Jianguo Su

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, 139.7xa0kb) covering 7.4× 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 121.5xa0kb with 2.8% insert-empty clones, thus providing genome coverage of 6.3× 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.

Genomic sequence comparison, promoter activity, SNP detection of RIG-I gene and association with resistance/susceptibility to grass carp reovirus in grass carp (Ctenopharyngodon idella).

As an intracellular pattern recognition receptor (PRR), retinoic acid-inducible gene-I (RIG-I) is responsible for detection of nucleic acids from pathogens in infected cells and activation of type I interferon (IFN). In the present study, the 5-flanking region, introns and single nucleotide polymorphisms (SNPs) of CiRIG-I (Ctenopharyngodon idella RIG-I) were identified and characterized. The genomic CiRIG-I was 12810 bp in length, consisted of an 1864 bp 5-flank region whose promoter activity was confirmed, 15 exons and 14 introns. By pooled DNA sequencing, two SNPs were detected in the 5-flanking region; 10 SNPs were discovered in introns; and one SNP was found in exons. After a challenge experiment, these SNPs were selected to analyze their association with the resistance/susceptibility of C. idella to grass carp reovirus (GCRV), using case-control study. Chi-square test was employed to assess the association. The result showed that -780 C/T, 4731 C/T, 4945 A/G, 8461 C/T, and haplotype 3428A-3432G were significantly associated with the phenotype (P<0.05). To confirm the correlation, another independent challenge experiment was performed, in which the cumulative mortality of -780 genotype CC, 4731 genotype CC and 4945 genotype AA were significantly lower than that of -780 genotype TT, 4731 genotype TT and 4945 genotype GG, respectively (P<0.05). In addition, the SNP-SNP interaction analysis revealed that there was no significant interaction among those SNPs (P>0.05). These significant SNPs and the haplotype might be potential genetic markers for the molecular selection of C. idella strains that are resistant to GCRV.

LGP2 plays extensive roles in modulating innate immune responses in Ctenopharyngodon idella kidney (CIK) cells

LGP2 (laboratory of genetics and physiology 2), RIG-I (retinoic acid inducible gene-I) and MDA5 (melanoma differentiation associated gene 5) constitute the RLR (RIG-I-like receptor) family. LGP2 plays a pivotal role in modulating signaling of RIG-I and MDA5 in innate immune responses. In this study, three representative overexpression vectors were constructed and transfected into C.u2009idella kidney (CIK) cell line to research functional characterizations of CiLGP2 (C.u2009idella LGP2). CiLGP2 overexpression led to the induction of CiRIG-I transcripts. After GCRV challenge, CiLGP2 enhanced CiMDA5 and CiIPS-1 to reinforce the immune response, however, impaired the expression of CiRIG-I. Meanwhile, antiviral activity assays showed that overexpression of CiLGP2 or its domains could inhibit GCRV replication and protect cells from death. Besides, CiLGP2 lingeringly induced CiRIG-I mRNA expression and inhibited CiMDA5 transcripts post poly(I:C) simulation. As a result, CiLGP2 suppressed the RLR-mediated signaling pathway against poly(I:C). Furthermore, CiLGP2 played active roles in RLR signaling response to bacterial PAMPs (LPS and PGN) stimulation. CiLGP2 altered the expression pattern of CiIPS-1 after LPS treatment, while it significantly enhanced the RLR signaling pathway against PGN stimulation. These results collectively suggested that CiLGP2 played a strikingly broad regulation in RLR mediated innate immune responses in C.u2009idella, responding to not only the dsRNA virus or synthetic dsRNA but also bacterial PAMPs, which contribute to the understanding of C.u2009idella LGP2 and RLR signaling pathways. In addition, these results lay a foundation for the further functional mechanism research of LGP2 in fishes.

Identification, characterization and immunological response analysis of stimulator of interferon gene (STING) from grass carp Ctenopharyngodon idella.

