α-法尼烯合成酶基因转化丰产梨的研究-植物学专业论文.docxVIP

山东农业大学硕士学位论文 中文摘要 α-法尼烯是一种存在于生物体中的倍半萜类物质，已知与植物的抗冷性、昆虫的 诱导及冷藏苹果和梨果实重大生理病害－虎皮病的发生有关，是苹果果实中重要的香 气成分之一。α-法尼烯的生物合成是通过类异戊二烯途径中的甲羟戊酸途径，α-法尼 烯合成酶是调控 α-法尼烯合成的关键酶，催化 FPP 转变为 α-法尼烯。 本实验根据 Genbank 中注册的 α-法尼烯合成酶基因全长序列设计特定引物，利 用荧光定量 PCR 和 Western 杂交的方法，鉴定初步获得转基因植株，并研究了生理 特性。主要结果如下： （1）构建了 pBI-AFS 反义和 RNAi 表达载体，并转化丰产梨，得到转 AFS 基因 的丰产梨植株。 （2）以丰产梨组培苗为材料，优化了丰产梨组培苗的生根及遗传转化体系，并 成功将转基因组培苗驯化移栽。最适宜丰产梨继代的培养基为 MS+BA1.0mg/L +IBA0.2mg/L+蔗糖 30.0g/L+琼脂 6.0g/L，生根培养基为 1/2 MS+ IBA0.3mg/L+蔗糖 30.0g/L+琼脂 6.0g/L. （3）转基因植株经过 RT-PCR 和荧光定量 PCR 分析表明，野生型中 α-法尼烯合 成酶基因的相对表达量最高，反义转化植株中有所降低，RNAi 转化植株中 α-法尼烯 合成酶基因的相对表达量最低。 （4）通过克隆青香蕉苹果的 α-法尼烯合成酶基因（AFS）片段，构建 pEASY E1-AFS-原核表达载体，转化大肠杆菌，制备抗体，进行了 Western 杂交分析，显示 该基因在蛋白水平成功表达。 （5）野生型丰产梨比转 AFS 基因丰产梨具有较高的耐热性，高温胁迫条件下野 生型丰产梨维持较高的光合能力。 关键词：α-法尼烯合成酶；载体构建；遗传转化；western 杂交；丰产梨 1 苹果 α-法尼烯合成酶基因转化丰产梨的研究 Genetic Transformation of Fertility Pear with Apple α- Farnesene Synthase Gene Abstract α-Farnesene, an acyclic sesquiterpene hydrocarbon that existed in organisms, has been proved to play an important role in attraction of codling moth chilling injury and development of scald on cold-storage apples. As one of aroma related volatiles in apple fruit, α-farnesene appears to be predominantly through classical mevalonate pathway. α-farnesene synthase (AFS) is the final, rate-limiting enzyme that converts farnesyl diphosphate (FPP) to α- farnesene in the mevalonic acid (MVA) pathway of terpene biosynthesis. Specific primers according to the full sequence of α-farnesene synthase gene were designed. Transgenic plant was got by Real time RT-PCR and Western Blot analysis，and physiological characteristic of transgenic plant was studied .The main resμLts were as follow: A recombinant plant expression vector pBI-AFS was constructed and was used to transform to ‘Fertility’ pear. Adventitious bud regeneration from the leaflet of fertility pear and efficient gene transformation system were optimized. The resμLts showed that the optimum medium for adventitious bud regeneration was MS+BA1.0mg/L+I