【作者】 王君丹；

【导师】 车代弟； 林忠平；

【作者基本信息】 东北农业大学 ， 园林植物与观赏园艺， 2004， 硕士

【摘要】 水资源匮乏严重地制约着农业和园林事业的发展。干旱是影响植物生长和发育的重要环境因子，植物在干旱、低温、盐和ABA等逆境的胁迫下，脱水素的表达水平明显升高，这表明脱水素可能对于增强植物对逆境胁迫的耐受力有重要的作用。本实验将源于耐旱植物厚叶旋蒴苣苔（Boea crassiffolia）的脱水素（Dehydrin）基因DBDN置于CaMV35S启动子的调控之下，并插入于表达载体pCAMBIA3300中，通过根癌土壤杆菌(Agrobacterium tumefaciens)介导导入矮牵牛(Petunia Hybrida)，经PCR及Southern杂交验证DBDN基因已整合入矮牵牛基因组中，Northern实验证明，DBDN基因在矮牵牛中在RNA水平有较强的表达。通过对转DBDN基因矮牵牛的保水力和低温离子渗漏的测定，初步验证转基因矮牵牛对水分胁迫的能力在一定程度上有所增加。本实验是培育耐水分胁迫的观赏园艺作物的一次尝试，也为将脱水素基因导入园林植物培育耐寒耐旱园林植物新品种做了一个铺垫。另一方面，将脱水素基因置于T7启动子的调控之下，插入原核表达载体pMXB-10中，在大肠杆菌ER2566中表达，超声破碎后，得到可溶的融合蛋白，经Chitin Beads纯化得到BDN1蛋白。将所得蛋白免疫兔子，得到抗血清，并用ELISA对抗血清进行效价测定，测定结果表明，抗血清中含有针对BDN1蛋白的抗体，所得的抗血清可用于转基因植物BDN1蛋白的检测，BDN1蛋白在植物中的表达定位实验尚在深入进行。更多还原

【Abstract】 Water deficiency is a serious limited factor in the agricultural and horticultural development. And drought is the critical factor to affect plant growth and development. Expression of dehydrin increases when plant is under the stess of drought, low tempreture, salt and ABA. This phenomenon indicate that dehydrin could have more important action in improve the tolerant of stress of plant.Dehydrin-like Gene (DBDN) from Boea crassifolia was placed under the control of CaMV35S promoter and inserted into binary vector pCAMBIA3300 for genetic transformation. The transgenic plants of Petunia hybrida were identified by molecular biology procedure and show strong expression of transgene of dehydrin at transcriptional level. The water conservation ability assay and leaf electrolyte leakage determination showed transgenic petunia plants are with strong tolerance to stress condition. Otherwise, put the gene BDN1 under the regulation of T7 promoter and into the prokaryotic expression vector pMXB-10. Express the protein BDN in E. coli ER2566. Collected solubility fusion protein after ultrasonication, cleavage and purification we get the protein BDN1. Use BDN1 immune cony to get antiserum. Potency test of antiserum with ELISA indicate there are antibody of BDN1 in antiserum. The antiserum can be used to test of protein BDN1 expressing in transgenic plant. The work on dehydrin distribution is under further study by immunological method.Master Candidate: Wang JundanMajor: Garden Plant and Ornamental HorticultureSupervisor: Pro. Che Daidi andPro. Lin Zhongping更多还原