Molecular Characterization And Expression Analyses Of The Complement Component C8α, C8β And C9 Genes In Yellow Catfish(Pelteobagrus Fulvidraco)

The complement system, which is made up of a series of distinct serum proteins and cell surface receptors, is an essential link between innate and adaptive immune responses that allows the host defense against bacterial invasion and inflammatory response. There are three pathways initiated by different triggers to activate the complement system: the classical, the alternative and the lectin. Yellow catfish(Pelteobagrus fulvidraco Richardson)(Teleostei: Bagridae) is an important commercial fish in the aquaculture industry of China due to its excellent meat quality. In recent years, artificial aquaculture of yellow catfish has been seriously affected by several kinds of bacterial diseases, including ascites disease, ulcerative syndrome, and red-head disease, all of which have led to substantial economic losses. It is thus important and necessary to understand expression characteristics and potential functions in innate immune system of C8α, C8β, and C9 genes from yellow catfish for disease controlling. In addition, it has provided basic data for further selective breeding of disease-resistant strain of yellow catfish.Therefore, sequences of the Pf_C8α, Pf_C8β, and Pf_C9 genes(Pf: abbreviation of Pelteobagrus fulvidraco) from yellow catfish were identified using rapid amplification of c DNA ends(RACE) based on transcriptome data. We investigated the tissue distributions of Pf_C8α, Pf_C8β and Pf_C9 genes in adults and their expression profiles during the early larval developmental stages using real-time quantitative PCR(q PCR). Furthermore, tissue-specific expressions were carried out to determine the responses of Pf_C8α, Pf_C8β and Pf_C9 genes to challenges of Aeromonas hydrophila. The main results are as follows:1. Characterization of Pf_C8α, Pf_C8β and Pf_C9 c DNA SequencesThe partial c DNA of the Pf_C8a gene(Gen Bank accession no. KT588317) was 1939 bp, and contains a 145 bp 3′-untranslated region(UTR) and a 1794 bp open reading frame(ORF). The ORF of the Pf_C8a gene encoded 597 amino acids. BLAST(Basic Local Alignment Search Tool) analysis in Gen Bank showed that the Pf_C8α amino acid sequence had the highest homology(54%) to that of Danio rerio, followed by Sinipercachuatsi(52%) and Oncorhynchus mykiss(51%).The partial c DNA of the Pf_C8β gene(Gen Bank accession no. KT588318) was 1767 bp, and contains a 31 bp 5′-UTR, a 1701 bp ORF, and a 35 bp 3′-UTR. The ORF of the Pf_C8β gene encoded 566 amino acids. BLAST analysis revealed that the Pf_C8β amino acids sequence had the highest homology(62%) to that of D. rerio, followed by Esoxlucius(61%) and O. mykiss(60%).The partial c DNA of the Pf_C9 gene(Gen Bank accession no. KT454382) was 1939 bp, and contains an 1806 bp ORF and a 133 bp 3′-UTR. The ORF of the Pf_C9 gene encoded 601 amino acids. BLAST analysis showed that the Pf_C9 amino acid sequence had the highest homology(52%) to that of Ctenopharyngodon idella, followed by O. mykiss(50%) and Oplegnathus fasciatus(49%). Like most other teleosts, the Pf_C8α, Pf_C8β and Pf_C9 genes of yellow catfish all have two TSP 1 domains, an LDLR-A domain, a MACPF domain and an EGF domain.2. Quantitative Analyses of Pf_C8α, Pf_C8β and Pf_C9 m RNA Expression Levels in Adult TissuesExpression levels of Pf_C8α, Pf_C8β and Pf_C9 genes were greatest in the liver, followed by a relatively low level in the head kidney, foregut, midgut, hindgut, kidney, heart, and spleen, and a lower level still in the other tissues.3. Quantitative Analyses of Pf_C8α, Pf_C8β and Pf_C9 m RNA Expression Levels during Embyonic and Early Developmental StagesThe expression of Pf_C8α m RNA was low until the neurula stage, and then increased significantly to the highest value at the heart beat stage, then significantly decreased to a moderate level at the blood circulation stage and was maintained in the newly hatched larval stage. The expression of Pf_C8α m RNA decreased significantly after hatching.The gene expression of Pf_C8β was moderate at the fertilized egg stage. It then significantly decreased to a low level at the cleavage stage and was maintained until the heart beat stage. It increased significantly to a moderate level at the blood circulation stage, and further increased significantly to its highest level at the newly hatched larval stage. After hatching, the expression of Pf_C8β m RNA decreased significantly to a low level at 5 dph. Subsequently, the m RNA expression increased significantly to a moderate level at 15 dph, was maintained until 25 dph.The gene expression of Pf_C9 was low during the early embryonic period from the fertilized egg to the blastula stage. It then increased to a moderate level at the gastrula stage. The expression of Pf_C9 m RNA increased significantly to a highest value at the newly hatched larval stage. After hatching, the expression of Pf_C9 m RNA decreased significantly to a moderate level from 1 to 25 dph, and then decreased gradually to a low level at 30 dph.4. Quantitative Analyses of Pf_C8α, Pf_C8β and Pf_C9 m RNA Expression Levels in Five Tissues after Challenges of A. HydrophilaExpression levels of the Pf_C8α gene in the spleen, head kidney, kidney, liver, and blood after the A. hydrophila challenge were measured by q PCR. In the spleen and kidney, expressions increased significantly to a peak level at 12 h post-injection. This peak appeared at 72 h post-injection in the liver. In the head kidney, the expression level was significantly up-regulated at 12 h and reached a peak at 24 h post-injection. The expression level in the blood increased significantly from 72 to 168 h and reached a peak at 168 h post-injection.Expression levels of the Pf_C8β gene in the spleen, head kidney, kidney, liver, and blood after the A. hydrophila challenge were measured by q PCR. In the spleen, kidney and blood, expressionswere significantly up-regulated at 120 h and reached a peak level at 168 h post-injection. This peak appeared at 120 h post-injection in the liver. The expression in the head kidney increased significantly to a peak level at 24 h post-injection.Expression levels of the Pf_C9 gene in the spleen, head kidney, kidney, liver, and blood after the A. hydrophila challenge were measured by q PCR. In the spleen and kidney, expressions increased significantly to a peak at 12 h post-injection. This peak appeared at 48 h post-injectionin the liver. The expression level in the head kidney was significantly up-regulated at 12 h and reached a peak at 24 h post-injection, whereas the expression level in the blood increased significantly from 72 to 168 h and reached a peak at 168 h post-injection.