叶用莴苣LsHsp70-3701基因过表达载体的构建及遗传转化体系的优化  

Over-expression vecter construction and Agrobacterium transformation optimization of leaf lettuce gene LsHsp70-3701

作　　者：苏振琪 韩莹琰[1] 李雅博[1] 刘然[1] 刘超杰[1] 郝敬虹[1] 范双喜[1] 

机构地区：[1]北京农学院植物科学技术学院,北京102206

出　　处：《中国农业大学学报》2018年第1期63-70,共8页Journal of China Agricultural University

基　　金：国家自然科学基金项目(31372057;31401883);北京市叶类蔬菜创新团队建设专项(blvt-02);北京市农业科技项目(20140134);北京市科技成果转化提升计划项目(PXM2015-D14207O00012);北京市自然科学基金B类项目

摘　　要：为研究叶用莴苣中Hsp70基因的功能,以提高其耐热性,构建LsHsp70-3701过表达载体,农杆菌介导法转化叶用莴苣。从叶用莴苣中克隆得到LsHsp70-3701,利用NcoⅠ和SpeⅠ双酶切LsHsp70-3701和植物表达载体pCAMBIA1304,回收目的片段并连接,经鉴定后获得过表达载体。以‘P-S11'叶用莴苣为转化材料,针对遗传转化关键因素:苗龄、植物激素浓度、预培养时间、侵染液OD值、侵染时间和共培养时间进行优化。试验结果表明最佳转化条件为:苗龄5d的子叶,6-BA 0.2mg/L+NAA 0.1mg/L,KT质量浓度为0.2mg/L,OD600为0.3,预培养2d,侵染10min,共培养2d。To study the function of a leaf lettuce Hsp70 gene,and improve plant thermo-tolerance,LsHsp70-3701 overexpression vector was constructed and transformed into lettuce by Agrobacterium transformation.LsHsp70-3701 was cloned from lettuce leaves.LsHsp70-3701 and plant expression vector of pCAMBIA1304 was digested by NcoⅠ and SpeⅠ.Target fragment was recovered and connected to plant expression vector.After identification,the expression vector was obtained.Using ‘P-S11＇leaves as material,transformation conditions was optimized.Key factors including seedling stage,plant hormone concentration,pre-culture time,infection OD,infection time and co-culture time were investigated.The results showed that：The optimized combination of hormones was 0.2 mg/L 6-BA,0.1 mg/L NAA and 0.2 mg/L KT;The optimized condition was pre-culture 2 d,10 min bacterial infection and 2 dco-culture.

关 键 词：莴苣 过表达载体 农杆菌 遗传转化 

分 类 号：S636.2[农业科学—蔬菜学]