杜氏盐藻核基质附着区的分离及特征性分析

Isolation and characterization of matrix attachment region from Dunaliella salina

采用0.5%TritonX 100破碎细胞,15%Percoll分离盐藻细胞核,25mM二碘水杨酸锂(lithiumdi iodosalicylate,LIS)抽提核蛋白,限制酶消化除去结合松弛的DNA,蛋白酶K SDS处理,酚/氯仿抽提,乙醇 沉淀提取核基质附着DNA,限制酶酶切连至pUC18载体上构建MARs文库。随机挑选6个克隆进行体外结 合实验筛选,筛选出一能与核基质结合的克隆,测序分析结果表明该序列具明显的MAR序列特征。

The Dunaliella salina cells were disrupted with 0.5% Triton X-100,purified by 15% Percoll. Nuclear proteins were removed using 25 mM lithium diiodosalicylate. After the nuclear matrices digested with restriction enzyme,the matrix attached DNAs were treated with proteinase K and SDS,extracted by phenol/chloroform and ethanol-precipitated, subsequently cut with restriction enzyme and ligated to the pUC18,transformed into E.coli JM109 and six colonies were picked randomly and screened through in vitro binding assay. One DNA fragments could specifically bind to the nuclear matrix and the sequence had the features of MAR typically.