Fish & Shellfish Immunology 42 (2015) 249e255

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Promoter analysis and transcriptional regulation of a Gig2 gene in grass carp (Ctenopharyngodon idella) Huarong Chen 1, Changgui Sun 1, Wenqun Liu, Meihui Gu, Gang Lin, Yong Liu, Yichuan Mi, Lihua Fan, Binhua Wang, Chengyu Hu* Department of Bioscience, College of Life Science, Nanchang University, Nanchang 330031, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 22 September 2014 Received in revised form 2 November 2014 Accepted 7 November 2014 Available online 15 November 2014

Grass carp reovirus (GCRV)-induced gene 2 (Gig2) is recognized as a new antiviral factor involved in response to viral infection. However, little is known about the mechanisms behind the transcriptional regulation of Gig2 when infected by virus. In this study, the upstream promoter region of grass carp (Ctenopharyngodon idella) Gig2 gene (CiGig2) was identified by homology cloning strategy. CiGig2 promoter sequence was found to be 859 bp in length and contained three scattered IFN-stimulated response elements (ISRE). In addition, some grass carp IRFs (CiIRF1, CiIRF2 and CiIRF3) ORF sequences were subcloned into the expression plasmids pET-32a and expressed in Escherichia coli BL21, then the expressed proteins were purified by affinity chromatography with the Ni-NTA His-Bind Resin. Gel mobility shift assay was employed to screen the transcriptional regulatory factor for CiGig2. The results revealed that the recombinant polypeptides of CiIRF1, CiIRF2 and CiIRF3 bound to CiGig2 promoter with high affinity; indicating that IRF1, IRF2 and IRF3 could be the potential transcriptional regulatory factors for Gig2. Subsequently, CiGig2 promoter sequence was cloned into pGL3-Basic vector and the ORFs of CiIRF1, CiIRF2 and CiIRF3 were cloned into the expression plasmids pcDNA3.1 (þ). Then, pGL3-CiGig2 promoter sequence and pcDNA3.1-CiIRFs were co-transfected into C. idella kidney (CIK) cells. The in vivo effects of CiIRFs on CiGig2 promoter were measured by dual-luciferase assays in the transfected CIK cells. Our results showed that the roles of CiIRFs were diversified in regulating CiGig2 transcription, e.g., CiIRF3 played a positive role in during this process; on the contrary CiIRF1 worked as a suppressor; however the effect of CiIRF2 on CiGig2 transcription was not obvious. For further study the roles of the three ISREs in CiGig2 transcription, we cloned three mutant CiGig2 promoters called ISRE1mut-luc (deleted ISRE1), ISRE2mut-luc (deleted ISRE2) and ISRE3mut-luc (deleted ISRE3), respectively. In vitro, gel mobility shift assays showed that all three mutant promoters also were combined with CiIRFs. CIK cells were cotransfected with CiGig2 promoter mutants (ISRE1mut-luc, ISRE2mut-luc or ISRE3mut-luc, respectively) and pcDNA3.1-IRFs. The results suggested that different ISRE played the diverse roles. ISRE2 is more important than ISRE1 and ISRE3 to the transcription of CiGig2 induced by CiIRF1. ISRE1 and ISRE3 are important to the transcription of CiGig2 induced by CiIRF2 and CiIRF3. © 2014 Elsevier Ltd. All rights reserved.

Keywords: Gig2 ISRE Transcription regulation IRF Innate immunity

1. Introduction The cytokines are secreted or membrane-bound proteins and involved in virtually every aspect of immunity and inflammation, including innate immunity, antigen presentation, bone marrow differentiation, cellular recruitment and activation, and adhesion

* Corresponding author. Tel.: þ86 791 8831 7270; fax: þ86 791 8396 9530. E-mail address: [email protected] (C. Hu). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.fsi.2014.11.008 1050-4648/© 2014 Elsevier Ltd. All rights reserved.

molecule expression [1]. The field of cytokine has undergone a quiet but vigorous expansion in the last decade [2]. Cytokines cover a wide range of branching, including tumor necrosis factor (TNF), interferon (IFN), interleukin (IL), colony stimulating factor (CSF), transforming growth factor-b family (TGF-b family), growth factor (GF) [3]. For the past few years many new cytokines have been continuously reported in different animal species. In recent years, significant progress in fish innate immune system has been made and many cytokines have been discovered [4e6]. In general, besides a series of cytokines similar to the counterparts of mammals, fish also has some unique innate

