| Construction of transformation-competent artificial chromosome libraries has been significantly improved and advanced technically since the PAC and YAC systems were established. Large genomic DNA fragments cloned in TAC vectors can be maintained stably in both Escherichia coli and Agrobacterium tumefadens, and transferred efficiently into the host genomes, faithfully transmitted to transgenic progeny. Once a genomic library of plant in the TAC vector is established, the target genes can be cloned by screening the library, and the library should be applicable in positional cloning. These TAC clones are available for direct introduction into plants with the Agrobacterium transformation system and conducted functional experiment in revertant mutants, so as to accelerate target genes practical processes.Two large-insert genomic DNA libraries of Leymus multicaulis (2n=4X=28, XmXmNsNs) that is one gramineous plant native to Xingjiang in China and possessed of many excellent genes conferring tolerance to stresses, were constructed in the transformation-competent artificial chromosome (TAC) vector, pYLTAC17 and pYLTAC747H, which accept and maintain large genomic DNA fragments stably in both Escherichia coli and Agrobacterium tumefaciens. Library in pYLTAC17 and library in pYLTAC747H consisted of 1.65×105 and 2.36 × 105 clones respectively. The genome coverage of the two libraries was totally estimated to be about three to five haploid genome equivalents, as size selection of genomic DNA fragments was approximately from 9 to 300 kbp. Clones of the Genomic libraries were collected as bulked pools each containing 500 clones or so, stored in twelve 96-deep-well plates and then were gridding in triplicate onto a high-density colony hybridization filter with a 3×3 pattern by GeneTACTMG3 after transferred manually into three 384-well plates. Meanwhile 2501 and 2890 clones of Library in pYLTAC17 and in pYLTAC747H were stored individually in forteen 384-well plates and then were gridding in duplicate onto a high-density colony hybridization filter with a 6×6 pattern after a replication of plates by GeneTACTMG3. Nineteen positive clones were detected by using the probe glutahione reductase gene of Leymus multicaulis. TAC libraries constructed here can be used to isolate genomic clones containing target genes, and to carry out genome walking for positional cloning. Once the target TAC clones were isolated, they could be immediately transferred into plant genomes with the Agrobacterium system.
|
PDF Full Text Request