Amphioxus Gnbp And Litaf Genetic Evolution, Functions And Immune Related Mirnas Transcriptome Analysis

Both morphological and molecular evidence show that amphioxus is the basal chordate and is a crucial organism for understanding the chordate evolution including the origin of the immune system, morphology and development. With the rapid development of comparative immunology, as well as the completion of the Branchiostoma floridae genome sequencing, the attention was focused on amphioxus immune increasingly. In this study, based on the ESTs of amphioxus, using molecular cloning, real-time PCR, microarray, comparative genomics and immunobiology technology, we studied the immune-related gene LITAF, GNBP and microRNAs. The results as follows:1. We have systematically compared amphioxus ESTs from five developmental stages. For the egg, gastrula, neurula, larva and adult stages, amphioxus ESTs were assembled, respectively, into3364,3230,10,299,4052and3866contigs, and193,183,933,178and151singlets.25,796ORFs were identified, of which6,529predicted ORFs have found matches from the UniProt database. The analysis suggested that there were significantly more GO terms assigned to the conreads of neurula stage. Meanwhile, we found20putative immune-related genes from these BLASTx results, providing the prilimilary data for functional study of amphioxus immunity. In addition, we also analyzed the target gene of these microRNAs. A web interface was developed for visualization of the contigs, miRNAs and annotations (http://www.rich.yunda.org/test/amphioxusest/).2. Gram-negative bacteria-binding proteins (GNBPs) are important pattern recognition proteins (PRPs), which can initiate host defense in response to pathogen surface molecules. In this study, we identified and characterized an amphioxus GNBP gene (designated as AmphiGNBP). The AmphiGNBP encodes a putative protein with558aa, which contains a conserved β-1,3-glucan recognizing and binding domain. Interestingly, AmphiGNBP encodes two extra WSC (cell wall integrity and stress response component) domains, which are unique in AmphiGNBP and not found in those known GNBPs from other species. The AmphiGNBP was mainly expressed in immune tissues, such as hepatic cecum and intestine. The expression of AmphiGNBP was affected after LPS stimulation. These studies disclose the particularity and universality of AmphiGNBP and will provide profound insights into the function and evolution of GNBP. 3. TNF-a is an important cytokine that can be stimulated by Lipopolysaccharide (LPS) to regulate the innate immunity. The lipopolysaccharide-induced TNF-a factor (LITAF) functions as a transcription factor for regulating the expression of TNF-a as well as various inflammatory cytokines in response to LPS stimulation. In this study, we identified and characterized an amphioxus LITAF gene (designated as AmphiLITAF). First, we identified the AmphiLITAF from the amphioxus and found that AmphiLITAF gene with～1.6kb in length has a827bp cDNA transcription product which encodes a putative protein with127amino acids containing conserved LITAF-domain, the deduced amino acid of AmphiLITAF shared37-60%similarity with the LITAFs from other species; second, we uncovered the spatial distribution of the LITAF in different tissues, the expression level of AmphiLITAF mRNA was the highest in hepatic cecum and intestine, moderate in muscles, gills and gonad, and the lowest in notochord; finally, we discovered that the expression of AmphiLITAF was affected after LPS stimulation. Our findings provide an insight into the innate immune response in the amphioxus and the evolution of the LITAF family.4. Using small RNA deep sequencing technology, a total of568microRNAs (miRNA) have been found in Branchiostoma belcheri.171microRNA sequences are consistent with those of Branchiostoma floridae, the rest of the497microRNAs are newly discovered in amphioxus. Among these newly discovered microRNAs,302microRNAs are conserved in other species, and95microRNAs are amphioxus specific microRNAs. We used microRNA microarray technology to analysis six microRNA transcriptomes prepared from amphioxus under PBS stimulation and amphioxus infection with Vibrio parahaemolyticus (PV) at different time points. By comparing the data from different treatment groups, we found18immune-related microRNAs. These microRNAs may be interaction with31immune-related genes. These results of our study suggest that miRNAs play an important role in the coordinated regulation of immune-related gene expression in the amphioxus.In this thesis, we built amphioxus EST database under the windows operation system. A web interface was developed for visualization of the contigs, microRNAs and annotations. The constructed AmphiEST database may serve as an essential resource for future studies of the development and evolution of amphioxus. Based on the EST database, we firstly identified and characterized LITAF and GNBP gene homolog from amphioxus, and our findings provide an insight into the innate immune response in the amphioxus and the evolution of the LITAF and GNBP family. Screening of amphioxus immune-related microRNAs will help with the development of new immune mechanism to prevent or treat Vibrio parahaemolyticus infections in the amphioxus.