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prokaryotic expression vector pET-32a (þ) were purchased from Invitrogen and Novagen respectively. During the process of cell culture, cells were washed with PBS and digested by 1 ml 0.25% trypsin for 3e5 min, afterwards, the trypsin digestion liquid was discarded and the cells were separated and suspended in 5 ml culture medium. After cells were resuspended, 2e3 ml of medium was transferred into a new culture flask and the medium was supplemented to a total volume of 5 ml.

immune factors, or anti-virus mechanism due to its aquatic environment [7]. Gig2 had been identified from UV-inactivated GCHVtreated Carassius auratus blastulae embryonic (CAB) cells by suppressive substractive hybridization [8] and identified as a novel gene specific to non-amniote vertebrates [9]. Gig2 transcript is highly up-regulated under the stimulation of GCHV or IRF7, indicating Gig2 plays a pivotal antiviral role [9,10]. In order to further understand the transcriptional regulatory mechanism of Gig2 in response to virus infection in fish cells, we cloned Gig2 promoter (KC196275) from grass carp (Ctenopharyngodon idella). The full-length of CiGig2 promoter sequence is 859 bp containing three putative ISRE. IRF1, IRF2 and IRF3 could be the potential transcriptional factors for CiGig2 by Non-radioactive EMSA. Co-transfected experiment indicated that IRF1 and IRF3 really regulate the transcription of CiGig2. CiIRF1 evidently decreased the transcriptional level of CiGig2; CiIRF3 is just the opposite. CiIRF2 could not regulate its transcription. The experimental results from ISRE mutations in CiGig2 promoter showed that ISRE play a different role in the transcription of CiGig2. ISRE2 is more important to the transcription of CiGig2 than ISRE1 and ISRE3 when induced by CiIRF1. ISRE1 and ISRE3 are important to the transcription of CiGig2 by CiIRF2 and CiIRF3.

Homologous primers Gig2-Pro-F and Gig2-Pro-R, designed according to the known C. auratus genomic DNA of CaGig2 (GQ181131) sequence, were used to clone grass carp Gig2 promoter sequences. Genomic DNA was purified from whole blood of obviously healthy grass carp using a Universal Genomic DNA Extraction Kit (TaKaRa). PCR cycling conditions were: 1 cycle of 94  C/5 min; 30 cycles of 94  C/30 s, 61  C/30 s, 72  C/30 s; and 1 cycle of 72  C/ 10 min. Overlapping PCR was used to clone ISRE1mut, ISRE2mut and ISRE3mut sequences (Fig. 5A). PCR cycling conditions were the same as above. The primers used in clone were listed in Table 1.

2. Materials and methods

2.3. Protein expression and purification

2.1. Cells, vectors

Grass carp IRF1, IRF2 and IRF3 coding sequences and their corresponding DBD-deleted sequences were digested with EcoR I/Xho I and subcloned into the EcoR I/Xho I site of pET32a (þ) expression vector. Recombinant plasmid pET32a/CiIRF1-ORF, pET32a/CiIRF2ORF, pET32a/CiIRF3-ORF and pET32a/CiIRF1-nDBD, pET32a/ CiIRF2-nDBD, pET32a/CiIRF3-nDBD were sequenced and transformed into competent E. coli BL21 cells respectively. Cells were cultured in incubator shaker (ZHWY-200H) at 37  C until the OD600

C. idellus kidney (CIK) cells were kindly provided by Professor Pin Nie, Institute of Hydrobiology, Chinese Academy of Sciences and maintained at 28  C in medium 199 supplemented with 10% fetal calf serum (FCS). Cells were seeded in 25 cm2 flasks. pGL3-basic, Escherichia coli strains DH5a and BL21 (DE3) pLys were bought from Promega. The eukaryotic expression vector pcDNA3.1 (þ) and the

2.2. Cloning of CiGig2 promoter and its mutants

Fig. 1. Promoter sequence of CiGig2 and their potential functional components. The promoter region and 50 UTR are shown in capital letters. The 50 UTR intron region is indicated with lower case italics. The three ISRE sites, TATA box are shaded in grey. The transcription start site of Gig2 gene is designated as position þ1. The start code of the ORF and the putative transcription start site are indicated with boxes.

