Genomic structure, expression pattern and polymorphisms of GILT in golden pompano Trachinotus ovatus (Linnaeus 1758)

Accepted Manuscript Genomic structure, expression pattern and polymorphisms of GILT in golden pompano Trachinotus ovatus (Linnaeus 1758)

Kecheng Zhu, Wenbo Yu, Huayang Guo, Nan Zhang, Liang Guo, Baosuo Liu, Shigui Jiang, Dianchang Zhang PII: DOI: Reference:

S0378-1119(18)30463-3 doi:10.1016/j.gene.2018.04.081 GENE 42805

To appear in:

Gene

Received date: Revised date: Accepted date:

7 October 2017 7 April 2018 26 April 2018

Please cite this article as: Kecheng Zhu, Wenbo Yu, Huayang Guo, Nan Zhang, Liang Guo, Baosuo Liu, Shigui Jiang, Dianchang Zhang , Genomic structure, expression pattern and polymorphisms of GILT in golden pompano Trachinotus ovatus (Linnaeus 1758). The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Gene(2017), doi:10.1016/j.gene.2018.04.081

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ACCEPTED MANUSCRIPT Genomic structure, expression pattern and polymorphisms of GILT in golden pompano Trachinotus ovatus (Linnaeus 1758) Kecheng Zhu

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, Wenbo Yu a, Huayang Guo

a,b,c

, Nan Zhang a,b,c, Liang

Guo a,b,c, Baosuo Liu a,b,c, Shigui Jiang a,b,c , Dianchang Zhang a,b,c * Key Laboratory of South China Sea Fishery Resources Exploitation and Utilization,

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a

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Ministry of Agriculture; South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, 510300, Guangzhou, Guangdong Province, The

Guangdong Provincial Engineer Technology Research Center of Marine Biological

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b

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People’s Republic of China;

Seed Industry, Guangzhou, Guangdong Province, The People’s Republic of China; Guangdong Provincial Key Laboratory of Fishery Ecology and Environment,

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c

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Guangzhou, Guangdong Province, The People’s Republic of China.

*Corresponding author: Dr. Dianchang. Zhang 231 Xingang Road West, Haizhu District, Guangzhou City, Guangdong 510300, PR China. E-mail address: [email protected] Tel.: +86 02089108316; fax: +86 02089022702

ACCEPTED MANUSCRIPT ABSTRACT: The interferon-g-inducible lysosomal thiol reductase (GILT) plays a significant character in the processing and presentation of MHC class II restricted antigen (Ag) by catalyzing disulfide bond reduction in mammals. To explore the function of GILT in the immune system of fish, we cloned a GILT gene homologue from Trachinotus ovatus, the full-length cDNA of GILT, which consisted of 2, 747 bp with a 771 bp

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open reading frame, encoding a protein of 256 amino acids. Moreover, similar to other species GILT gene, 7 exons and 6 introns were identified in T. ovatus, the

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deduced protein also possessed a representative characteristic of known GILT proteins. The result of real-time quantitative PCR showed that GILT mRNA was

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dramatically expressed in immune-associated tissues, such as spleen (p < 0.01) and kidney (p < 0.05). Bacterial challenge revealed that GILT mRNA level remarkably

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up-regulation in liver, spleen, kidney and intestine after induction with Photobacterium damsela. Furthermore, based on cloned sequences and genome

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BLAST, only one SNP site (ToGILT-S1-g.148C>G) was identified, and the allele C was significantly associated with high-susceptibility (HS) group, nevertheless, the allele G was dramatically associated with high-resistance (HR) group, indicating

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potential application for disease resistant breeding selection in T. ovatus.

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Abbreviations

GILT, interferon-g-inducible lysosomal thiol reductase; Ag, antigen; HS, highsusceptibility; HR, high-resistance; M6PR, mannose-6-phosphate receptor; APCs,

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antigen-presenting cells; MAS, molecular assisted selection; UPGMA, unweighted

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pair-group method with arithmetic; ORF, Open Reading Frame;

Keywords: Trachinotus ovatus; GILT; SNP site; Gene expression; Bacterial challenge.

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1. Introduction Antigen (Ag) processing plays an important role in activating specific T-cell

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immune responses. The decrease of disulfide bonds in exogenous antigens is a critical step among the MHC class II Ag processing pathway in which a IFN-γ-inducible

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lysosomal thiol reductase (GILT) has been involved (Collins et al. 1991; Watts, 1997).

