微绿球藻DNA质粒文库的构建  

DNA library of Nannochloropsis oculata constructed with plasmid vector pUC18

作　　者：赵大显[1] 周志刚[1] 

机构地区：[1]上海水产大学农业部水产种质资源与养殖生态重点开放实验室,生命科学与技术学院,上海200090

出　　处：《海洋科学》2006年第5期87-91,共5页Marine Sciences

基　　金：国家转基因植物研究与产业化开发专项(JY03-B-20);上海市属高校自然科学研究资助项目(01H04)

摘　　要：微绿球藻（Nannochloropsis oculata）DNA被提取纯化并经超声波处理后，将所得大小在1．6～3kb之间的DNA片段用T4DNA聚合酶补平，再与Sma I酶切消化并经去磷酸化处理的质粒栽体pUCl8连接。转化至DH10B大肠杆菌（Escherichiacoli）的感受态细胞中。建立的DNA质粒文库容量含2×10^4个克隆，其中重组子占90％。随机挑选白色菌落并培养，抽提的质粒经XbaI和SacI双酶切鉴定，显示重组的质粒中均含有大小不等的DNA插入片段。The genomie DNA isolated and purified from Nannochloropsis oculata was sheared after a treatment with an ultrasonic processor. The treated DNA was blunt ended with T4 DNA polymerase and then was collected from 1.6 kb to 3 kb in size. The target blunt ended DNA was cloned into plasmid vector pUC18 which was previously digested with Sma I and dephosphorylated with calf alkaline phosphatase. The recombinant plasmid was transformed into Escherichia coli DH10B competent cells and the genomie library was constructed. There were about 2 × 10^4 clones in the genomie library of N. oculata, and the percentage of recombinants was about 90%. It was confirmed that all randomly selected plasmids of white colonies contained target DNA inserts from 1.6 kb to 3 kb in size after restriction mapping with Xba I and Sac I endonucleases.

关 键 词：微绿球藻(Nannochloropsis oculata) 超声处理 质粒载体pUC18 DNA文库 

分 类 号：Q78[生物学—分子生物学]