Transcriptomic Analysis Of Immune Relevant Genes And Crystallization Of MHC Class I Of The Goose

The avian have many immunological mechanisms in common with the mammals, but avian genomes is about third in size of that of the human and have evolved separately in a number of distinct strategies from the mammalian genomes for about310MYA (million years ago). After availability of the chicken, zebra finch, turkey and duck genomes, explores the evolutionary events between birds and other vertebrates. For that, the Chinese goose (Anser cygnoides) is well suited for further insight in these fields. Interest in the goose immune system comes not only from its importance as a food animal species, but also for its role as a natural reservoir of many avian viruses, such as influenza virus. Due to lack of genomic information, the nature and regulation of the innate and adaptive immune systems of this waterfowl species are not completely understood. After the high-throughput RNA sequencing of the transcriptome, it became one of the most convenient method to obtain the overall genomic information. Here, present the first genetic architecture of the goose PBLs transcriptome, which explore their immune relevant genes and immune related pathways. Additionally, pMHC I complex of goose has been cloned, expressed, crystallized and solved three dimensional structure.In this study, we pooled RNA from normal healthy geese and performed deep sequencing using the Illumina-solexa platform. In total,211,198unigenes were assembled from the69.36million cleaned reads in the goose PBLs transcriptome. The average length, N50size and the maximum length of the assembled unigenes were687bp,1,298bp and18,992bp, respectively. A total of36,854unigenes showed similarity by BLAST search against the NCBI non-redundant (Nr) protein database. For functional classification,163,161unigenes were comprised of three Gene Ontology (Go) categories and67subcategories. A total of15,334unigenes were annotated into25eukaryotic orthologous groups (KOGs) categories. Kyoto Encyclopedia of Genes and Genomes (KEGG) database, annotated39,585unigenes into six biological functional groups and308pathways. Among the2,757unigenes that participated in the17immune system KEGG pathways,125of the most important immune relevant genes were summarized and analyzed by STRING analysis to identify gene interactions and relationships. Of these125unigenes,109unigenes, approximately87%, were not previously identified in the goose. Moreover,10genes of the innate, adaptive immune system and signaling pathways identified in this study, which were confirmed by PCR and further analysis their sequences to other species.125novel genes of the immune system of goose were compared to genes in the duck, chicken, turkey and zebra finch. Of these125unigenes of the goose PBLs transcriptome,104genes shared to the duck, and21genes (including TLR13, CC26, IL8, IFR3, and IFR7) were not found in duck.8genes (including CCL14, CCL26, IFR3, C4-1) were not shared in the chicken,43genes (including TAPBP, HSP70, HSP90A, TLR3, TLR13, LY96, C8G, BAFF) were not found in the turkey and30genes (including TAPBP, TLR13, TLR15, CCL14, CCL19, IL9R, IL23R, C2, MBL) were not found in zebra finch. This comparison of our sequencing results to other species provides more genomic and transcriptomic information and contribute to the study of the avian transcriptome. Among the2,757unigenes that participated in the17immune system KEGG pathways,11pathways along with their large number genes have been identified in this study.The cloning, expression, purification and crystallization of Goose (Ancy)-$2m and-MHC Ⅰ complex with avian influenza virus was also determined in this study and the goose MHC Ⅰ complex was resolved at2.9A. The secondary structure of Ancy-MHC class Ⅰ complex and Ancy-P2m were determined by Circular Dichroism (CD) spectroscopy which shows that the Goose MHC Ⅰ complex contains38%and18%of a-helix and β-sheet, respectively, whereas goose p2m structure contains36%β-sheet. It provide basic information for the development of some structural analysis in the three dimensional structure of βm and MHC Ⅰ of the goose.The de novo transcriptome analysis of goose PBLs will provide important Chinese goose sequence information. In addition, our data highlights the value of new gene discovery, immune pathways description, defense system gene identification and gene interaction relationships as useful tools to understand the goose immune system. The crystallization studies of goose P-MHC I-P2m will help us for the understanding of peptide presentation of goose MHC I and promote the development of goose virus-derived epitopes based vaccines.