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Fig. 2. CiIRFs and CiIRFs-nDBD fusion peptides were analyzed by 12 % PAGE M: protein molecular weight marker (Fermentas). Lane 1: non-induced; lane 2: IPTG induced; lane 3: purified of IRF1 (50 kD), IRF1-nDBD (37 kD), IRF2 (54 kD), IRF2enDBD (40 kD), IRF3 (69 kD), and IRF3-nDBD (43 kD) fusion peptides.

Fig. 3. Screening the transcriptional regulatory factor for CiGig2. Grass carp Gig2 promoter and the mutants were incubated with CiIRF1, CiIRF2 and CiIRF3 (shown in lanes 2, 3, 4. respectively). Shifted complexes are shown in lanes 2, 3 and 4. Negative control is shown in lane 1. Positive control is shown in lanes 5, 6, 7. M: denotes protein molecular weight marker (TaKaRa).

reaching 0.6e0.8 and then added 1 mM IPTG for 4 h. Later, the cells were harvested, centrifuged, resuspended by binding buffer (20 mM Trise HCl, 500 mM NaCl, 5 mM imidazole, pH 7.9) completely. The suspensions were broken by sonication and centrifuged for 15 min (4  C, 12,000 rpm). Then the supernatant was purified by affinity chromatography with Ni-NTA resin (Novagen). The purified recombinant proteins were electrophoresed on a 12% SDS-PAGE gel. The primers used in the test were listed in Table 1. 2.4. Screening the transcriptional regulatory factor for CiGig2 by gel mobility shift assays Non-radioactive EMSA was utilized in scanning transcriptional regulatory factors for CiGig2. The concrete methods are as follows: the 10 ml reaction system for gel mobility shift assays contained

about 20 ng promoter sequences and about 1 mg of purified CiIRF polypeptides; DNA-protein complexes were incubated on ice for 45 min, and then analyzed on a 1% agarose gel. After that, the gels were stained with ethidiumbromide (0.5 mg/ml) and photographed. 2.5. Transfection and luciferase activity assay A luciferase reporter gene assay was applied to determine the impact of CiIRFs on CiGig2 promoter. IRF1, IRF2 and IRF3 -ORF sequences were amplified and subcloned into the EcoR I/Xho I sites of the expression vector pcDNA3.1 (þ). Gig2 pro-Luc was obtained with insertion of 50 flanking region (þ43/859) into MIu I/Xho I sites of pGL3-Basic luciferase reporter vector. ISRE1mut, ISRE2mut and ISRE3mut were cloned into pGL3-Basic luciferase reporter vector in the same sites as Gig2 pro-Luc. All the recombinant

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three parallel wells (each with 25 ml mixtures). After 24 h posttransfection, the cells were lysed and collected. Luciferase activities of all samples were measured using the Luciferase Assay System (Promega) according to the manufacturer's instructions. The luciferase activity was measured on a fluorescence spectrophotometer (Hitachi). The means of triplicates were representative of more than three independent experiments and were subjected to Student's t-test. Differences were considered significant at p < 0.05 and highly significant at p < 0.01. 3. Results 3.1. The organization of CiGig2 promoter Fig. 4. Impact of CiIRFs on CiGig2 promoter. CIK cells were transiently transfected with pGL3-basic or Gig2pro-luc, or co-transfected with Gig2pro-luc and pcDNA3.1 or pcDNA3.1-CiIRFs (namely pcDNA3.1-CiIRF1, pcDNA3.1-CiIRF2 and pcDNA3.1-CiIRF3) in 24 - well plates, respectively. Luciferase activities were measured after 24 h posttransfection using dual-luciferase reporter assay system. The mean of three replicates are shown with S.D. The level of statistical significance in comparison with pGL3Basic or pGL3-CiGig2 & pcDNA3.1 is indicated. Asterisks indicate a significant difference between the experimental group and control group (*p < 0.05, **p < 0.01).

plasmids were transformed into E. coli DH5a and sequenced. The bacteria containing the correct plasmids would be used to extract the plasmids without endotoxin. For the transient transfection assays, 4  104 cells per well were seeded in 24-well plates and maintained at 28  C. Transfection experiments were carried out until the cells reached 90e95 % confluence. Before transfection, the 1.7 mg plasmids firstly incubated with 75 ml medium for 5 min. Then the mixture incubated with Fugene 6.0 for the other 5 min. The ratio of pRL-TK to the total expression construct was 10: 1. The final mixture was added to

