鲑鱼降钙素基因植物表达载体的构建及遗传转化研究

【作者】 赵吉强；

【导师】 程智慧；

【作者基本信息】 西北农林科技大学 ， 蔬菜学， 2001， 硕士

【摘要】 降钙素（Calcitonin，CT）在临床上主要用于治疗骨质增生、各种原因引起的骨骼疼痛症状、Paget’S疾病、高钙血症等。现临床应用的主要是从鲑鱼鳃和猪甲状腺分离纯化的降钙素，来源有限，价格昂贵。尽管研究者已在原核细胞表达系统对鲑鱼降钙素的表达进行了研究，但由于原核表达系统缺乏翻译后的加工修饰等缺点，限制了其应用。因此，利用植物表达系统大规模生产降钙素，满足社会对降钙素日益增长的需求，具有重要的实际意义。本研究旨在由鲑鱼降钙素（sCT）基因的链霉菌表达载体构建sCT基因的植物表达载体；并将sCT基因转入生菜等作物，使其成为降钙素的新型生物反应器，为降钙素的生产提供一条经济有效的新途径。主要研究结果如下：1. 通过限制性内切酶BamHI/HindIII酶切sCT基因的链霉菌表达质粒pMS680获得sCT基因，并将其克隆至植物表达载体pART27，获得sCT基因的植物表达载体pART27-sCT，并通过电击转化法成功转化土壤根癌农杆菌C58。2. 通过基因枪微弹射入法将sCT基因导入胡萝卜愈伤组织；酶联免疫法检测sCT蛋白的瞬时表达情况，结果表明sCT基因能够在胡萝卜愈伤组织中表达。3. 通过研究不同激素浓度对大速生散叶生菜子叶外植体再生的影响，确定其适宜的诱芽培养基为：MS+0.1mg/L6-BA+0.05mg/LNAA，生菜子叶外植体的再生率为60.3%；抗生素敏感性试验表明，适宜的Km选择压为75mg/L，Carb的浓度为300mg/L。4. 通过农杆菌介导的叶盘法，将sCT基因转入烟草及生菜，经在含75mg/LKm的诱芽培养基和1/2MS生根培养基筛选，获得Km抗性烟草40株，生菜118株；经PCR检测，得到转基因烟草11株，转基因生菜29株。更多还原

【Abstract】 Calcitonin (CT) mainly be used in curing osteoporosis, all kinds of ostalgia , Paget’ S disease , hypercalcemia and etc in clinical practice . The calcitonin being used in clinic is the product that separated and purified from salmon’s gill lamella or pig’s thyroid gland, but the calcitonin produced in this way is too limited and expensive. Although it has been expressed in the prokaryotic cell system, its application is restricted for its deficiency in modification after translation. Therefore, taking the transgenic plant as a bio-reactor to produce calcitonin in large scale has a great meaning in meeting the increasing demand of calcitonin. The aim of this study was to construct the expression vector of salmon calcitonin (sCT) gene, and to transfer sCT gene into lettuce genome and other crops. The main results are as following:1. The sCT gene coding sequence was obtained from its streptomyes vector pMS680 by restriction enzyme BamHI and HindIII , and be cloned into expression vector pART27 to produce a plant expression vector pART27-sCT. The recombinant plasmid be transferred into Agrobacterium tumafaciens C58 by electroporation successfully.2. Particle bombardment was used to transform sCT gene into carrot callus, ELISA analysis of proteins extracted from callus showed the transient expression of sCT gene has achieved. 3. The effects of different hormone concentration on shoot regeneration from cotyledon explants was investigated, the results show that MS medium supplied with 0.1mg/L 6-BA and 0.05mg/L NAA is the suitable shoot regeneration medium; The experiment on sensitivity of cotyledon to kanamycin and carbenicillin shows the suitable kanamycin concentration for selecting transgenic tissue is 75mg/L and the carbenicillin concentration is 300 mg/L.4. sCT gene was transferred into tobacco and lettuce mediated by Ti plasmid of Agrobacterium tumafaciens. Forty tobacco and 118 lettuce which resistance kanamycin was obtained after sifted on shoot regeneration medium and root induce medium supplied with 75mg/LKm; PCR amplification of genomic DNA from kanamycin resistance tobacco and lettuce leaf tissue shows sCT gene was integrated into the genome of transgenic tobacco and lettuce, 11 transgenic tobacco and 29 transgenic lettuce was obtained .更多还原