Construction Of Genomic BAC Library Of Chinese A Higator And Contigs Of MHC Class Ⅱ And OR Gene

Chinese alligator (Alligator sinensis) is a rare and endangered species endemic to China. For a long time, due to the environment deterioration, habitat loss and the influence of human activitiy, wild Chinese alligator population has dropped sharply to less than120. At present it appears restricted to southeastern Anhui Province of China, possibly very low numbers in Zhejiang and Jiangsu Provinces. To protect this species,in1972the Chinese Government listed Chinese alligator as a Class I endangered species. Anhui Research Center for Chinese Alligator Reproduction, Anhui National Nature Reserve for Chinese Alligator and Changxing Breeding and Research Center for Chinese Alligator were established in1978,1982,1979, separately.In this study, for preserve the genetic resources, we constructed a high-quality bacterial artificial chromosome (BAC) library of the Chinese Alligator with fresh blood, which was provided by Changxing Breeding and Research Center of Chinese Alligator.Then further researchs show that this genomic BAC library is high quality and effective by constructing two contigs of MHC class II and OR genes of Chinese alligator. The main results are as follows:1. The Chinese Alligator bacterial artificial chromosome (BAC) library consisting of216,238clones has been constructed using the whole blood and pCC1BAC vector. The average insert size was calculated to be90kb based on the examination of150randomly selected clones, indicating that the Chinese Alligator library contained6.8-fold genome equivalents. About5.53%non-insert clones were observed in the150clones tested (10out of174). The probability of any Chinese alligator gene being found in this library should be about99.93%.2. Construction of high-effective rapid screening libraries by4D-PCR. The216,238BAC clones were divided into46superpools and1,564(46x34) sub-superpools of1D,2D,3D,4D. We can obtain the desired positive BAC clones by any primers or probes through80PCR reactions. 3. Using7Chinese Alligator speciese specific SSR PCR primers for screening the BAC library,6.1positive clones were got an average. The PCR screening results were thus in good agreement with an expected6.8-fold genomic coverage of the library.4. We have designed two MHC class II PCR primers and one OR gene PCR primers by the homologous sequences.5. Two contigs of Chinese Alligator MHC class II and OR genes were constructed by screening the genomic BAC library using the three primers in point4, BAC end sequencing and chromosome walking.6. Though sequencing the nine BAC clones in the OR gene contig, we confirmed the high-quality of the BAC library. Then we conducted a preliminary analysis of OR genes in Chinese alligator genome.