Screening And Identification Of Differentially Expressed Genes Treated With DHV-I

China is the dominant country of duck rearing and consumption. But with the increasing breeding amount and rearing density, diseases are also increasing and becoming more complex year by year, which causes huge economic losses. Duckling viral hepatitis of type I caused by duck hepatitis virus mainly infected ducks in0-3weeks, characterized by rapid spread, acute onset, high infectivity and mortality. After infection, the body can induce changes in genes expression through multiple signaling pathways. But so far, which genes expression changed by DHV-I was not clear. Therefore, looking for genes closely related with replication of virus in body cells can help elucidate the molecular mechanism of virus infection. This study built a suppression subtractive hybridization (SSH)-cDNA library of full-sib ducklings injected duck hepatitis virus and the same quantity of saline artificially to screen differentially expressed cDNA and identify responsive genes to the DVH. Results are as follows in detail.1. Screening, identification and sequence analysis of genes treated with DHV-IThis trial has established0.4ml DHV-I (provided by Professor Peng Daxin at Yangzhou University) muscle injection model. Concentrated death phenomenon is obvious during24-48hours after infection. The vast majority of ducklings appeared poor mental status, activity reduced, neck stiffened before death. Necropsy revealed that, swollen liver with yellow or yellow-brown spots on, kidney hyperemia and swelling, spleen enlargement in experimental ducklings. Primers were designed based on the sequence of DHV-I to amplify the target fragment. The infection group obtained the aim strip of440bp, the control group showed negative. Suppression subtractive hybridization was used to screen differentially expressed ESTs under two conditions of injection DHV-I and equal volume of saline, dot hybrization was used for identification ESTs.Sequence the PCR products when differences of signal value was higher than twice. Sequence homology comparison was performed using the BLASTn and BLASTx programs. When at least150bp matched, with an E-value less than e-10, the cDNA sequence was considered homologous with a certain gene in Gene Bank. Uniprot database analysis was performed based on screened sequences. Also, Gene Ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) analysis was carried out based on the BLAST analysis. The results showed that222and77positive clones were respectively choosed from the forward and backward libraries.299positive clones were sequenced to obtain299ESTs, which were corresponding to70genes including52up-regulated genes and18down-regulated genes in NCBI. Cluster analysis showed that of all the ESTs,12%was related with DNA replication;12%was related with fatty acid metabolism;48%with regulation of abnormal life activities in drugs, poisons, metalion and pyridoxal phosphate;3%with protein chelate; the other25%ESTs participated in other life activities regulatory role. Therefore, the occurrence and development of DVH-I was a multi-step process involved many genes. These genes participated in69pathways, including8metabolic pathways,6endocytic pathways,4Focal adhesion,4Lysosome,3ECM-receptor interaction,3Glyoxylate and dicarboxylate metabolism,3MAPK signaling pathway,3Apoptosis and the other35pathways.2. Cloning the full-length cDNA of ALB gene using RT-PCR combined with RACEClone CDS of ALB using RT-PCR and then get the full cDNA sequence using RACE. The sequencing results showed that the full-length cDNA of ALB in ducks was2107bp including5’UTR (47bp),3’UTR (212bp) and ORF (1848bp).The mRNA expression of ALB was concentrated in livers.The ducks ALB gene encoded615amino acid, respectively9,8,9,10,9and1amino acid residues more than people, rats, cattle, pigs and chickens. Amino acids of ducks was highest homologue with chickens (85.4%), followed by rats (47.2%), people (46.6%),cattle (46.6%)and pigs(45.8%).The phylogenetic tree based on the sequence of amino acid showed that the protein of ducks and chickens are clustered together having the closest kinship while mammalian belong to another class.ALB protein mainly consist of alpha helix (66.02%), followed by random coil (25.37%), extended strand (4.39%) and beta turn (4.23%). 3. Detecting differentially expressed genes using RT-qPCR&ELISAExcept in leg muscles, the expression level of ALB and TLR7genes in susceptible group is highly significantly less than that in resistant group and control group(P＜0.01), and the expression level in resistant group is highly significantly or significantly less than that in control group (P＜0.01or P＜0.05).The expression of PSPH and WSB1genes in susceptible group is highly significantly greater than that in control group(P<0.01), but the difference between resistant group and control group is not significant(P＞0.05).Detecting ALB protein by using ELISA displayed that ALB protein in susceptible group was significant or very significant lower than that of control group but higher than that of resistant group(P＜0.01or P＜0.05).Whereas there was no significant differences in the other tissues among the three treatments.The results of serum biochemical parameters showed that ALT in the infected group increased very significantly (P＜0.01) and Urea nitrogen increased significantly (P＜0.05); blood GLU and IgG had a certain amount of decline compared with control group but the difference was not significant (P＞0.05); AST, TG, UA, IgA, IgM was slightly elevated at the time of onset (P＞0.05).