Construction And Characterization Of Genomic BAC Library And Physical Map Of MHC Class Ⅱ Of Giant Panda

The giant panda (Ailuropoda melanoleuca) panda is rare and endangered species endemic to China. Unfortunately, due to habitat fragmentation, the population size of giant panda decline rapidly. Now, the wild giant pandas distribute six completely isolated mountain ranges such as Qinling Mountain, Minshan Mountain, Qionglai Mountain, Xiangling Mountain and Liangshan Mountain. Moreover, the giant panda population shows significant genetic differentiation and yield two evolutionarily significant units (ESU), a new Qinling subspecies from the nominate Sichuan subspecies.In order to protect this rare and endangered species, the Chinese government implements the gene resources conservation project followed by the in-situ and ex-situ conservation projects of giant panda in 2003. One goal of the project is conserves the genetic resources by constructing the genomic DNA library and provide the gene resources platform for related scientific research.As the main part of giant panda gene resources conservation project, the present study is to construction and characterization of high coverage genomic DNA library. Then further researchs show that this genomic BAC library is high quality and effective by constructing physical map of MHC class II of giant panda. The main results are as follows:1. The giant panda bacterial artificial chromosome (BAC) library consisting of 205,800 clones has been constructed using the whole blood and pCC1BAC vector. The average insert size was calculated to be 97 kb based on the examination of 164 randomly selected clones, indicating that the giant panda library contained 6.8-fold genome equivalents. About 5.74% non-insert clones were observed in the 174 clones tested (10 out of 174). The probability of any giant panda gene being found in this library should be about 99.93%.2. Two or more positive clones (2 ~ 16) were found for these 16 loci, with an average of 6.4 positive clones per locus. The PCR screening results were thus in good agreement with an expected 6.8-fold genomic coverage of the library.3. Construction of high-effective rapid screening libraries by 4D-PCR. The 205,800 BAC clones were divided into 43 superpools and 1462 (43x34) sub-superpools of 1D, 2D, 3D, 4D. We can obtain the desired positive BAC clones by any primers or probes through 77 PCR reactions.4. The giant panda metaphase chromosomes were prepared from fibroblast cells of giant panda. The results indicate that the MHC is located on the chromomse 9q by fluoresecence in situ hybridization.5. We have designed 10 giant panda MHC class II gene PCR primers by the homologs counterpart of the MHC class II in the GenBank database. All the primers were amplified successfully and the PCR products of BTL2, DRA, DQA, DRB1, DOB, LMP2, DMA, DMB, COL11A2 and DAXX were submitted to GenBank.6. The physical map of MHC class II of giant panda was constructed by screening the genomic BAC library using 10 MHC class II gene primers. The end sequencing primers were designed according to positive BAC clones and screen further the giant panda genomic BAC library to cover the gaps of physical map. According to the restriction fingerprint analysis and test, the whole physical map of MHC class II region was spanning about 650 kb.