Construction And Characterization Of A BAC Library From Brassica Rapa Subsp. Pekinensis And Screening And Analysis Of BAC Clones Related CO Gene

As an important vegetable belonged to the genus Brassica of the family Brassicaceae, Brassica rapa subsp. pekinensis acts as the most widely planted leaf vegetable in China. With the development of plant molecular biology, the structure and function of genome and the mechanism of gene expression regulation have become hot topics which attract a lot of vegetable researchers. The construction of genomic libraries containing large segments is an essential tool for genomic studies. The BAC library of Chinese cabbage has been constructed internationally, however, the research about the genome structure and function of the genus Brassica is still in initial stage. Therefore, it is important to construct a BAC library of Chinese cabbage cultivar. The edible part of Chinese cabbage is its nutritorium. Early bolting is a major problem of cultivation in spring which seriously decreases the amount and quality of Chinese cabaage. So it is a big task to cultivate the late bolting variety to meet the need of vegetable market. The research on the bloom-related genes and mechanism of gene expression regulation will provide a theoretical basis for cultivating new Chinese cabbage cultivars.In the study, using Chinese cabbage inbred line'85-1'as the material, pIndigoBAC5 as the vector, Hindâ…¢as the restriction enzyme, a Chinese cabbage BAC library was successfully constructed through the production of high molecular weight DNA, connection of DNA and vector, and transformation process. By constructing first- and second-level pools, we screened the BAC clone with CO gene by the method of PCR and analysed SNP loci in the sequence of amplified product. The study lay solid foundations on analysing the sequence character of CO gene and difference with other Brassica plants.The results are as follows:1) The BAC library contains 57600 clones in total and the clones were stored in 150 384-well plates in the form of monoclone; According to the haploid genome size of 550 Mb, the library represented approximately an equivalent of 10.3 fold size of Chinese cabbage haploid genome. Therefore the possibility to find a single-copy gene in this library is 99.99%. Two hundred clones were selected randomly and digested by Notâ… restriction enzyme. The average insert size is 98.4 Kb and the majority clones are during 90~120 Kb. 85% of BAC clones is longer than 90 Kb, which showed a good uniform of segment size. The clone numbers without insert segment are three among 200 clones, so the rate of empty clone is 1.5%; We selected six BAC clones randomly which were cultured for five days successively. The plasmid was completely digested by restriction enzyme Hindâ…¢. The result shows that DNA fingerprints of every clone have not changes after about 125 generations. Therefore the genetic stability of insert segment is perfect. Connecting the 100~200 Kb DNA with vector and transforming, we got about 5000 clones, while only about 800 clones were abtained though connecting the 200~300 Kb DNA. So we concluded that the efficiency of connection and transformation is closely related to the length of DNA segment. The longer the segment is, the lower the connection efficiency is.2) We designed six pairs of primers according to homologous sequence of the CO gene from Arabidopsis thaliana, and screened the clones from 18048 clones in 47 384-well plates by PCR amplification. In total three positive CO clones were obtained through three steps of PCR screening with 167 reactions. We named them BrCO97A1, BrCO140N21, BrCO147H21 respectively.3) The BAC clones contain CO gene which could code constans-like protein. Comparing the sequence of amplification product in the website NCBI, we found that the sequence similarity among the PCR product and Brassica rapa(AA), Brassica napus(AACC) and Brassica juncea(AABB) was high, next to Brassica oleracea(CC), and the third Brassica nigra(BB). So the similarity with genome C is higher than genome B.4) By comparing with 18 sequences containing CO gene found in the website http://brassicadb.org, the rusults show that the sequence similarity is 96% between the PCR pruducts and Bra008668. In the readable sequences, we also found 12 SNP locus, in which there are six C/T variants, two G/A variants, two A/T variants, one A/C variants, one T/G variants. There is one SNP locus per 51 bp. It is not a very representative number, but reflected the richness of SNP locus in genome.