To further investigate the transcriptional regulation of CiGig2, the homology cloning was carried out to clone the upstream promoter region of CiGig2 according to the promoter sequence of CaGig2 (GQ181131). The length of CiGig2 promoter is 859 bp. Sequence analysis showed that the promoter region has three putative ISRE (designated as ISRE1, ISRE2 and ISRE3 respectively) (Fig. 1). ISRE1 and ISRE2 are close to each other and locate at the position from 306 to 294 and 328 to 316 respectively. The third ISRE motif (ISRE3) is found at terminal position from 619 to  605. Different with the proximal ISRE1 and ISRE2, the distal ISRE3 has two GAAA sequences interspaced with three instead of one nucleotide. 3.2. Expression and purification of recombinant CiIRFs and their CiIRFs-nDBD The constructed plasmids pET-32a/CiIRFs and their CiIRFs-nDBD were transformed into E. coli BL21 (DE3). After induction with

Fig. 5. ISRE element is an important component for the transcription of CiGig2. (A) Schematic of Gig2pro-luc, ISRE1mut-luc, ISRE2mut-luc and ISRE3mut-luc. The four fragments were identical through sequencing. The Gig2pro and the mutants were cloned in front of luciferase reporter gene. (B) CIK cells were transiently co-transfected with pGL3CiISRE1/2/3mut and pcDNA3.1-CiIRF1/2/3 in 24-well plates, respectively. 24 h later, cells were harvested and the luciferase activities were measured using the Dual-Luciferase Reporter Assay System. The mean of three replicates is shown with ±S.D. The level of statistical significance between control (pGL3-CiGig2-pro & pcDNA3.1-IRFx) and treatment groups are determined by t-test (*p < 0.05; **p < 0.01).

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Table 1 Primers used for all the experiment. Primer name

Primer sequence (50 /30 )

Usage

Gig2-Pro-F Gig2-Pro-R Gig2-Pro-F1 Gig2-Pro-R1 ISRE1mut-F ISRE1mut-R ISRE2mut-F ISRE2mut-R ISRE3mut-F ISRE3mut-R IRF1-ORF-F IRF1-ORF-R IRF1-nDBD-F IRF2-ORF-F IRF2-ORF-R IRF2-nDBD-F IRF2-nDBD-R IRF3-ORF-F IRF3-ORF-R IRF3-nDBD-F

TTATCAGTGGCACAACAGTTTTCTC TGCCTTACCTCTGAGTTTGCTTCTA CGACGCGTTTATCAGTGGCACAACAGTTTTCTCTTTGCGCTC CGCTCGAGAAAGACAAAGTGGTGCCTTACCTCTGATGTTGC GCGTTTTTCTCACAAAGCTAAGTTTCATTTCTGCTG CTTAGCTTTGTGAGAAAAACGCACATGAATTTC GGAAACAGTCCTCTACAGTAGCTTTGTTTGAAATG CAAACAAAGCTACTGTAGAGGACTGTTTCCTGT ACCCAGAACCTAAAGTTCTACCTCTAGATC GAGGTAGAACTTTAGGTTCTGGGTTTTAATCCGC CGGAATTCATGCCTGTGTCCAGAATGC CGCTCGAGTCAGAGAGGACACATGGTCG CGGAATTCAAGAGACCCAAGGGTCGTG CGGAATTCATGCCGGTAGAGAGAATGCGTA CGCTCGAGTCTGAGGTTTTCATGATGACGCT CGGAATTCAAGTTAAAGGTGGTGCGAGCG CGCTCGAGGGTGTCATCATCAGCAGCTCTTG CGGAATTCATGACCCATCCAAAACCG CGCTCGAGTCACTTGGTGTCACACAACTC CGGAATTCCATCATTCTCCAGCATATCAG

Promoter cloning Construction of promoter plasmid

Eukaryotic/Prokaryotic vector construction

Nucleotides shown in underline indicate restriction sites.

1.0 mM IPTG, all of the cell lysates (non-induced, induced) and the purified proteins were subjected to SDS-PAGE analysis. The protein sizes are 50 kD (CiIRF1), 54 kD (CiIRF2), 69 kD (CiIRF3), 37 kD (CiIRF1-nDBD), 40 kD (CiIRF2-nDBD) and 43 kD (CiIRF3-nDBD), respectively (Fig. 2, lane 3). These sizes are reasonably close to the theoretical molecular mass of recombinant fusion protein. The 6  histidine-tagged recombinant proteins were purified by the Novagen Ni2 þ affinity chromatography system as described by the manufacturer. The concentration of the purified CiIRF1 (950 mg/ml), CiIRF2 (1.5 mg/ml), CiIRF3 (900 mg/ml), CiIRF1-nDBD (854 mg/ml), CiIRF2-nDBD (1.23 mg/ml) and CiIRF3-nDBD (862 mg/ml) were determined using the Bradford reagent.

