Construction And Application Of BAC (Bacterial Artificial Chromosome) Library Of Guanximiyou

Guanximiyou [Citrus grandis (L.) Osbeck cv.Guanximiyou], originated from Pinghe county Fujian Province, is one of the most famous pummelos with high economic value. It has been cultivated for 4 hundred years. Juice sac granulation effected edible quality severely and income of farmers. Genomics research mainly focuses on plants with significantly economical value, and the study results offer the theoretical basis to solve the issues on production. Few researches on Guanximiyou genomics have been reported.A BAC library was constructed for the first time in this study. The library was screened by probe EST M4. Some BAC end sequences were sequenced, and bioinformatics were used to analyze the BESs. The study revealed first insights into the organization of Guanximiyou genome.The main results were indicated as follows:1. High molecular weight nuclear DNA(HMW-DNA) from leaves of Guanximiyou was prepared by two methods (improved Zhang's and Peterson's method) and the extracting effects of the two preparing methods were compared, the results showed that improved Zhang's was more suitable for preparation of HMW-DNA in Guanximiyou. In the improved Zhang's method, the cell walls were broken down by gentle physical homogenization to keep integrity of nuclei under suitable osmolarity. Debris of cell walls and most of polysaccharides were removed by filtering with miracloth and centrifugation under slow speed, the isolated nuclei were proved to be intact, pure, and high-quality by observation under microscope and were embedded with low-melting-point agarose to preserve the isolated nuclei in plugs for keeping stability of nuclei. The optimum digestion time of plugs was 48 hours, the pulse field gel electrophoresis (PFGE) results showed that the isolated HMW-DNAs were more than 1 Mb in size and were digested completely or partially by restriction enzymes, therefore, Zhang's method was suitable for construction of BAC library of Guanximiyou.2. Method for constructing Guanximiyou BAC library was improved. The result showed that DNA fragments obtained by one step electrophoresis protocol were suitable for library construction. The library consists of 26112 clones. A random sample of 100 clones indicated an average insert size of 120 kb, corresponding to 8 genome equivalents. Less than 1% of the clones do not contain inserts. Screening of BAC library with chloroplast DNA probes indicated that less than 1% of the clones contained organelle-derived DNA.3. The BAC library was screened with a pair of primers deprived from EST related to granulation by PCR and Southern blot. A positive clone was obtained, proving that the library had deep coverage of Guanximiyou genome. A 1088 bp DNA fragment was amplified by PCR with the primer from EST M4. The sequence contained two introns with the length 122 bp and 172 bp respectively. An about 4000 bp and an about 400 bp DNA fragments were amplified by I-PCR with a pair of I-PCR primers from EST M4 using self-ligations as DNA template. The 4000 bp DNA fragment was cloned and sequenced, and the exactly length was 3984 bp. The potential coding regions were predicted by GENSCAN and FGENESH. The results showed that the coding regions predicted by these two softwares were almost consistent. The coding sequence has 85% and 71% identities with multicopper oxidase cDNA sequences of Ricinus communis and Populus trichocarpa respectively, furthermore, it has 76% and 73% identities with pectinesterase cDNA sequences of Annona cherimola and Arabidopsis thaliana respectively. According to the characterization of BAC clones and the results of screening, the library is suitable for functional genome research on Guanximiyou .4. One hundred and twenty BAC clones were picked up from the library, and a total length of 105664bp end sequence was obtained. These sequences were analyzed by bioinformatics. The results showed that approximately 0.2% of Guanximiyou genome contained simple sequence repeats (SSRs), 17.8% of BESs contained transposable elements and most of these elements were retrotransposons. The average GC content of the Guanximiyou genome was 39.17%, and the content of the predicted coding regions of BESs was 46.7%. It was estimated that Guanximiyou genome contained 64.02 Mb coding regions, and gene number was 320001.