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GILT, originally is described as IP30, is a novel gamma-interferon derivable glycoprotein which is transmitted to the endosomal/lysosomal system of the mannose-

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6-phosphate receptor (M6PR) in Homo sapiens (Arunachalam et al., 2000; Lackman et al., 2007). It is synthesized as a 35 kDa precursor enzyme, which constituted 261 amino acids with a 37 amino acid signal peptide. GILT proteins contained four

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representative features such as signature CQHGX2ECX2NX2EXC sequence, the active site CXXC motif, more than one assumed Asn-linked glycosylation site and 10-11 conserved cysteines.

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In GILT-absence mice, the Ag processing in antigen-presenting cells (APCs) was

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to be deficient (Maric et al., 2001), suggesting it played an important role in the immune system. Subsequently, GILT, had been studied in different species of fish, was conformed to resist pathogens, lipopolysaccharides (LPS) or polyriboinosinic

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polyribocytidylic acid [poly (I: C)]. The expression pattern of GILT gene was found to up-regulate after pathogen challenge in some immuno-related tissues (Liu et al., 2013;

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Song et al., 2014; You et al., 2018). Those findings revealed that fish GILT had crucial role in their antibacterial immune response. The golden pompano T. ovatus (Linnaeus 1758), Carangidae, Perciformes, are broadly cultivated in the Asia-Pacific region. The fish was a delicacy that has been popular with its rapid growth and higher quality rate, and was considered as an important aquaculture species in China (Sun et al., 2014; Zhen et al., 2014). Along with the deterioration of culture environment, T. ovatus was susceptible to several kinds of virus, bacteria, and protozoan pathogens, leading to mortalities and other disadvantages (Su et al., 2012). Thus, T. ovatus with high resistance to diseases need to be developed in aquaculture. Moreover, marker-assisted selection (MAS) had been

ACCEPTED MANUSCRIPT applied in breeding and had supplied lots of useful genetic markers in breeding (Yue 2014). T. ovatus GILT had been identified and had possessed active disease resistance. However, the polymorphisms of GILT are scarcely studied, and relevance between polymorphism and disease resistance/susceptibility was still unknown. Hence, in our study, firstly we identified and analyzed GILT genome DNA sequence from T. ovatus,

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then GILT transcripts in different tissues and in response to P. damselae were assessed, respectively. Moreover, to estimate the relevance of single nucleotide polymorphism (SNP) and disease resistance in T. ovatus, we analyzed the

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polymorphisms of GILT and investigated its possible association with disease

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resistance or susceptibility to P. damselae in T. ovatus population. This study should

2. Materials and methods 2.1 Fish rearing and challenge test

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be useful in MAS of disease-resistant in T. ovatus.

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T. ovatus juvenile fish (body weight: 250 ± 12.60 g) were collected from Linshui Marine Fish Farm in Hainan Province. Samples were maintained in fresh seawater

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supply at 22 ± 1 °C and dissolved oxygen > 6 mg/L under recirculating aquaculture

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system, and were reared with no bacteriumor infection in Tropical Fisheries Research and Development Center, Lingshui City, Hainan Province, China. The fish were fed a commercial diet according to standard feeding scheme two weeks before injection. The experiments contained two groups (control and bacterial challenge group). Each

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group was divided into three replicates. In the challenge experiment, 480 fish were intraperitoneally injected with 100 µL of P. damselae resuspended in phosphate-

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buffered saline (PBS) to 109 CFU/mL; 50 fish as a control group were injected with 100 µL of PBS (Su et al., 2012). After the treatment, all fish were returned to pools and then reared for 20 days. Mortality was recorded daily in each tank, and no fish in the control group died (Zhu et al., 2018). To determine spatio-temporal distribution of GILT, firstly, 200 fish as treatment group were intraperitoneally injected with 109 CFU/mL P. damselae (100 µL), 50 fish as control group were injected with 100 µL PBS. Those fish (both control and treatment) at 0, 1, 3, 6, 12, 24, 48 and 72 h were anesthetized using MS222 (0.1 g L−1; Sigma, Alcobendas, Spain) after infection, then liver, spleen, kidney and intestine

ACCEPTED MANUSCRIPT were gathered from all groups and promptly stored in RNA later (Life Technologies, Waltham, MA, USA) reagent until used. Secondly, tissue samples of healthy adult T. ovatus (n =5) including heart, stomach, brain, eye, gill, fin, skin, muscle, liver, spleen, kidney, intestine and blood were obtained. Total RNA was extracted from tissues using TRIzol reagent (TaKaRa, Dalian, China) according to the manufacturer. 2.2. Sample collection and DNA isolation

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Dead and living individuals were defined as high-susceptibility (HS) and highresistance (HR) group in the first 20 days, respectively. To examine whether GILT

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genome DNA of alleles were associated with HS/HR to P. damselae, fin clips were acquired from all groups of fish and stored in bottle with alcohol.

