Molecular cloning and preliminary functional analysis of six RING-between-ring (RBR) genes in grass carp (Ctenopharyngodon idellus)

Ubiquitination is a post-translational modification of proteins that is widely present in eukaryotic cells. There is increasing evidence that ubiquitinated proteins play crucial roles in the immune response process. In mammals, RING-between-RING (RBR) proteins play a key role in regulating immune signaling as the important E3 ubiquitin ligases during ubiquitination. However, the function of RBR in fish is still unclear. In the present study, six RBR genes (RNF19A, RNF19B, RNF144AA, RNF144AB, RNF144B and RNF217) of grass carp (Ctenopharyngodon idellus) were cloned and characterized. Similar to mammals, all six members of RBR family contained RING, inbetween-ring (IBR) and transmembrane (TM) domains. These genes were constitutively expressed in all studied tissues, but the relative expression level differed. Following grass carp reovirus(GCRV) infection, the expression of six RBR genes in liver, gill, spleen and intestine significantly altered. Additionally, their expression in Ctenopharyngodon idellus kidney (CIK) cells was significantly increased after GCRV infection. And deficiency of RNF144B in CIK with small interference RNA (siRNA) up-regulated polyinosinic:polycytidylic acid poly(I:C))- induced inflammatory cytokines production, including 1FN-I, TNF-alpha, IL-6, and transcription factor IRF3, which demonstrated that RNF144B was a negative regulator of inflammatory cytokines. Our results suggested that the RBR might play a vital role in regulating immune signaling and laid the foundation for the further mechanism research of RBR in fishes

Construction and Characterization of Two Bacterial Artificial Chromosome Libraries of Grass Carp

Bacterial artificial chromosome (BAC) library is an important tool in genomic research. We constructed two libraries from the genomic DNA of grass carp (Ctenopharyngodon idellus) as a crucial part of the grass carp genome project. The libraries were constructed in the EcoRI and HindIII sites of the vector CopyControl pCC1BAC. The EcoRI library comprised 53,000 positive clones, and approximately 99.94% of the clones contained grass carp nuclear DNA inserts (average size, 139.7 kb) covering 7.4x haploid genome equivalents and 2% empty clones. Similarly, the HindIII library comprised 52,216 clones with approximately 99.82% probability of finding any genomic fragments containing single-copy genes; the average insert size was 121.5 kb with 2.8% insert-empty clones, thus providing genome coverage of 6.3x haploid genome equivalents of grass carp. We selected gene-specific probes for screening the target gene clones in the HindIII library. In all, we obtained 31 positive clones, which were identified for every gene, with an average of 6.2 BAC clones per gene probe. Thus, we succeeded in constructing the desired BAC libraries, which should provide an important foundation for future physical mapping and whole-genome sequencing in grass carp

Isolation and expression of grass carp toll-like receptor 5a (CiTLR5a) and 5b (CiTLR5b) gene involved in the response to flagellin stimulation and grass carp reovirus infection

Toll-like receptor 5 (TLR5), a member of Toll-like receptors (TLRs) family and is responsible for the bacterial flagellin recognition in vertebrates, play an important role in innate immunity. In the study, two TLR5 genes of grass carp (Ctenopharyngodon idellus), named CiTLR5a and CiTLR5b, were cloned and analyzed. Both CiTLR5a and CiTLR5b are typical TLR proteins, including LRR motif, transmembrane region and TIR domain. The full-length cDNA of CiTLR5a is 3054 bp long, with a 2646 bp open reading frame (ORF), 78 bp 5' untranslated regions (UTR), and 330 bp 3' UTR. The full-length cDNA of CiTLR5b is 3326 bp, with a 2627 bp ORF, 95 bp 5' UTR, and 594 bp 3' UTR. Phylogenetic analysis showed that CiTLR5a and CiTLR5b were closed to the TLR5 of cirrhinus mrigala, cyprinus_carpio, and danio redo. Subcellular localization indicated that CiTLR5a and CiTLR5b shared similar localization pattern and may locate in the plasma membrane of transfected cells. Real-time quantitative PCR revealed CiTLR5a and CiTLR5b were constitutively expressed in all examined tissues, whereas the highest expressed tissue differed. Following exposure to flagellin and GCRV, CiTLR5a and CiTLR5b were up-regulated significantly. Moreover, the downstream genes of TLR5 signal pathway such as MyD88, NF-kappa B, IRF7, IL-1 beta, and TNF-alpha also up-regulated significantly, whereas the I kappa B gene was down-regulated, suggesting that CiTLR5a and CiTLR5b involved in response to flagellin stimulation and GCRV infection. The results obtained in the study would provide a new insight for further understand the function of TLR5 in teleost fish. (C) 2015 Elsevier Ltd. All rights reserved

