Construction Of Binary Bacterial Artificial Chromosome Library Of Dongxiang Wild Rice

Construction of binary bacterial artificial chromosome has been significantly improved and advanced technically since the YAC and PAC systems were established. Large genomic DNA fragments cloned in BIBAC vectors can be maintained stably in both Escherichia coli and Agrobacteriua tumefadens, and transferred efficiently into host genomes, faithfully transmitted to transgenic progeny. Once a genome library of plant in the BIBAC vector is established, the target can be cloned by screening the library, and the library should be applicable in positional cloning. These BIBAC clones are available for direct introduction into plants with Agrobacteriua transformation system and conducted functional experiment in revertant mutants, so as to accelerate the procession of molecular breeding.The Dongxiang wild rice (2n = 24) is the O. rufipugona with the northernest latitude in the world (located on east longitude 116° 36', north latitude 28° 14 ' ) , it still could safely survive in the winter under -12. 8~0℃ low temperatures conditions with perennial root (perennial), ratooning ability and the strong reproduction (asexual). It posese many favourable traits such as drought resistance, bore barren and resistances and many favourable genes such as male sterile gene, wide-compatible gene, restores gene, the disease and insect resistance gene.This research has referred to the predecessor in BIBAC, TAC 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 BIBAC library constructs the system.Using the BIBAC library which establishes constructs the system, take BIBAC2 as the carrier, constructs the Dongxiang wild rice BIBAC2 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 14 592 recombinant cloneso We selects ranomdly 26 clones and extracts recombinant plasmid through alkaline lysis. The result of PFGE indicates the clone reorganization rate is 92.2 % ,averages insertions fragment is 65 kb, covers Dongxiang wild rice genome 2 times. The test of stable to the library, finally the results indicates the Dongxiang wild rice genome can stabilize in BIBAC2. Four kind of paddy rice has inoculanted in the NB culture medium to induct callus , finally demonstrated Japonica rice TP309 and oryza sativa M86 suits the acceptor material which transforms as the heredity. With includes Dongxiang wild rice DX13F15 the recombinant plasmidAgrobacterium LB4404 to mediate callus of TP309, examination of transferred callus have the GUS instant expression response. Recombinant plasmid comes from Dongxiang wild library that can transform the paddy rice through the Agrobacteriurn LB4404 to callus.