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According to the manufacturer's instructions, fin samples of both HR and HS families were extracted total genomic DNA using the Ezgene™ tissue gDNA kit

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(Biomiga, USA). DNA integrity was estimated by electrophoresis on 0.8 % agarose gel. Furthermore, the quality and quantity (concentration) of DNA was measured by

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NANODROP 2000 spectrophotometer. The DNA specimens were adjusted to 100 ng/μL and stored at -20 °C. 2.3 Gene structure analyses

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According to genomic data of T. ovatus (Accession No. PRJEB22654 under

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ENA; Sequence Read Archive under BioProject PRJNA406847, unpublished), a fulllength sequence of T. ovatus GILT gene was obtained (GenBank accession No. MF929063). To verify the accuracy of the genome sequence of GILT, gene-specific

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primers were designed (Table 1). The full-length gene sequence of GILT was assembled by SeqMan software. Furthermore, to contrast the gene structure among in

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the T. ovatus, H. sapiens, M. musculus, D. rerio, T. obscurus, L. crocea and T. rubripes, the model of exons and introns were structured. 2.4. Discovery of SNPs and genotyping For SNP discovery, genomic DNA of 117 individuals from the test population were initially used for PCR amplification using the primers ToGILT-SF1-6/ToGILTSR1-6, which amplified all sequences of exons and introns (Table 1). The PCR reactions were performed in total 20 µL volume mixtures containing 50 ng cDNA, 0.3 µM of each primer pair and PCR Master Mix (Toyobo, Osaka, Japan) included dNTP and buffer. The PCR amplification program consisted of an initial denaturation at 95

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C for 5 min, following by 25 cycles of amplification 30 s at 95 oC, 30 s at specific

annealing temperatures, 40 s at 72 oC and final extension for 8 min at 72 oC. To examine for integrity of the PCR products, 0.8 % agarose gel was used. Moreover, imaging was utilized by Molecular Imager Gel Doc XR system (Biorad). PCR products were directly sequenced and were aligned using Clustal X to identify polymorphisms.

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2.5 Bioinformatics analysis The GILT of nucleotide and amino acid sequence similarity searches were

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performed using the BLAST program (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The ORF finder (http://www.ncbi.nlm.nih.gov/projects/gorf/) was used for predicting the

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coding sequence of GILT. The molecular weight, theoretical isoelectric point and features of the protein were obtained referring to the ExPASy analysis system

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(http://us.expasy.org/tools), and the SignaIP 4.1 server was used for signal peptide prediction (http://www.cbs.dtu.dk/services/). Phylogenetic tree was constructed by

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MEGA 6 software using the Maximum Likelihood (ML) method (Tamura et al., 2013).

2.6 Three-dimensional model analyses

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To explore the location of amino acids of SNP loci in GILT, three-dimensional

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analysis of the residue location was performed through structure prediction by similarity modeling as implemented in SWISS-MODEL (http://swissmodel.expasy.org/ ).

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2.7 Gene expression analysis by quantitative real-time PCR (qRT-PCR) The mRNA levels of GILT were analysed by qRT-PCR (Bustin et al., 2009).

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Specific primer pairs for GILT and the reference gene EF-1α (elongation factor 1, alpha) were developed (Table 1). The qRT-PCR was performed in 20 µL total volume containing 10 µL SYBR Green qPCR Master Mix (Toyobo, Osaka, Japan), 0.3 µM of each primer, 5 µL RNase-free H2O, and 2 µL cDNA. The qRT-PCR program consisted of an initial denaturation at 95 oC for 3 min, followed by 40 cycles of amplification 7 s at 95 oC, 10 s at specific annealing temperatures 56 oC, 15 s at 72 oC, and final extension for 10 min at 72 oC in a Light Cycler® 480 II (Roche, Basel, Switzerland). Relative expression was determined using the 2–ΔΔCT method (Livak and Schmittgen, 2001). 2.8 Statistical analysis

ACCEPTED MANUSCRIPT All statistical analyses were performed with the SPSS 13.0 software (IBM, USA). Statistical analysis of allele frequencies between the HR and HS groups were performed by using chi-squared test (X2 test), and the levels of GILT mRNA between control and challenge groups were performed by using Student’s t-test. The results were considered statistically significant at P < 0.05.

