Full-Length Transcriptome Sequencing and the Discovery of New Transcripts in the Unfertilized Eggs of Zebrafish (Danio rerio).

Understanding early gene expression in zebrafish embryos is a prerequisite for developmental biology research. In this study, 1,629,447 polymerase reads were obtained from the unfertilized eggs of zebrafish via full-length transcriptome sequencing using the PacBio RS II platform first. Then, 102,920 unique isoforms were obtained by correction, clustering and comparison with the zebrafish genome. 12,782 genes in the genome were captured, accounting for 39.71% of the all annotated genes. Approximately 62.27% of the 12,782 genes have been alternatively spliced. GO and KEGG annotations revealed that the unfertilized eggs primarily stored genes that participate in RNA processing and nuclear protein complex composition. According to this PacBio data that aligned with the genome, 3,970 fusion genes, 819 ncRNAs, and 84 new transcripts were predicted. Illumina RNA-seq and RT-qPCR detection found that the expression of two new transcripts, PB.5289.1 and PB.10209.1, were significantly up-regulated at the 2-cell stage and down-regulated rapidly thereafter, suggesting their involvement in minor ZGA during early embryonic development. This study indicated that the unfertilized eggs of zebrafish may have retained genes directly related to cell division and development to initiate the subsequent development in a limited space and time. On the other hand, NTRs or new transcriptome regions in the genome were discovered, which provided new clues regarding ZGA of MZT during early embryonic development in fish.

Expression and localization of grass carp pkc-θ (protein kinase C theta) gene after its activation.

Haemorrhagic disease caused by grass carp reovirus (GCRV) can result in large-scale death of young grass carp, leading to irreparable economic losses that seriously affect large-scale breeding. Protein kinase C (PKC, also known as PRKC) represents a family of serine/threonine protein kinases that includes multiple isozymes in many species. Among these, PKC-θ (PKC theta, also written as PRKCQ) is a novel isoform, mainly expressed in T cells, that is known to be involved in immune system function in mammals. To date, no research on immunological functions of fish Pkc-θ has been reported. To address this issue, we cloned the grass carp pkc-θ gene. Phylogenetic and syntenic analysis showed that this gene is the most evolutionarily conserved relative to zebrafish. Real-time quantitative PCR (RT-qPCR) indicated that pkc-θ was expressed at high levels in the gills and spleen of healthy grass carp. Infection with GCRV down regulated pkc-θ expression in the gills and spleen. Gene products that function upstream and downstream of pkc-θ were up regulated in the gill, but were down-regulated in the spleen. These results suggest that direct or indirect targeting of pkc-θ by GCRV may help the virus evade host immune defences in the spleen. Phorbol ester (PMA) treatment of Jurkat T cells induced translocation of grass carp Pkc-θ from the cytoplasm to the plasma membrane. This response to PMA suggests evolutionary conservation of an immune response function in fish Pkc-θ, as well as conservation of its sequence and structural domains. This study expanded our knowledge of the fish PKC gene family, and explored the role of pkc-θ in function of the grass carp immune system, providing new insights which may facilitate further studies of its biological functions.

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, in-between-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 IFN-I, TNF-α, 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.

Cloning of six serpin genes and their responses to GCRV infection in grass carp (Ctenopharyngodon idella).

Grass carp, an economically important aquaculture fish, is very sensitive to Grass Carp Reovirus (GCRV). Haemorrhagic disease caused by GCRV infection can cause large-scale death of first-year grass carp, thereby severely restricting the intensive culture. Serpins (serine protease inhibitors) belong to the protease inhibitor gene family and are involved in numerous physiological and pathological processes, particularly coagulation and anticoagulation. Reports on grass carp serpins are scarce. Thus, we cloned six grass carp serpin genes (serpinb1, serpinc1, serpind1, serpinf1, serpinf2b and serping1) in this study. Molecular evolution showed that serpins between grass carp and zebrafish or carp are the closest relatives. SERPIN domains in these 6 serpins and reactive centre loop (RCL) along with their cleavage sites of 5 serpins (serpinb1, serpinc1, serpind1, serpinf2b and serping1) were predicted. Real-time quantitative PCR (RT-qPCR) showed that these serpins displayed tissue significance. Among them, serpinc1, serpind1, serpinf2b and serping1 had the highest expression levels in the liver. After GCRV infection, RT-qPCR showed that the liver-enriched serpins were significantly changed. Key procoagulant factor genes (kng-1, f2, f3a, f3b and f7) and anticoagulant genes (tpa, plg, thbd, proc and pros) also showed significant changes on the mRNA level. Comprehensive comparative analysis showed that the up-regulated expression of key clotting factor genes was more prominent than that of main anti-coagulation factor genes. Thus, the function of coagulation may be more dominant in grass carp during the GCRV infection, which may cause overproduction of thrombi. The serpins were involved in GCRV infection and liver-enriched serpins participate in the interaction between coagulation and anticoagulation. This study provided new insights into further research on the biological functions of grass carp serpins and clarifying the molecular mechanism of GCRV affecting the homeostasis of grass carp blood environment.