[The construction and screening of genomic library based on transformable artificial chromosome (TAC) vector in Lotus...

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 III-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 muL 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. These data showed that this genomic library was reliable for further molecular research in Lotus japonicus.