3. Results

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3.1 Sequence characteristics

The full-length cDNA of the GILT gene (GenBank accession No. KY963347) is

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2, 747 bp, including 232 bp 5’ UTR, 771 bp encoding region and 1, 744 bp 3’ UTR

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region. The 771 bp Open Reading Frame (ORF) is detected to encode 256 amino acid residues, with a theoretical isoelectric point of 5.79 and a predicted molecular weight

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of 28.6 kDa. Signal peptide and SAP-A homodomain domains are found in sites 1-21 and 19-59, respectively (Fig.1). Moreover, the CXXC motif (CPGC, 74-77), sequence (CQHGX2ECX2NX4C, 120-134) and cysteines are also highly conserved in GILT.

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These results indicated that the T. ovatus GILT protein likely had similar regulatory functions as observed in mammalian counterparts.

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Similar to other species GILT genes, 7 exons and 6 introns are determined in GILT

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genomic DNA which was a full-length 6, 837 bp (Supplementary sequence 1). Furthermore, exons are particularly conserved, while the sequence difference of introns were tremendous in T. ovatus, H. sapiens, M. musculus, D. rerio, T. obscurus,

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L. crocea and T. rubripes GILT gene (Fig. 2). A phylogenetic tree analysis of GILT of T. ovatus and other metazoans was

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constructed (Fig. 3). It was revealed that T. ovatus GILT was closely grouped together with Oplegnathus fasciatus (Perciformes). The homologous relation with T. ovatus GILT from near to far was other fishes, reptiles, mammalia and insecta. This result conformed to conventional taxonomy. 3.2 A single SNP and its relation with the resistance/susceptibility to P. damselae Infection Base on the tolerance of P. damselae in T. ovatus, 58 fish that died in the first 20 days were regarded as HS group. Nevertheless, 59 fish that survived after P. damselae

ACCEPTED MANUSCRIPT infection until they were sampled at 20 days, these fish were regarded as HR group. All fish in the control group were alive all the time. For analyzing polymorphisms of GILT, 117 individuals were used. PCR products were amplified by six primer pairs spanning the exons and introns (Table 1). According to multiple alignments of GILT sequences from 59 individuals of the HR group and 58 individuals of HS group, only one difference site was detected by the

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primer pairs ToGILT-SF1/SR1 (Table 2 and Fig. 5 A), and two genotype CC and CG were authenticated (GenBank accession No. KY963348-KY963349).

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Association analysis revealed that the SNP ToGILT-S1-g.148C>G was obviously correlation with high-susceptibility, and individuals with genotype CC had

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higher susceptibility to P. damselae compared with genotype CG (Table 2). Nevertheless, the SNP ToGILT-S1-g.148G>C was observably associated with high-

compared with genotype CC (Fig. 4).

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resistance, and individuals with genotype CG had higher resistance to P. damselae

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The SNP site was investigated further by locating the sites in the 3D model of their corresponding gene (Fig. 5). The 3D protein structure revealed that the

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SAP-A homodomain domain.

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functional domains of T. ovatus GILT. Moreover, the SNP site (29 aa) located in

3.3 Constitutive and inductive expressions Tissues expression spectrum revealed that GILT ubiquitously expressed in

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various organs (Fig. 6). In healthy T. ovatus, GILT dramatically expressed in spleen (p < 0.01) and kidney (p < 0.05) compared with that in other tissues (Fig. 6). Moreover,

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low expressions were present in the muscle and brain. To obtain the modulation of T. ovatus GILT transcripts based on P. damselae challenge, we conducted qRT-PCR on liver, spleen, kidney and intestine tissues to assess GILT mRNA levels during 72 h stimulation. Time course analysis of gene expression indicated that GILT transcripts were remarkably highest at 24 h post-P. damselae challenge in liver, spleen and intestine (Fig. 7A, B, D). However, in kidney, the mRNA levels of GILT memorably expressed at 12 h post-P. damselae challenge (Fig. 7C). Compared to control groups, the expressions of GILT in injection groups were dramatically higher at each time.

