Construction And Screening Of A Transformation-Competent Artificial Chromosome (TAC) Library Of Zizania Latifolia (Griseb.)

Zizania lattfolia (Griseb.) is included in the Oryzeae tribe and possesses numerpus traits valuable for rice breeding, such as disease and insect resistance, cold and flood tolerance, and high grain quality. The genetic and breeding of Zizania lattfolia are still in their infancy.Construction of TAC has been significantly improved and advanced technically since the YAC and PAC systems were established. Large genomic DNA fragments cloned in TAC vectors can be maintained stably in both Esherichia coli and Agrobacterium tumefadens and transferred efficiently into host genomes, faithfully transmitted to transgenic Progeny. Once a genome library of Plant in the TAC vector was established, the target 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 Agrobacterium transformation system and conducted functional experiment in revertant mutants, so as to accelerate the procession of molecular breeding.This research has referred to the predecessor in the library method, established an own set to be possible to transform the big fragment library to construct the technical system. Including the carrier preparation, high molecular weight DNA withdraw, the determination of the enzyme cuts, the fragment size choice, the connection and the transformed system, finally obtained has needed the size the insertion fragment , the reorganization rate high TAC library constructs the system. Using the TAC17 vector as the carrier, the Zizania genome TAC library was constructed. It contained 91,584 TAC clones with average insert size of approximately 45 kb, covered five haploid Zizania genome equivalents. very low signals after hybridization with chloroplast and mitochondrial genes indicated that the TAC library was predominatly composed of nuclear DNA. The TAC clones were stable in E.coli for 100 generations. Clones containing the resistance resistance gene analogs (RGA) FZ14(GenBank:DQ239432)gene were screeded by pooled PCR. Two posive clones was retained,they be used for Zizania gene cloning and functional analysis. The library will be useful in studies of genome structure, gene cloning and evolution of rice.