Functional characterizations of IPS-1 in CIK cells: Potential roles in regulating IFN-I response dependent on IRF7 but not IRF3

IPS-1, as the sole adaptor of RIG-I and MDA5, plays a central role in innate antiviral immunity. In this study, we investigated potential roles of IPS-1 in innate immunity and the domain-requirement of IPS-1 for its signaling in grass carp (Ctenopharyngodon idella). Overexpression experiment showed that CiIPS-1 mediated IFN-I signal possibly dependent on CiIRF7 but not CiIRF3. Post GCRV challenge, CiIPS-1 could enhance antiviral immune responses. CARD and TM domains were crucial for antiviral function of CiIPS-1, and TRAF motif played an assistant role. PRO domain seemed as a negative regulator but was pivotal for the initiation of CiIFN-I and CiMx1. Post viral/bacterial PAMPs stimulation, CiIPS-1-mediated signaling was tightly controlled. CARD domain of CiIPS-1 could significantly elicit poly I:C/LPS/PGN-mediated signaling. PRO domain negatively regulated CiIRF7 and CiIFN-I but was indispensable for inductions of CiMx1 and CiIL-1 beta. TRAF motif and TM domain regulated the signaling presumably in a cooperative fashion. Post poly I:C stimulation, TRAF motif negatively regulated CiIRF7, CiIFN-I and CiIL-1 beta at a relative early time while TM domain functioned at a relative late time. TRAF motif was indispensable for the production of CiMx1, while TM domain slightly negatively regulated the expression. Post LPS and PGN stimulation, TRAF motif excited an assistant and persistent negative role on CiIFN-I, CiIRF7 and CiIL-1 beta induction, but was crucial for induction of CiMx1. TM domain slightly negatively regulated LPS- and PGN-triggered signaling. Taken together, CiIPS-1 not only exerted important functions in antiviral immune response but also participated in viral/bacterial PAMPs-triggered immune response which was tightly controlled to prevent harmful effects resulting from excessive activation. This study provided novel insights into the pivotal role of IFS-1 in innate immunity. (C) 2015 Elsevier Ltd. All rights reserved

RNA-seq profiles from grass carp tissues after reovirus (GCRV) infection based on singular and modular enrichment analyses

Hemorrhagic disease of the grass carp, Ctenopharyngodon idella, is a fatal disease in fingerlings and yearlings caused by a reovirus, GCRV. RNA-seq data from four diseased grass carp tissues (gill, intestine, liver and spleen) were obtained at 2 h before and six times after (2 h, 24 h, 48 h, 72 h, 96 h and 120 h) GCRV challenge. A total of 7.25 +/- 0.18 million (M) clean reads and 3.53 +/- 0.37 M unique reads were obtained per RNA-seq analysis. Compared with controls, there were 9060 unique differentially expressed genes (DEGs) in the four tissues at the six time points post-GCRV challenge. Hierarchical clustering analysis of the DEGs showed that the data from the six time points fell into three branches: 2 h, 24 h/48 h, and 72 h/96 h/120 h. Singular (SEA) and modular enrichment analyses of DEGs per RNA-seq dataset were performed based on gene ontology. The results showed that immune responses occurred in all four tissues, indicating that GCRV probably does not target any tissue specifically. Moreover, during the course of disease, disturbances were observed in lipid and carbohydrate metabolism in each of the organs. SEA of DEGs based on the Kyoto Encyclopedia of Genes and Genomes database was also performed, and this indicated that the complement system and cellular immunity played an important role during the course of hemorrhagic disease. The qPCR of pooled samples of duplicate challenge experiment were used to confirm our RNA-seq approach. (C) 2014 Elsevier Ltd. All rights reserved

Transgenic common carp do not have the ability to expand populations.

