| 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5 d的子叶，6-BA 0.2 mg/L+NAA 0.1 mg/L，KT质量浓度为0.2 mg/L，OD600为0.3，预培养2 d，侵染10 min，共培养2 d。 |
| 关键词: 莴苣 过表达载体 农杆菌 遗传转化 |
| DOI：10.11841/j.issn.1007-4333.2018.01.08 |
| 投稿时间：2017-03-06 |
| 基金项目: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 |
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| Over-expression vecter construction and Agrobacterium transformation optimization of leaf lettuce gene LsHsp70-3701 |
|
SU Zhenqi, HAN Yingyan, LI Yabo, LIU Ran, LIU Chaojie, HAO Jinghong, FAN Shuangxi
|
| (College of Plant Science and Technology, Beijing University of Agriculture, Beijing 102206, China) |
| Abstract: |
| To study the function of a leaf lettuce Hsp70 gene, and improve plant thermo-tolerance, LsHsp70-3701 over-expression 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 Nco Ⅰ and Spe Ⅰ. 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 0.2mg/L KT;The optimized condition was pre-culture 2 d, 10 min bacterial infection and 2 d co-culture. |
| Key words: lettuce over-expression vector Agrobacterium genetic transformation |
