Constructing Plant Artificial Chromosome Using Genetic Resources Of Arabidopsis

The construction and introduction of plant artificial chromosomes into plant cells would be an effective way to understand the essential elements necessary for maintaining chromosome function and structure in plants. The successfully constructed artificial chromosome would also can be used as a new vector to introduce chromosome segments of hundreds Kb to Mb length into plant cells. Because of its fully sequenced genome and the most complete, contiguous nucleotide sequence of centromeric DNA in plants, Arabidopsis was chose as the base to construct plant artificial chromosome. We identified two candidate YAC clones, CIC8H8 and CIC6F6, which are both proved to have the 178 bp Arabidopsis centromeric repeat sequences. Other chromosome elements, telomere and ARS, have also been cloned for constructing artificial chromosome. Arabidopsis telomere was cloned from PCR products using telomere repeat primers without other template. A 2000bp fragment of ARS was released from Arabidopsis Genomic BAC clone T14A4 by ClaIdigestion and subcloned into ClaI digested Pbluescript.In order to construct plant artificial chromosome, plasmid vectors have been constructed to integrate necessary elements into both right and left YAC arms. The right arm vector is constructed with Arabidopsis telomere sequence, kanMX4 gene of G418 selection marker and truncated TRPI gene. The left arm vector carries Arabidopsis telomere sequence, ADE2 gene of selection marker, ARS sequence, GFP reporter gene, and truncated URA3 gene. Retrofitting YAC clones carrying Arabidopsis centromere segment by homologous recombination with the constructed right and left arm vectors was performed and confirmed in yeast, thus the rudiment of Plant Artificial Chromosome has been formed in yeast cells. Isolation and purification of modified YACs from yeast strain have been carried out by pulsed-field gel electrophoresis. The modified YACs are expected to generate plant artificial chromosome after being transferred into Arabidopsis protoplast by liposome-mediated method.