以可转化人工染色体(TAC)载体为基础的百脉根基因组文库的构建及筛选  

The Construction and Screening of Genomic Library Based on Trans- formable Artificial Chromosome (TAC) Vector in Lotus japonicus

作　　者：郭熙志[1] 刘耀光[2] 罗达[1] 

机构地区：[1]中国科学院上海生命科学研究院植物生理生态研究所植物分子遗传国家重点实验室,上海200032 [2]华南农业大学生物技术学院,广州510642

出　　处：《植物生理与分子生物学学报》2004年第2期234-238,共5页Journal Of Plant Physiology and Molecular Biology

基　　金：国家自然科学基金项目(No.39825102)资助~~

摘　　要：可转化人工染色体(transformation-competentartificial chromosome,TAC)载体是具有克隆和转移大片段DNA特征的新型载体,是植物基因克隆和转化的有效工具。该研究把它用于豆科植物百脉根(Lotus japonicus)基因组文库的构建。此文库由1.8×105个克隆组成,平均插入片段大小为15 kb左右,约覆盖百脉根基因组6倍。文库保存在12块96孔板中,每个孔中约含150个不同的重组克隆。用与花发育相关的同源基因Ljcenl片段为探针,筛选得到6个阳性克隆,酶切后验证这些阳性克隆,结果表明这些克隆含有同一个基因片段。此基因组文库可直接用于植物转化,为百脉根功能基因组的研究打下基础。Many vectors, especially artificial chromosome vectors, have been developed for genome-scale mapping and sequencing. As the first artificial chromosome, YAC has been extensively used in the genomic library construction and mapping. Shizuya et al. (1992) devised the bacterial artificial chromosome (BAC) originated from F factor of E.coli, which is much easy to handle and isolated with large harboring capacity. Liu et al. (1999) designed the transformation-competent artificial chromosome (TAC) vector, derived from PAC vector. TAC could shuttle a large-scale DNA fragment between bacteria and Agrobaceria. Further, it could integrate a large targetted DNA fragment into plant genome, as being documented in Arabidopsis and rice genome research (Liu et al. 2000,2002). The time-saving virtue of TAC should be significant in genomics research in Lotus japonicus, a model plant of legume. Using a nuclei-based method of Liu and Whittier, high molecular weight DNA was isolated from Lotus japonicus (Gifu ecotype). The DNA was digested partially with Hind Ⅲ and size-fractionated in the 10- to 20-kb size range as described (Liu and Whittier 1994). The partially digested and size selected DNA fragments were ligated with Hind Ill-digested pYLTAC7 and then used for transformation of E. coli DH10B by electroporation. Transformants carrying inserts were selected on LB agar plates containing 25 mg/L kanamycin and 5% sucrose. The library, 6 haploid genome equivalents, was pooled in 12 96-well microtiter plates at about 150 transformants per well. The TAC library was then arrayed in nylon membranes and subjected to screening. The probe, a homolog fragment of CEN gene controlling the structure of inflorescence Antirrhinum, was used for screening. 0.5 μL solution from the positive pool was titered and the secondary screening was conducted in the same way. Finally, these positive clonies were confirmed by Southern blot (Figs.l, 2). These data showed that this genomic library was reliable for further molecular research in Lotus japonicus.

关 键 词：基因组文库 TAC载体 百脉根 同源基因 

分 类 号：Q785[生物学—分子生物学]