The Construction And Screening Of Genomic Library Based On Transformable Artificial Chromosome (TAC) Vector In Orazy.Minuta

Construction of the Transformationable artificial chromosome libraries 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 Escherichia 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 is 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.Oryza.minuta (4n = 48,BBCC)derives from Asia.It is a wild rice with kinds of resistances and a fine material for the improvement of O.sativa. By now taking O.minuta as material to the O.sativa improvement, researchers has get the rice plants which have resistences to the Bland,BB diseases。This research has referred to the predecessor in TAC, BIBAC and in the BAC method, established an own set to be possible to transform the big fragment library to construct the technical system. Including the carrier preparation, macromolecular weight DNA withdraw, the enzyme cuts the condition the determination, the fragment size choice, the goal big fragment recycling, the connection and the transformed system, finally obtained has needed the size the insertion fragment, the reorganization rate high TAC library constructs the system.We design probe according to the sequences of cloned resistance gene.Using the TAC library which establishes constructs the system, take pYLTAC27 as the carrier, constructs the O.minuta TAC library, the choice wild rice young tender leaf blade, withdraws macromolecular weight DNA, chooses the appropriate enzyme quantity, selects about 50-100 kb the scope fragment. So far we have obtained 7 000 recombinant clones. We select ranomdly 36 clones and extracts recombinant plasmid through alkaline lysis. The result of PFGE indicates the clone reorganization rate is 97.3%, averages insertions fragment is 45 kb.The test of stable to the library, finally the results indicates the O.minuta genome can stabilize in pYLTAC27.With the colony hybridization method, the genome library was screened and two candidate clones were obtained. The result of PCR test shows that these clones have the same conserved positions as the cloned resistence gene.