Characterization Of A BAC Library And Screening Of Flowering Time Related Gene FLC1 In Brassica Campestris L.ssp. Pekinensis Olsson

Bacterial artificial chromosome (BAC) libraries are the most convenient, reliable and widely used genomic libraries in genome research of organisms with highly complex genomes. It can also serve for chromosomal location of target genes, constructing of physical map, comparative genomic and map-based cloning and function, sequencing of large DNA fragment, verification of supposed studying of transgene and so on. Construction of BAC library and selection of special clones in Chinese cabbage lay a foundation for utilization of BAC-FISH to distinguish alien chromosomes in alien addition line, locating gene and selection of founctional gene and study relation between comparetive study on brassion genomes between A and others.In this study, a constructed Chinese cabbage inbred line'85-1'BAC library was used. 200 white clones picked randomly were digested by NotI restriction enzyme, the average insert fragment size, coverage rate and the empty clone of BAC library were analysed through pulsed-field gel electrophoresis. 6 postive clones picaked randomly were digested by Hindâ…¢and assayed by serial culture of more than 100 generations for 5 days. The primary and secondary pools of a BAC library in Chinese cabbage were constructed using improved BAC pooling strategy. The homologeous sequence of flowering time gene FLC1 were screened from those BAC clones by PCR amplification. The main results are as follows:1.The library consists of 57 000 clones. The library covers 10.4-fold genome size assuming that the genome size is 500Mb. Then the possibility to find a single-copy gene in this library is 99.996%.2.A number of 200 BAC clones were picked at random, it indicated an average insert size of 98.4kb .The ratio of negative clone is 1.5% and fragment size between 90 to 110kb covers more than 85% in this library. Generally, the size of DNA fragments in this library is unbiased and could meet the demand of further study.3. The BAC clone assayed by serial culture of more than 100 generations for 5 days. The electrophoretic patterns revealed that no apparent rearrangements were observed and the Chinese cabbage inserts in the BAC clones were stable in the vector for long culture. The result indicats that this Chinese Cabbage BAC library is sufficient for target gene isolation.4. In this paper, the improvement methods of screening PCR that we only constructed row pools to compare with traditional method. After improving it enable the reduction of storage space and PCR reations, Furthermore, rate of contamination be lower in 384-well microriter plate.5 .In total two positive FLC1 clones were obtained through three steps of PCR screening with 114 reactions, 50 primary plate, 16 secondary plate and 48 single clones. Sequence analysis of FLC1 clones showed that these fragments has 99% of homology with the FLC1 in Chinese cabbage, indicating that the flowering time gene FLC1 is contained in the BAC clones. The obtaining of positive clones with flowering time gene FLC1 will lay a foundation for further research on functional analysis of the flowering gene in Chinese cabbage.