Stimulator of interferon gene (STING), an important adapter responsible for RLR pathway, plays a pivotal role in both viral RNA- and DNA-triggered induction of IFNs in mammals. To understand the roles of STING in piscine immune system, STING gene (CiSTING) was identified from grass carp (Ctenopharyngodon idella). The genomic sequence of CiSTING was of 8548 base pairs (bp), including 899 bp 5 flank region, 7 exons and 6 introns. Promoter region was predicted and promoter activity was verified. The CiSTING cDNA was of 1358 bp with an open reading frame of 1185 bp, encoding a polypeptide of 394 amino acids with a signal peptide and three transmembrane motifs in the N-terminal region. mRNA expression of CiSTING was widespread in fifteen tissues investigated, and was up-regulated by GCRV in vivo and in vitro. Meanwhile, the transcription of CiSTING was inhibited at early stage, and then up-regulated at late phase upon poly(I:C) or PGN stimulation in vitro. Interestingly, CiSTING had little impact on LPS in vitro. In CiSTING over-expression cells, CiTBK1, CiIRF3 and CiIRF7 were significantly up-regulated post GCRV or viral/bacterial PAMPs stimulation. In addition, post GCRV or PGN stimulation, the transcription of CiIFN-I was remarkably inhibited while CiMx1 was up-regulated; as for poly(I:C) stimulation, mRNA expressions of CiIFN-I and CiMx1 were inhibited at early stage while enhanced at late phrase; after LPS stimulation, both CiIFN-I and CiMx1 were inhibited. Furthermore, antiviral activity of CiSTING was manifested by the inhibition of GCRV yield. Taken together, these results demonstrated that CiSTING may be involved in board innate immune responses via the TBK1-IRF3/IRF7 cascade, responding to not only dsRNA analogue in an IFN-dependent pathway, but also virus and bacterial PAMPs in an IFN-independent pathway. This study provided novel insights into the essential role of STING in innate immunity.

Cloning and preliminary functional studies of the JAM-A gene in grass carp (Ctenopharyngodon idellus)

Grass carp (Ctenopharyngodon idellus) is a very important aquaculture species in China and other South-East Asian countries; however, disease outbreaks in this species are frequent, resulting in huge economic losses. Grass carp hemorrhage caused by grass carp reovirus (GCRV) is one of the most serious diseases. Junction adhesion molecule A (JAM-A) is the mammalian receptor for reovirus, and has been well studied. However, the JAM-A gene in grass carp has not been studied so far. In this study, we cloned and elucidated the structure of the JAM-A gene in grass carp (GcJAM-A) and then studied its functions during grass carp hemorrhage. GcJAM-A is composed of 10 exons and 9 introns, and its full-length cDNA is 1833 bp long, with an 888 bp open reading frame (ORF) that encodes a 295 amino acid protein. The GcJAM-A protein is predicted to contain a typical transmembrane domain. Maternal expression pattern of GcJAM-A is observed during early embryogenesis, while zygote expression occurs at 8 h after hatching. GcJAM-A is expressed strongly in the gill, liver, intestine and kidney, while it is expressed poorly in the blood, brain, spleen and head kidney. Moreover, lower expression is observed in the gill, liver, intestine, brain, spleen and kidney of 30-month-old individuals, compared with 6-month-old. In a GcJAM-A-knockdown cell line (CIK) infected with GCRV, the expression of genes involved in the interferon and apoptosis pathways was significantly inhibited. These results suggest that GcJAM-A could be a receptor for GCRV. We have therefore managed to characterize the GcJAM-A gene and provide evidence for its role as a receptor for GCRV.

Gene-based polymorphisms, genomic organization of interferon-β promoter stimulator 1 (IPS-1) gene and association study with the natural resistance to grass carp reovirus in grass carp Ctenopharyngodon idella.

Interferon-β promoter stimulator 1 (IPS-1) plays a pivotal role in the production of type I interferon (IFN) and pro-inflammatory cytokines. Though its function in innate immunity was widely studied, its genetic polymorphisms and evolution information were rarely known. Herein, the present study firstly identified and characterized the genomic organization of CiIPS-1 (IPS-1 gene of grass carp, Ctenopharyngodon idella) and its nucleotide polymorphisms. CiIPS-1 consists of 8789 bp, with a 314 bp 5-flanking region, 6 exons and 5 introns. After a challenge experiment, statistical analysis was performed to assess the association between the 17 polymorphisms of CiIPS-1 and the natural resistance of C. idella to grass carp reovirus (GCRV), which revealed that -3741 C/T in the intron 1, 933 C/G in the coding sequence and 2299 G/T in the last intron were significant (P < 0.05). The subsequent secondary challenge test further confirmed that -3741TT group were more resistant than -3741CC group to GCRV infection, while 933GG and 2299GG stocks were respectively more susceptible than 933CC and 2299TT stocks (P < 0.05). In addition, haplotype and polymorphism-polymorphism interaction analyses uncovered four significant haplotypes (P < 0.05) and two notable polymorphisms. These findings may provide basic data for the further functional research of CiIPS-1, and genetic markers for molecular and transgenic breeding of C. idella.

