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第二部分：载体的构建 Construction of vector

第二部分：载体的构建 Construction of vector. Kpn I. Sma I. BamH I. EcoR I. Sac I. Xba I. Sal I. Pst I. Hind III. 35 S. Nos. Sac I. Kpn I. BamH I. Xba I. Pst I. Sal I. Sma I. 一、目前实验室最常用载体的选择及注意事项 1． 超表达的载体主要有2个： 35 S-1300 与 Ubi -1300

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第二部分：载体的构建 Construction of vector

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  1. 第二部分：载体的构建 Construction of vector

  2. Kpn I Sma I BamH I EcoR I SacI Xba I Sal I Pst I Hind III 35S Nos SacI Kpn I BamH I Xba I Pst I Sal I Sma I 一、目前实验室最常用载体的选择及注意事项 1．超表达的载体主要有2个：35S-1300与Ubi -1300 35S-1300：将35S插入在Cambia1300的EcoRI与SacI之间 将Nos终止子插入到PstI与Hind III之间。多克隆位点单酶切位点非常丰富有：SacI, Kpn I, Sma I , BamH I, Xba I, Sal I, Pst I共7个。具体信息如下： gagctcggtacccggggatcctctagagtcgacctgcag

  3. Ubi –1300：超表达载体 Cambia Ubiquitin 1300 载体介绍 多克隆位点单酶切位点序列： KpnI SmaI BamHI PstI atgctcaccctgttgtttggtgttactcggtacccggggatcctctagagtcgacctgcagagctttcgttcgtatcatc

  4. 2.注意事项 • 千万注意：在双酶切时当用到BamHI酶切位点时，先用37℃切其它位点，再用30℃切BamHI。否则构出的质粒会缺失包括右边界在内的大片段。已经有过惨痛教训。

  5. 目的基因的克隆与鉴定 生理检测 转化载体的构建 纯化 E.Coli转化，质粒 抽提与鉴定 移栽 分子检测 继代繁殖 农杆菌的转化与活化 选择培养 外植体制备 浸 染 共培养 转基因技术的一般实验流程

  6. 实验流程参考图 PCR扩增LOB29 与pUCm-T连接 转化感受态菌DH5a 筛选鉴定 提质粒pUCm-T-LOB29 提质粒pCAMBIA1300 KpnII +BamHI/XbaＩ酶切 HindIIII+BamHI/XbaＩ酶切 回收NAC2片断 连接 回收线性pCAMBIA1300 目的载体pCAMBIA1300-LOB29 转化感受态菌DH5a 筛选鉴定 转化感受态农杆菌 转基因

  7. 四、植物增强表达载体的构建。 ---------------OsLoB29的35S增强型载体 将OsLoB29基因ORF构建在pCAMBIA1301中的35S启动子后，示意图如下： OsLOB29 35S Prom NOS

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