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
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(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 |