SNP detection of TLR8 gene, association study with susceptibility/resistance to GCRV and regulation on mRNA expression in grass carp, Ctenopharyngodon idella.

Toll-like receptor 8 (TLR8), a prototypical intracellular member of TLR family, is generally linked closely to antiviral innate immune through recognizing viral nucleic acid. In this study, 5-flanking region of Ctenopharyngodon idella TLR8 (CiTLR8), 671bp in length, was amplified and eight SNPs containing one SNP in the intron, three SNPs in the coding region (CDS) and four SNPs in the 3-untranslated region (UTR) were identified and characterized. Of which 4062 A/T was significantly associated with the susceptibility/resistance to GCRV both in genotype and allele (P < 0.05), while 4168 C/T was extremely significantly associated with that (P < 0.01) according to the case (susceptibility)-control (resistance) analysis. Following the verification experiment, further analyses of mRNA expression, linkage disequilibrium (LD), haplotype and microRNA (miRNA) target site indicated that 4062 A/T and 4168 C/T in 3-UTR might affect the miRNA regulation, while the exertion of antiviral effects of 4062 A/T might rely on its interaction with other SNPs. Additionally, the high-density of SNPs in 3-UTR might reflect the specific biological functions of 3-UTR. And also, the mutation of 747 A/G in intron changing the potential transcriptional factor-binding sites (TFBS) nearby might affect the expression of CiTLR8 transcriptionally or post-transcriptionally. Moreover, as predicted, the A/G transition of the only non-synonymous SNP (3846 A/G) in CDS causing threonine/alanine variation, could shorten the length of the α-helix and ultimately affect the integrity of the Toll-IL-1 receptor (TIR) domain. The functional mechanism of 3846 A/G might also involve a threonine phosphorylation signaling. This study may broaden the knowledge of TLR polymorphisms, lay the foundation for further functional research of CiTLR8 and provide potential markers as well as theoretical basis for resistance molecular breeding of grass carp against GCRV.

Grass carp SARM1 and its two splice variants negatively regulate IFN-I response and promote cell death upon GCRV infection at different subcellular locations.

Sterile alpha and Toll/IL-1R motif containing 1 (SARM1) negatively regulates TRIF-dependent TLR signaling in mammals. However, its immune function remains unclear in teleost. Here, a grass carp Ctenopharyngodon idella SARM1 (CiSARM1) gene and its two novel splice variants (CiSARM1s1 and CiSARM1s2) were identified. CiSARM1s1 and CiSARM1s2 are generated by intron retention mechanism, and they only retain N-terminal HEAT/armadillo motifs. In C. idella kidney (CIK) cells, CiSARM1 and CiSARM1s1 are located in mitochondria, whereas CiSARM1s2 distributes in the whole cell. All the three transcripts are ubiquitously expressed in 15 investigated tissues. They were responsive to GCRV in vivo and in vitro and to viral/bacterial PAMPs in vitro, implying they participate in both antiviral and antibacterial immune responses. By overexpression experiment, CiSARM1 and its two isoforms affected each others expression in CIK cells. CiSARM1 inhibited GCRV-triggered IFN-I response by affecting the expressions of CiTRIF, CiMyD88, CiIPS-1, CiTRAF6, CiTBK1, CiIRF3 and CiIRF7 in TRIF-, MyD88- and IPS-1-dependent pathways; CiSARM1s1 and CiSARM1s2 inhibited GCRV-triggered IFN-I production through suppressing the expressions of CiMyD88, CiIPS-1, CiTRAF6, CiTBK1, CiIRF3 and CiIRF7 in MyD88- and IPS-1-dependent pathways. Moreover, antiviral activity assays indicated that all the three genes promote GCRV-induced cell death. These results were further verified by RNAi experiments. Thus, CiSARM1 and its two splice variants jointly prevent excessive activation of the host immune response. These findings uncover the regulatory mechanisms of SARM1 in teleost and lay a foundation for further functional and evolutionary researches on SARM1.

