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

草鱼PKC-θ基因活化后的表达和定位

由GCRV引起的出血病可导致幼龄草鱼大面积死亡，造成难以挽回的经济损失，严重影响草鱼的规模化养殖。蛋白激酶C（Protein kinase C，PRKC，也称为PKC）是一类由多种同工酶组成的丝/苏氨酸蛋白激酶家族。其中的Protein kinase C, theta（PKC-θ，也称为PRKCQ）是主要表达于T细胞的PKC亚型，涉及重要的获得性免疫，但关于鱼类PKC-θ基因的相关免疫学功能研究仍然未有报道。本研究克隆了草鱼PKC-θ基因的序列。分子进化和同源共线性分析显示该基因在草鱼和斑马鱼间的进化保守性最高。定量PCR提示PKC-θ在健康草鱼的鳃和脾中具有高水平的组织分布；GCRV感染后，PKC-θ在鳃和脾中的表达均下调，其上下游基因在腮中的表达均上调，在脾中的表达则全部下调，结果提示PKC-θ可能是GCRV在脾脏组织中逃避宿主免疫防御的间接或直接作用靶点。佛波酯（PMA）处理Jurkat T细胞后，草鱼PKC-θ从胞质集中转位到胞膜，说明草鱼PKC-θ也可以响应PMA的应答，提示鱼类PKC-θ的免疫应答功能在进化过程中具有一定的保守性。综上，本研究丰富了鱼类PKC基因家族成员，初步探讨了草鱼PKC-θ基因的免疫学功能，为进一步研究草鱼PKC-θ基因的相关生物学功能提供了新的见解

Expression and localization of grass carp pkc-theta (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-theta (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-theta has been reported. To address this issue, we cloned the grass carp pkc-theta 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-theta was expressed at high levels in the gills and spleen of healthy grass carp. Infection with GCRV down regulated pkc-theta expression in the gills and spleen. Gene products that function upstream and downstream of pkc-theta were up regulated in the gill, but were down-regulated in the spleen. These results suggest that direct or indirect targeting of pkc-theta 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-theta from the cytoplasm to the plasma membrane. This response to PMA suggests evolutionary conservation of an immune response function in fish Pkc-theta, 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-theta in function of the grass carp immune system, providing new insights which may facilitate further studies of its biological functions

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

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 (gm, pig, 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