One of three IRFs (pcDNA3.1-CiIRF1, pcDNA3.1-CiIRF2 and pcDNA3.1-CiIRF3) each time was co-tranfected with ISRE1mut-luc, ISRE2mut-luc and ISRE3mut-luc into CIK cells, respectively. As is shown in Fig. 5B, the luciferase activity of ISRE1mut-luc, ISRE2mutluc and ISRE3mut-luc were up-regulated by CiIRF1 compared to the control. Specially, CiIRF1 increased the luciferase activity obviously in ISRE2mut-luc group. So, ISRE2 is important to the transcription of CiGig2 induced by CiIRF1. However, the luciferase activity of ISRE1mut-luc and ISRE3mut-luc changed significantly when induced by CiIRF2 and CiIRF3, suggesting ISRE1 and ISRE3 are essential to the transcription of CiGig2 induced by CiIRF2 and CiIRF3.

3.3. Affinity of CiIRFs with CiGig2 promoters

4. Discussion

Non-radioactive EMSA was employed to analyze the interaction between CiIRFs and CiGig2 promoter sequence, as presented in lanes 2, 3 and 4 (Fig. 3A). This result indicated that they were the potential transcriptional factors for CiGig2. IRF-nDBD did not hamper the shift of CiGig2 promoter in gel (lanes 5, 6, 7), hence DBD domain of IRF plays an important role in the combination of CiGig2 promoter sequence and CiIRFs. In further experiments, we studied the affinity of IRFs with three CiGig2 promoter mutants (Fig. 3B, C and D), it showed that CiIRFs still hampered the shift of three CiGig2 promoter mutants in agarose gel.

Scientists used different methods, e.g., yeast one-hybrid, phage display technique, EMSA, to study the interaction between DNA and protein. Although these methods are sensitive to analyze the interaction of proteineDNA, there are some discommodiousness. For example, the integrated reporter constructs is cumbersome in yeast one-hybrid [11]. The phage display technique needs to insert the protein sequences and DNA sequences to phage [12,13]. The conventional EMSA technique should deal with hazardous 32P-labeled DNA probes [14]. Nonconventional (non-radioactive) EMSA was successfully developed in recent years [15,16]. It only needs to mix the definite concentration of protein and DNA and analyze by agarose gels [17]. Therefore, in this paper non-conventional EMSA was carried out to scan the potential transcriptional regulatory factor for Gig2 in vitro. IRF is a class of transcriptional mediator of virus-, bacteria- and IFN-induced signaling pathways and as such play critical roles in antiviral defense, immune response, cell growth regulation and apoptosis [18]. For now, many kinds of IRFs have been indentified not just in mammals but in fish. IRFs play various roles in regulating IFN and ISG transcription. For example, IRF1 can positively regulate the transcription of type I IFN gene both in grass carp and mice [19,20]. IRF2 is just the opposite in mice [21]. AsIRF2 showed a rather interesting activation curve, activating the atlantic salmon IFNa1 promoter at low plasmid concentrations and inhibition at high [22]. Overexpression of mouse IRF3 causes a marked increase in virus-induced IFN-b mRNA [23]. Gold fish IRF3 has a similar

3.4. Cell transfection and luciferase activity analysis The plasmid pcDNA3.1-CiIRF1, pcDNA3.1-CiIRF2 and pcDNA3.1CiIRF3 were co-transfeted with CiGig2 pro-Luc into CIK cells respectively. Luciferase activities were detected in cell lysates 24 h post-transfection. As is shown in Fig. 4, the co-tranfection of pcDNA3.1-CiIRF1 and CiGig2 pro-Luc significantly decreased the luciferase activity compared with the control group. The change of luciferase activity was unconspicuous in cells co-transfected pcDNA3.1-CiIRF2 and CiGig2 pro-Luc. However, co-transfection of pcDNA3.1-CiIRF3 and Gig2pro-Luc into cells yielded highly luciferase activity, being 2 -fold over the control. ISRE is essential for the transcriptional activation of corresponding gene. Three deletion mutations of CiGig2 promoter (ISRE1mut-luc, ISRE2mut-luc and ISRE3mut-luc) were constructed to clarify which ISRE is necessary for the transcription of CiGig2.

