT control. Therefore, it may come to a conclusion that MdTTGl works together with MdMYB1, MdbHLH3and MdbHLH33to form WBM complex and is involved in the regulation of anthocyanins biosynthesis.3. MdMYB9and MdMYB11were involved in the regulation of anthocyanins and proanthocyanidins biosynthesisIn this study, two new MYB transcription factor genes named MdMYB9and MdMYB11were isolated from apple fruit. Transgenic callus overexpressing MdMYB9and MdMYB11, respectively, generated more transcripts of structural genes associated with flavonoids biosynthesis, and therefore produced more anthocyanins and proanthocyanins than control. ChIP-PCR assay demonstrated that MdMYB9and MdMYB11bind to the promoters of the structural genes, respectively, which leads to anthocyanins and proanthocyanins accumulation in transgenic callus.4. JA regulates the accumulation of anthocyanins and proanthocyanins by modulating the expression MdMYB9and MdMYB11The expression levels of MdMYB9and MdMYB11were induced by MeJA. Both MdMYB9and MdMYB11transgenic calluses accumulated more anthocyanins and proanthocyanins under MeJA treatment, compared with untreatment. In addition, MeJA enhanced the binding of MdMYB9and MdMYB11to the promoters of anthocyanins and proanthocyanins structural genes. Therefore, MdMYB9and MdMYB11are involved in the JA-induced anthocyanins and proanthocyanins accumulation in apple.更多还原