Guo Xi-Zhi, Liu Yao-Guang, Luo Da
National Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China.
Zhi Wu Sheng Li Yu Fen Zi Sheng Wu Xue Xue Bao. 2004 Apr;30(2):234-8.
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.
为了进行基因组规模的图谱绘制和测序，人们开发了许多载体，尤其是人工染色体载体。作为第一个人工染色体，酵母人工染色体（YAC）已被广泛应用于基因组文库的构建和图谱绘制。Shizuya等人（1992年）设计了源自大肠杆菌F因子的细菌人工染色体（BAC），它易于操作且具有较大的容纳能力。Liu等人（1999年）设计了源自P1人工染色体（PAC）载体的可转化人工染色体（TAC）载体。TAC能够在细菌和农杆菌之间穿梭大规模DNA片段。此外，它能够将一个大型靶向DNA片段整合到植物基因组中，如在拟南芥和水稻基因组研究中所记载的那样（Liu等人，2000年、2002年）。TAC省时的优点在豆科模式植物百脉根的基因组学研究中应该是很显著的。采用Liu和Whittier基于细胞核的方法，从百脉根（Gifu生态型）中分离出高分子量DNA。如所述（Liu和Whittier，1994年），用Hind III对DNA进行部分消化，并在10至20 kb大小范围内进行大小分级分离。将部分消化并经大小选择的DNA片段与经Hind III消化的pYLTAC7连接，然后通过电穿孔用于转化大肠杆菌DH10B。在含有25 mg/L卡那霉素和5%蔗糖的LB琼脂平板上筛选携带插入片段的转化子。该文库包含6个单倍体基因组当量，被汇集到12个96孔微量滴定板中，每孔约150个转化子。然后将TAC文库排列在尼龙膜上并进行筛选。使用控制金鱼草花序结构的CEN基因的同源片段作为探针进行筛选。对来自阳性池的0.5 μL溶液进行滴定，并以相同方式进行二次筛选。最后，通过Southern杂交对这些阳性克隆进行确认。这些数据表明，该基因组文库对于百脉根进一步的分子研究是可靠的。