ACCEPTED MANUSCRIPT 4. Discussion GILT functions as an immunological surveillance-related factor are in both innate and adaptive immunity (Ma et al., 2017). Innate immunity system may be a forceful parclose to prevent teleosts from the attack of pathogenic germs. Moreover, the GILT plays a role in facilitating the processing and presentation of MHC class IIrestricted antigens and is also involved in MHC I-restricted antigens in adaptive

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immunity catalyzing disulfide bond reduction. In recent years, several studies showed that GILT gene might be involved in the immune response to bacteria challenge and

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maintained first line of innate immune defense. GILT mRNA levels were obviously up-regulated in splenocytes and the cells from head kidney after induction with LPS

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in Oncorhynchus mykiss (Liu et al., 2013). Moreover, recombinant protein of GILT could defend against pathogenic microorganisms and played the function of

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immunization in Perciformes and other teleosts (Li et al., 2014; Song et al., 2014; Yang et al., 2015; You et al., 2018). According to previous studies, GILT gene can be

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considered as a disease resistance gene in teleosts, and it may be used as one of the markers against diseases in molecular breeding. In present study, we amplified the GILT in T. ovatus because of crucial biological defense role of GILT in innate

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immunity. Only one SNP was detected in the first exon of GILT gene. Then,

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resistance and susceptibility groups were obtained after injecting P. damselae, and the genotypes of resistance/susceptibility groups were significantly different. We speculated that this SNP might be related to disease resistance.

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In previous study, the polymorphisms of genes were detected to associate with susceptibility/resistance to some diseases. The polymorphisms of MHC in different

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fish, including P. olivaceus, Hippocampus erectus, Epinephelus coioides, O. mykiss and T. ovatus were demonstrated to associate with resistance to variety of diseases (Xu et al., 2010; Luo et al., 2016; Yang et al., 2016a; Yang et al., 2016b; Yang et al., 2016; Zhu et al. 2018). The polymorphisms of Mx2, MDA5, TLR22 and RIG-I in Ctenopharyngodon idella were associated with GCRV (Grass carp reovirus, GCRV) (Su et al., 2011; Wang et al., 2011; Wan et al., 2012; Wang et al., 2012). Moreover, SNP loci in the chicken-type lysozyme gene of P. olivaceus were observed to associate with the disease resistance against Listonella anguillarum (Liu et al., 2017). Nevertheless, no more information was related to polymorphisms of GILT to date. In present study, the GILT gene was chosen as a target gene for researching the

ACCEPTED MANUSCRIPT connection between T. ovatus and resistance to P. damselae challenge. Only one SNP site (ToGILT-S1-g.148C>G) was identified in the first exon of GILT gene. The allele C was significantly associated with HS group, while the allele G was dramatically associated with HR group. The SNP (ToGILT-S1-g.148 G>C) was a nonsynonymous mutation (changing Alanine acid to Proline acid) which might transform the structure and function of SAP-A homodomain in GILT (Marchler-Bauer et al.,

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2015; Marchler-Bauer et al., 2017). Actually, this mutation was not included in the main functional domain. These results suggested that this SNP was the candidate loci associated with resistance/susceptibility to P. damselae.

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GILT is ubiquity expressed in APCs and is inducible by IFN-γ in other type cells

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(Arunachalam et al., 2000; O’Donnell et al. 2004; Hastings et al. 2011). GILT mRNA was detected in various tissues (heart, liver, gill, spleen, kidney, intestine and muscle)

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from some bony fish (S. chuatsi, O. mykiss, Carassius auratus, Microptenus salmoides) (Liu et al., 2013; Song et al., 2014; Li et al., 2015). In our study, the spatial expression patterns showed that GILT was pervasively expressed in all

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detected organs, with high transcription levels observed in fish related-immune tissues, such as: spleen, kidney, liver and intestine, low expressions were present in the

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muscle and brain. This result was similar to that in S. chuatsi and O. mykiss (Liu et al., 2013; Song et al., 2014) which indicated that the expression pattern of GILT gene was

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well conserved in fish related-immune tissues. Furthermore, conform to the function of GILT in MHC class II antigen processing pathway, GILT mRNA levels dramatically increased in liver, spleen, kidney and intestine after induction with P.

