Amphioxus REL, NFAT, TGFBI Cloning And Expression Of TLR Signaling Pathway Evolutionary Studies

Amphoxus, as an ancestral species of vertebrate animals, play important roles in the evolution of vertebrate morphogenesis, development, immunity. With the development of whole genome sequencing finished, the rapid development of the comparative immunology and the completion of Branchostoma florida genome sequencing which could provide the material basis for systematically studying the evolution and the origin of vertebrate immune system. Therefore, we studied the function and evolution of amphoxus(Branchostoma belcheri) REL, NEAT, TGFBI genes based on the amphioxus EST database (http://rich.yunda.org/test/amphioxusest/) and some public database and then we used the comparative genomics research methods to study the evolution and origin of TLR signaling pathway in order to reveal the relationship between the natural selection and gene duplication, the shifted of gene structure, and the changed of gene domains. The detail results as following:1. NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells), as a very important transcription factor, play an important roles in cell signalling, apoptosis, innate immunity and adaptive immunity and so on. We cloned a NF-κB gene from Branchostoma belcheri genome (named as AmphiREL). Its full-length has2700bp and encodes a putative protein with773aa, which including two conserved domains:RHD domain and IPT domain. AmphiREL could involve in amphioxus innate immunity after LPS stimulation, which gene expression was significantly up-regulated up to2.4fold (P<0.01) at6h after LPS challenge. The results of gene synteny and gene structure analysis have shown that AmphiREL gene may be orthologous to human RELB, whereas human RELA and REL are paralogous genes of the AmphiREL gene. The conserved motif of Rel subfamily genes has regular change and all genes under very strong negtive selection in the evolutionary history. Together, our results provide important clues for understanding the evolution and function of Rel subfamily genes.2. NFAT (Nuclear Factor of Activated T cells), which paly an important roles in immunity system and development of vertebrate, was first identified in nuclear extracts of activated T cells as an inducible DNA-binding factor that binds to IL-2. We cloned a NFAT gene from Branchostoma belcheri genome (named as AmphiNFAT) which full-length has3547bp, encodes a putative protein with605aa. The polypeptide has a highly conserved DNA-binding domain (Rel homology region, RHR), a moderately conserved regulatory domain (NFAT homology region, NHR), and a non-conserved carboxyterminal domain. The AmphiNFAT could response to LPS-induced innate immunity of amphioxus. Tissue expression pattern of AmphiNFAT gene has shown that the expression of AmphiNFAT was high in the muscles and notochord, moderate in gonad and gills, and low in the intestine and hepatic cecum. The NFAT family genes were present in a common ancestor with cnidaria, and NFAT1-4paralogs were lost early in Branchiostoma and Strongylocentrotus genomes. Besides, we discovered that NFAT family genes underwent strong purifying selection. Finally, we propose a model of the relationship between natural selection and NFAT genes duplication. Taken together, our findings provide an insight into the innate immune response of amphioxus and the evolution of the NFAT gene family.3. TGFBI (transforming growth factor β induced) encoding an extracellular matrix (ECM) protein, was identified from a TGFβ1treated lung adenocarcinoma cell line. We cloned a TGFBI gene from Branchostoma belcheri genome (named as AmphiTGFBT), which full-length has3095bp, encodes a putative protein with867aa. The AmphiTGFBI contains a signal peptide (SP), an EMI domain, five Fasl domains (Fasla-e) compared to no more than4Fasl domains in other animals. The tissue expression patterm has shown that AmphiTGFBI was detected in all tissues. The TGFBI family genes were present in a common ancestor with Amphimedon queenslandica and the gene structures and conserved motifs of invertebrate TGFBIs were found to present regular changes during the evolutionary history. Positive selection and Fasl domain loss might cause the regular changes of gene structures and conserved motifs in invertebrate TGFBI during the evolutionary history. Together, our findings provided an insight into the function and evolution of the TGFBI family.4. The TLR signaling pathway, which plays a central role in innate immunity from Drosophila to mammal, is highly conserved in structure and function from insects to vertebrates. Otherwise, genes carry out their biological functions through pathways in complex networks consisting of many interacting molecules. Therefore, we studied the evolution and origin of animal TLR signaling pathway revealed by network-level molecular evolutionary analyses. All genes in TLR signaling pathway were highly conserved and underwent strong purifying selection and the selection constraint of genes was negatively correlated with its position along TLR signaling pathway. The TLR signaling pathway might present in a common ancestor of sponges and eumetazoa, and evolve via the TLR, IKK, IκB and NF-κB genes underwent duplication events as well as adaptor molecular enlargement, and gene structure and conservation motif of NF-κB genes shifted in their evolutionary history. Our results will improve our understanding on the evolutionary history of animal TLR signaling pathway as well as the relationship between the network architecture and the sequences evolution of individual protein.In this thesis, the function and evolutionary history of amphioxus REL, NFAT, TGFBI genes and the evolution and origin of animal TLR signaling pathway have been studied. We first revealed the proccess of evolution and origin of Rel subfamily, NFAT family, TGFBI family and TLR signaling pathway, and discussed the relationship between natural selection and gene duplication, the change of gene structure and the change of conserved domain. Our results will improve our understanding on the relationship between natural selection and new function obtained of proteins.