CpA/CpG methylation of CiMDA5 possesses tight association with the resistance against GCRV and negatively regulates mRNA expression in grass carp, Ctenopharyngodon idella

Melanoma differentiation-associated gene 5 (MDA5) plays a crucial role in recognizing intracellular viral infection, activating the interferon regulatory factor pathways as well as inducing antiviral response. While the antiviral regulatory mechanism of MDA5 remains unclear. In the present study, CiMDA5 (Ctenopharyngodon idella MDA5) against grass carp reovirus (GCRV) would be initially revealed from the perspective of DNA methylation, a pivotal epigenetic modification. Two CpG islands (CGIs) were predicted located in the first exon of CiMDA5, of which the first CpG island was 427 bp in length possessed 29 candidate CpG loci and 34 CpA loci, and the second one was 130 bp in length involving 7 CpG loci as well as 10 CpA loci. By bisulfite sequencing PCR (BSP), the methylation statuses were detected in spleen of 70 individuals divided into resistant/susceptible groups post challenge experiment, and the resistance-association analysis was performed with Chi-square test. Quantitative real-time RT-PCR (qRT-PCR) was carried out to explore the relationship between DNA methylation and gene expression in CiMDA5. Results indicated that the methylation levels of CpA/CpG sites at +200, +202, +204, +207 nt, which consisted of a putative densely methylated element (DME), were significantly higher in the susceptible group than those in the resistant group. Meanwhile, the average transcription of CiMDA5 was down-regulated in the susceptible individuals compared with the resistant individuals. Evidently, the DNA methylation may be the negative modulator of CiMDA5 antiviral expression. Collectively, the methylation levels of CiMDA5 demonstrated the tight association with the resistance against GCRV and the negative-regulated roles in mRNA expression. This study first discovered the resistance-associated gene modulated by DNA methylation in teleost, preliminary revealed the underlying regulatory mechanism of CiMDA5 transcription against GCRV as well as laid a theoretical foundation on molecular nosogenesis of hemorrhagic diseases in C. idella.

Two novel homologs of high mobility group box 3 gene in grass carp (Ctenopharyngodon idella): Potential roles in innate immune responses

High mobility group box 3 (HMGB3) protein is a universal sentinel in the activation of innate antiviral immune responses in mammalian cells of limited tissues. However, the underlying immune functions of HMGB3 responding to viruses and viral/bacterial pathogen-associated molecular patterns (PAMPs) are still unknown in teleosts. In the present study, two novel homologs of grass carp (Ctenopharyngodon idella) HMGB3 (designated as CiHMGB3a and CiHMGB3b) were identified and characterized. Quantitative RT-PCR analysis showed that CiHMGB3a and CiHMGB3b were widely expressed in tissues. The mRNA expressions of CiHMGB3a and CiHMGB3b were induced by grass carp reovirus (GCRV) challenges both in tissues and in cells, and CiHMGB3a played a more active role in antiviral immune responses. Viral PAMP stimulation evidenced that CiHMGB3a and CiHMGB3b mediated immune responses in CIK (C.xa0idella kidney) cells. Interestingly, CiHMGB3a had little impact on bacterial PAMPs (LPS and PGN), whereas CiHMGB3b was critical responding to bacterial PAMPs stimulation. In overexpressions of CiHMGB3a and CiHMGB3b cells, the transcriptional levels of CiHMGB3a, CiHMGB3b, CiTRIF, CiIPS-1, CiIFN-I and CiMx1 were remarkably induced. In addition, CiMyD88 had vital impact on antiviral signaling channels in overexpression of CiHMGB3b cells. Furthermore, 96-well plate staining assay, virus titer test and GCRV quantitative analysis collectively indicated CiHMGB3a and CiHMGB3b exhibited substantial antiviral activity. These results suggest that CiHMGB3a and CiHMGB3b exert important functions in antiviral immune responses by TLRs and RLRs signaling pathways. Taken together, current study provides the first evidence that HMGB3 participates in broad antiviral and antibacterial immune responses in teleosts.