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damselae (Fig. 7). It indicated that GILT might be involved in the immune response oppose bacterial challenge in T. ovatus (Song et al., 2014).

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To promote comprehending of both evolution of GILT genes and immunological function in T. ovatus, the GILT genomic structure was obtained. 7 exons and 6 introns which were analogous to other reported vertebrate GILT gene were acquired in GILT genomic sequence. Moreover, exons were particularly conserved indicated that the function of T. ovatus GILT was similar to that in other species (Liu et al., 2013). Furthermore, to further understand structural features in fish family genes, the cDNA sequence of GILT from T. ovatus was obtained. Several typical structural features were conservative in GILT amino acid sequence (Arunachalam et al., 2000; Liu et. al, 2007; Song et al., 2014; Li et al., 2015). The sequence alignment showed that GILT was highly homology with other reported GILT. Higher sequence similarity of GILT

ACCEPTED MANUSCRIPT between bony fish and mammalian indicated that they might have a conservative function in immunology. Protein alignments with other vertebrates indicated that the T. ovatus GILT shared the highest homology with O. fasciatus, consistented with the fact that T. ovatus and O. fasciatus were members of the Perciformes superfamily. In conclusion, in this study, GILT was identified and characterized in T. ovatus, and exons were quite conserved, while the sequence divergence of introns was

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tremendous. Moreover, high expressions of GILT were in immune-associated tissues (spleen and kidney), also GILT mRNA levels dramatically increased in liver, spleen, kidney and intestine after challenge with P. damselae. Sequence analysis showed that

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only one SNP site was observed in GILT gene, and association between SNP and

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resistance/susceptibility to P. damselae analysis revealed that the SNP associated with resistance could be used as candidate markers and might be contributed to the

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selective breeding of T. ovatus.

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Acknowledgments

This work was supported by Fishing Port Construction and Fishery Development Special Funds for Guangdong Province (Sci-tech Popularization, 2017A0008), China

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Agriculture Research System (CARS-47), the Central Public-interest Scientific

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Institution Basal Research Fund, CAFS (NO. 2016HY-JC0304), the Special Scientific Research Funds for Central Non-profit Institute, Chinese Academy of Fishery Sciences (2016A11JC03) and National Infrastructure of Fishery Germplasm

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Xu, T.J., Chen, S.L., Zhang, Y.X., 2010. MHC class IIa gene polymorphism and its association with resistance/susceptibility to Vibrio anguillarum in Japanese flounder (Paralichthys

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ACCEPTED MANUSCRIPT Yang, Q., Zhang, J.X., Hu, L.L., Lu, J., Sang, M., Zhang, S.Q., 2015. Molecular structure and functional characterization of the gamma-interferon-inducible lysosomal thiol reductase (GILT) gene in largemouth bass (Microptenus salmoides). Fish Shellfish Immunol. 47(2), 689-696. You, X.L., Liu, L., Li, X.Y., Du, H.J., Nie, D.S., Zhang, X.G., Tong, H.B., Wu, M.J., Gao, Y.T., Liao, Z.Y., 2018. Immune response of interferon-γ-inducible lysosomal thiol reductase (GILT) from Chinese sturgeon (Acipenser sinensis) to microbial invasion and its antioxdative activity in lipopolysaccharides-treated mammalian dentritic cells. Fish Shellfish Immunol. 72,

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356-366.

Yue, G.H., 2014. Recent advances of genome mapping and marker-assisted selection in

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aquaculture. Fish fish. 15(3), 376-396.

Zhen, P.L., Ma, Z.H., Guo, H.Y., Li, Y.N., Zhang, D.C., Jiang, S.G., 2014. Ontogenetic

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development of caudal skeletons in Trachinotus ovatus larvae. South China Fish. Sci. 10, 4550.

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Zhu, K.C., Yu, W.B., Guo, H.Y., Zhang, N., Jiang, S.G., Zhang, D.C., 2018. The polymorphisms of MHCIIß gene of Trachinotus ovatus and their association with resistance/susceptibility to

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Photobacterium damsela. Aquaculture 485, 160-165.

ACCEPTED MANUSCRIPT Fig. 1. Comparison of deduced amino acid sequences of Trachinotus ovatus GILT with published GILT in other species, respectively. Blue and red boxes represent Signal peptide domain and CXXC motif, respectively; SAP-A homodomain domain is underline; Glycosylation sites are boxed by ellipse. Conservative sequence and Conservative cysteines are marked by orange and yellow, respectively; Dashes represent gaps created to maximize the degree of identity among all compared sequences. The accession numbers of the GILT sequences used are listed in

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supplement table 1.

