作　　者： ; ; ; (徐碧玉）; (金志强）;

机构地区： 海南大学海洋学院

出　　处： 《科学技术与工程》 2002年第6期40-43,共4页

摘　　要： 用改良CTAB法提取巴西橡胶树幼嫩叶片总DNA,通过限制性内切酶Sau3A I不完全酶切,回收并纯化后得到15～23kb的DNA片段.以来源于λ噬菌体的EMBL3为载体DNA,用T4DNA连接酶将载体与DNA片段连接成为重组DNA.经体外包装形成完整的噬菌体颗粒,转染受体菌E.coli LE392,在LB平板培养基上获得重组子的效率为1.1×106个/μg DNA,达到构建文库的要求.随机挑取一定数量的克隆子,酶切分析结果发现有88%的克隆子携带外源DNA. A genomic library of the Hevea brasiliensis was constructed by using the broad-host-range vector λ EMBL3.The λ EMBL3 containing the multiple restriction enzyme coloning sites was digested with restriction enzyme BamH Ⅰ,and the linear DNA of λ EMBL3 was treated with bovine intestine alkaline phosphorylase.Total DNA of Hevea brasiliensis was extracted by CTAB method,and was partly digested with restriction enzyme Sau3Al and the 15 to 23 kb fragments were isolated and collected from Low-point-melting gel( 0.4% agrose) by electrophoresis.The fragments of DNA were ligated with vectors under the role of T_4 DNA ligase.The recombinant DNA moleculars were packed by the λ phage shell protein in vitro to form active phages.After infected by the packed phage with different dilution,the LB agar plate containing E.coli fonned phage plaques. About 1.1×10~6 plaques were obtained from 1μg DNA,and eighty- eight percent of the clones contained with insert DNA.

关 键 词： 巴西橡胶树 基因组 片段

领　　域： [农业科学] [农业科学]