微绿球藻DNA质粒文库的构建

DNA library of Nannochloropsis oculata constructed with plasmid vector pUC18

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.