The ecological safety of transgenic organisms is an important issue of international public and political concern. The assessment of ecological risks is also crucial for realizing the beneficial industrial application of transgenic organisms. In this study, reproduction of common carp (Cyprinus carpio, CC) in isolated natural aquatic environments was analyzed. Using the method of paternity testing, a comparative analysis was conducted on the structure of an offspring population of "all-fish" growth hormone gene-transgenic common carp (afgh-CC) and of wild CC to evaluate their fertility and juvenile viability. Experimental results showed that in a natural aquatic environment, the ratio of comparative advantage in mating ability of afgh-CC over wild CC was 1∶1, showing nearly identical mating competitiveness. Juvenile viability of afgh-CC was low, and the average daily survival rate was less than 98.00%. After a possible accidental escape or release of transgenic CC into natural aquatic environments they are unable to monopolize resources from eggs of natural CC populations, leading to the extinction of transgenic CC. Transgenic CC are also unlikely to form dominant populations in natural aquatic environments due to their low juvenile viability. Thus, it is expected that the proportion of afgh-CC in the natural environment would remain low or gradually decline, and ultimately disappear

Different responses in one-year-old and three-year-old grass carp reveal the mechanism of age restriction of GCRV infection

Grass carp is an important fish species in Chinese aquaculture, and can be afflicted by a hemorrhagic disease caused by the grass carp reovirus (GCRV). Interestingly, the affects of GCRV infection of grass carp are age-restricted, meaning that one-year-old grass carp can be infected and can suffer hemorrhagic disease, but three-year-old carp are not so afflicted. In this study, we investigated the mechanism responsible for this age-restricted pathology. We evaluated the relative copy number of GCRV RNA, the expression levels of proteins in blood, and changes in DNA methylation in carp from the two age groups after infection with GCRV. After GCRV infection, the relative copy number of GCRV RNA in three-year-old grass carp was significantly lower than in one-year-old carp. The differences in circulating protein levels mainly occurred in concentrated in complement and coagulation proteins, and the expression levels of these proteins were significantly higher in three-year-old grass carp than in one-year-old carp. Moreover, the expression levels of DNA methylation-related genes in the liver and spleen of one-year-old grass carp were significantly higher than those of three-year-old carp. These results suggested that as age of grass carp increases, faster and more efficient response of the immune system after viral infection, especially the complement system, and differences in DNA methylation may be important factors that affect the age restriction observed in GCRV infection. Our study provides new insights into the mechanisms underlying age restriction of GCRV infection

ITGB1b-Deficient Rare Minnows Delay Grass Carp Reovirus (GCRV) Entry and Attenuate GCRV-Triggered Apoptosis

Integrin β-1 (ITGB1) is a transmembrane protein belonging to the integrin family and it plays an important role in viral entry. In this study, the itgb1b gene of the rare minnow, Gobiocypris rarus, was cloned and analyzed. To investigate the possible role of itgb1b on grass carp reovirus (GCRV) infection, we generated an ITGB1b-deficient rare minnow (ITGB1b−/−) using the CRISPR/Cas9 system. Following stimulation with GCRV, the survival time of the -ITGB1b−/− rare minnows was extended in comparison to the wild-type minnows. Moreover, the relative copy number of GCRV and the level of clathrin-mediated endocytosis-associated and apoptosis-related gene expression in the ITGB1b−/− rare minnows was significantly lower than that of the wild-type minnows. These results suggested that the absence of itgb1b reduced viral entry efficiency and the expression of apoptosis-related genes. Moreover, the data suggested that itgb1b played an important role in mediating the entry of viruses into the cells via clathrin. Therefore, these findings provide novel insight into the function of itgb1b in the process of GCRV infection