Fig. 2. Comparison of the genomic structure of GILT among the T. ovatus, H.

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sapiens, M. musculus, D. rerio, T. obscurus, L. crocea and T. rubripes. Different

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colour boxes and the lines represent exons and introns, respectively. The boxes and the length of lines also indicate exons and introns, respectively.

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Fig. 3. Phylogenetic tree analysis of GILT in metazoan. Sequence alignment of GILT was analyzed using the MEGA 6 software with Maximum Likelihood (ML) method.

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Different colours represented various orders. Black dot represented T. ovatus. The accession numbers of the sequences used in the phylogenetic analysis are listed in supplement table 1.

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Fig. 4. Distribution of GILT alleles in the high-resistance groups (HR) and high-

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susceptibility groups (HS) of T. ovatus during bacterial challenge. Significant difference between the HR and HS are indicated with the asterisk (* represented p < 0.05, ** represented p < 0.01).

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Fig. 5. Compare to the SNP sequences (A); Three-dimensional mapping of SNP sites

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of the T. ovatus GILT protein (B). Fig. 6. Gene transcripts of GILT in various tissues of T. ovatus. Different letters indicate significant differences. Fig. 7. Temporal expression of GILT in liver (A), spleen (B), kidney (C) and intestine (D) after bacterial challenge with Photobacterium damselae for the indicated times.

EF-1α expression was used as an internal control for real-time PCR. The data are expressed as the mean ± SE. Significant difference between the challenge groups and control groups are indicated with the asterisk (* represented p < 0.05, ** represented p < 0.01).

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Table 1 Primers used in this experiment. Primer

Amplification

Sequence (5’-3’)

target

AAAGTTGCTCAGGACAGTAGTTCGT

DNA fragment

ToGILT-R1

CTCAGTGTTGCTTATTTAGTGGC

DNA fragment

ToGILT-F2

GAATGGGGTTCCTGTATCCTCCTCT

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ToGILT-F1

DNA fragment

GGAAGAGTGATGGAACCTGGAG

ToGILT-SF1

ATTTAAAGTTGCTCAGGACA

SNP

ToGILT-SR1

CTTTTACATGAACATTACTT

SNP

ToGILT-SF2

TATTGCTGGTGTCTGTTTGTCTGC

SNP

ToGILT-SR2

CAAACAACAATGGAAATGAGCACAC

SNP

ToGILT-SF3

CACTAAATAAGCAACACTGAGGAC

SNP

ToGILT-SR3

CACTGTGAACTTAAACGACCCTGC

SNP

ToGILT-SF4

TAAATAGAAAGGTTGTAGGCAGCAG

SNP

ToGILT-SR4

ATGGGAACAAGACAACTGGGAAGG

SNP

ToGILT-SF5

AGGTGGGAGGTTTCTTGTGTGATT

SNP

AGGTGGGAGGTTTCTTGTGTGATT

SNP

ToGILT-SF6

GTTGCTCAGGACAGTAGTTCGTTGG

SNP

ToGILT-SR6

ATGAACATTACTTACCCCGCACTGG

SNP

EF-1α-F

CCCCTTGGTCGTTTTGCC

qRT-PCR

EF-1α-R

GCCTTGGTTGTCTTTCCGCTA

qRT-PCR

ToGILT-qF1

ATGAAGACCCCTCTGCTGCT

qRT-PCR

ToGILT-qR1

TGAGCACCACTTTGACGGAGG

qRT-PCR

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ToGILT-SR5

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ToGILT-R2

DNA fragment

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Table 2 Distribution of single-nucleotide polymorphism (SNP) site of the exon 1 of GILT sequences. Resistant no (%)

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CG

24 (14.3)

4 (61.2)

CC

34 ( 85.7)

55 (38.8 )

GG

0 (0)

0 (0)

Allele Frequency no.(N=117) G

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Susceptible no (%)

C

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Genotype

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Locus

0.189 0.811

ACCEPTED MANUSCRIPT Highlights 1. 7 exons and 6 introns were identified in T. ovatus GILT. 2. GILT expression remarkably up-regulation in four tissues after induction. 3. The allele C was significantly associated with high-susceptibility (HS)

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group.

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Figure 7