Transcriptome Profiling And Digital Gene Expression Analysis Of Nile Tilapia And Expression Analysis Of Siglecs

Nile Tilapia (Oreochromis niloticus) belongs to Percifomes, Cichlidae,Tilapia. It has been recommended to countries worldwide as an excellentaquaculture species because of its good adaptability, fast growth rate,strong reproductive capacity and omnivorous feeding habit. China is themajor country of tilapia farming where the annual yield of1.2million tonsof tilapia accounts for almost50percent of global production. However,infectious disease caused by Streptococcus agalactiae has been severe inrecent years due to the excessive breeding density and degradingenvironment, resulting in great economic loss and threatening the healthydevelopment of tilapia aquaculture. This study conducted transcriptomeand digital gene expression (DGE) analysis of tilapia before and after S.agalactiae challenging in order to investigate transcriptomic changes intilapia caused by bacterial infection. Then we selected the importantimmune receptor Siglecs for cloning and expression level study. These willfurther our understanding in the immune response mechanism of tilapiaagainst pathogen invasion and provide scientific basis for effectiveprevention and control of tilapia streptococcal disease and sustainabledevelopment of tilapia aquaculture.1. Transcriptome profiling and Digital Gene Expression analysis ofNile tilapia infected by S. agalactiaeThis study employed Illumina RNA-seq technology to analysis tilapiatransciptome. We obtained82,799Unigenes (mean size:618bp) afterSOAPdenovo assembly. Unigenes were annotated by comparing againstdatabases including Nr, Swissprot, COG (Cluster of Orthologous Groups ofproteins), KEGG (Kyoto Encyclopedia of Genes and Genomes), and GO(Gene ontology) using BlastX. Taking the assembled transcriptomeUnigenes as reference sequences, combining with DGE technology, weinvestigated changes in the tilapia transcriptome before and after S. agalactiae infection. A total of774significantly up-regulated and625significantly down-regulated unigenes were identified, among which293were mapped to181signaling pathways including17immune-relatedpathways involving65differentially expressed genes. We also selected sixdifferentially expressed genes in the Toll-like receptor signaling pathway toconduct quantitative real-time PCR and the results validated the reliabilityof our DGE data. Large amounts of assembled transcriptome sequencesenriched the gene sequence resources of tilapia and the generateddifferentially expressed genes and related signaling pathways providedvaluable resource for further research into the mechanism of tilapia againstStreptococcus infection and other immune related researches.2. Cloning and tissue expression analysis of Nile tilapia Siglec genesTwo Nile tilapia Siglec genes, Siglec-1and Siglec-14were cloned.ORF of Siglec-1is1350bp in length and encodes449amino acids. Proteinprimary structure prediction shows that Siglec-1contains a signal peptide,three Ig-like domains, a transmembrane domain and one ITIM domain inthe cytoplasmic region. We can see from the phylogenetic tree that fishSiglec-1gathered into a branch while that of mammalian gathered intoanother branch. ORF of Siglec-14is921bp in length and encodes306amino acids. Protein primary structure prediction shows that Siglec-14contains a signal peptide, two Ig-like domains and a transmembranedomain. Results of quantitative real-time PCR showed that Siglec-1andSiglec-14are all widely expressed in tilapia tissues. The most highlyexpression tissue is spleen and then gill and heart, liver is the leastexpression tissue. This research has laid the foundation for relevant studiesof interactions between S. agalactiae and Nile tilapia Siglecs.