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capability, displaying strong ability to trigger IFN transcription [24,25]. Interferon (IFN) and interferon-stimulated genes (ISG), such as Mx [26e29], IRF [30,31], major histocompatibility complex (MHC) class I genes [32,33], vig-2 [34] and ISG15, have typical ISRE motifs within their promoter [35]. Interestingly, many non-IFN system genes also have ISRE in their promoter. For example, both of HIV-1 LTR and murine IL-12p40 promoter carry an ISRE-like element [36,37]. Human CXCL10 promoter also has ISRE [38]. Structurally, all IRF members share extensive homology in Nterminal DNA binding domain (DBD). The DBD forms a helix-turnhelix motif and recognizes the IRF-E/ISRE. IRF can regulate the transcription of gene with ISRE in their promoter regardless of whether it belongs to ISG. For instance, IRF1 stimulates the transcription of HIV-1 LTR through ISRE-like element both in the presence and absence of Tat [36]. ICSBP and IRF1 synergistically stimulate murine IL-12p40 promoter activity through this novel site that resembles ISRE [37]. IRF1, IRF2, IRF3, IRF7 also regulate the transcription of CXCL10 via ISRE [38]. Through binding to ISRE/IRFE, IRF either up-regulate or down-regulate the transcription of the corresponding ISRE-associated genes [22]. In this paper, in vitro experiments showed CiIRF1, CiIRF2, CiIRF3 bound to CiGig2 promoter with high affinity through its DBD (Fig. 3A). In vivo experiments suggested that CiIRF1 really reduced the transcription of CiGig2; while CiIRF3 increased it (Fig. 4). CiIRF2 did not affect the transcription of CiGig2. The human MxA promoter contains 3 ISRE (ISRE1, ISRE2 and ISRE3). Among them, ISRE1 and ISRE2 are functional [39,40]. The MxA ISRE1 appears to be essential for IFN-a-induced transcription through binding to IRF3 complex, whereas the ISRE2 appears to greatly enhance the overall inducibility of MxA promoter [39]. ISRE mutation in gold fish IFN promoter revealed that two ISRE sites (ISRE1 and ISRE2), particularly ISRE2, serve to control IRF3dependent induction of fish IFN transcription [24]. In the same manner, the mutation of ISRE2 within the region (14 to þ66) of TRIM22 promoter abolishes IFN-g inducibility, indicating that ISRE2 is crucial for IFN-geinduced transcriptional activity via interaction with IRF-1 in human [41]. In our experiment, the functions of different ISREs were tremendously complex. ISRE at different position of promoter performed different roles in CiGig2 transcription when induced by different IRF (Fig. 5B). Of couse, the relationship between ISRE and gene transcription need to be explored further. Acknowledgments This work was supported by research grants from the National Natural Science Foundation of China (31360515), the Science & Technology Project of Jiangxi Province of China (20111BBF60020) and the earmarked fund for Jiangxi Agriculture Research System, the earmarked fund for Jiangxi Agriculture Research System (JXARS-04) and the Science & Technology Foundations of Education Department of Jiangxi (KJLD14002). References [1] Borish LC, Steinke JW. 2. Cytokines and chemokines. J Allergy Clin Immunol 2003;111(2):460e75. [2] Perez OA, Berman B. Cytokines and chemokines. Clin Basic Immunodermatol 2008:3e16. [3] Chung KF. Cytokines in chronic obstructive pulmonary disease. Eur Respir J 2001;18(34 Suppl.):50e9. [4] Savan R, Sakai M. Genomics of fish cytokines. Comp Biochem Phys D 2005;1(1):89e101. [5] Zhang YB, Gui JF. Molecular regulation of interferon antiviral response in fish. Dev Comp Immunol 2012;38:193e202. [6] Gui JF, Zhu ZY. Molecular basis and genetic improvement of economically important traits in aquaculture animals. Chin Sci Bull 2012;57:1751e60.

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Promoter analysis and transcriptional regulation of a Gig2 gene in grass carp (Ctenopharyngodon idella).

Grass carp reovirus (GCRV)-induced gene 2 (Gig2) is recognized as a new antiviral factor involved in response to viral infection. However, little is k...
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