青岛农业大学 毕 业 论 文（设计）
题 目: 葡萄逆境胁迫诱导启动子的克隆
及其表达载体的构建
姓 名: 刘丽雪 学 院: 生命科学学院 专 业： 生物技术 班 级： 2007级4班 学 号： 20072833 指导教师： 薛仁镐
2010年6月17日
目 录
中文摘要···························································································································1 Abstract······························································································································2 1、引言······························································································································3 2、CAN启动子的克隆·······································································································5 2.1 材料····························································································································5 2.2 方法····························································································································5 2.2.1 组培培育葡萄幼苗·································································································5 2.2.2 DNA的提取·············································································································6 2.2.3 目的基因PCR扩增·································································································6 2.2.4 目的基因与T--A载体连接及产物转化································································7 2.2.5 重组质粒的提取及酶切鉴定·················································································8 2.2.6重组质粒测序鉴定···································································································9 2.3 结果与分析··············································································································10 2.3.1 DNA提取鉴定·······································································································10 2.3.2 PCR扩增结果········································································································10 2.3.3 质粒提取鉴定·······································································································11 2.3.4 双酶切鉴定···········································································································11 2.3.5 克隆启动子的序列分析·······················································································13 3、植物表达载体pBI1391Z- CAN的构建·····································································15 3.1材料····························································································································15 3.2方法····························································································································16 3.2.1植物表达载体pBI1391Z- CAN的构建·································································17 3.2.2植物表达载体pBI1391Z- CAN转入农杆菌·························································17 3.3 结果与分析··············································································································17 3.3.1 表达载体pBI1391Z- CAN的酶切鉴定·······························································17 4、农杆菌介导的烟草遗传转化及转基因植株分析·····················································18 4.1 实验材料··················································································································18
4.1.1 供试烟草品种·······································································································18 4.1.2 菌株与载体···········································································································18 4.1.3 培养基···················································································································18 4.1.4 主要生化试剂及其配制·······················································································18 4.2 试验方法··················································································································18 4.2.1 农杆菌活化及菌液制备·······················································································18 4.2.2 根癌农杆菌介导的烟草遗传转化·······································································19 4.3 结果··························································································································19 5、讨论与展望···············································································································20 致谢·································································································································21 参考文献·························································································································22
葡萄逆境胁迫诱导启动子的克隆及其表达载体的构建
生物技术 刘丽雪 指导教师 薛仁镐
摘要：本文以葡萄幼苗为材料，依据基因组序列，设计引物，以叶片基因组DNA为模板，利用PCR技术，对葡萄逆境胁迫诱导型启动子进行了扩增，获得了约1.35 kb的启动子序列，将启动子片段回收后连接到T载体，酶切鉴定结果表明，启动子构建到T-A载体，对重组质粒进行测序，结果表明，测序的启动子序列与预知序列只有少数碱基的差异，同源性达到97%，表明已克隆到葡萄启动子。将克隆到的启动子与pBI1391Z连接，转入大肠杆菌细胞， 提取质粒进行酶切鉴定。结果表明，植物表达载体构建完成并将其命名为pBI1391Z-CAN。将其转入农杆菌细胞内，并利用农杆菌介导法对烟草进行了遗传转化。葡萄逆境胁迫诱导型启动子的克隆对下一步抗逆调控分子机制研究及作物分子育种具有重要理论和实际意义。 关键词：逆境胁迫；启动子；PCR扩增；克隆；表达载体；
1
Cloning of Adversity Stress-Induced Promoter in Grapes
Student majoring in Biotechnology Lixue Liu
Tutor Name Ren-Gao Xue
Abstract: The 1.35 kb promoter of a gene was amplified by PCR technique using genomic DNA of grapes as template in this study. The fragment of promoter was then ligated to the T cloning vector and confirmed by digestion with endonuclease and sequencing. The results of sequencing showed that there were only a few differences between the sequencing promoter sequence and predicted sequence, homology of bases reached to 97%. The fragment of promoter was ligated to vector pBI1391Z and introduced into the E.coli cells, then the construct was extracted and confirmed by digestion with enzyme. The recombined plants express vector was named as pBI1391Z-CAN. The pBI1391Z-CAN was introduced into Agrobacterium cells, and then transformation of tobacco was done using Agrobacterium–mediated transformation method. Cloning of stress-induced promoter has important significance on molecular regulation mechanism of adversity and genetic breeding in crop. Key words: Stress tolerance；promoter；PCR amplification；Cloning；expression vector
2