Subcellular Localization of Large Yellow Croaker(Larimichthys crocea) TLR21 and Expression Profiling of Its Gene in Immune Response

1 Introduction

Innate immunity is the first defense in fish against various pathogens (Rauta et al., 2014; Zhu et al., 2013).As a member of the immunity system, toll-like receptor(TLR) is an important pattern recognition receptor family which can recognize highly conserved microbial products known as pathogen associated molecular patterns (PAMPs),including LPS, lipoproteins, flagellin, double- and singlestrand RNAs, etc. (Akira et al., 2006). Typically, TLRs include three different domains, an N-terminal leucinerich repeat (LRR) domain, a transmembrane domain and a cytoplasmic Toll/IL-1 receptor (TIR) domain (Zhu et al.,2013; Akira et al., 2006). The extracellular LRR region plays a role in ligand recognition, whereas the cytoplasmic TIR domain is necessary for binding adapter molecules participated in activating downstream signal transduction (Palti, 2011). More importantly, the TLR signaling pathway plays a crucial role in bridging native and adaptive immune responses (Kawai and Akira, 2010).

Till now, at least 13 TLRs (TLR1-13) have been identified from mammals (Hopkins and Sriskandan, 2005).However, more than 20 TLRs have been identified and characterized in teleost fishes (Rauta et al., 2014). Some of them, such as TLR5S, TLR14 and TLR18-27, have not been identified in mammals (Rauta et al., 2014; Palti,2011; Pietretti and Wiegertjes, 2014). TLR21, a novel non-mammalian TLR, was initially identified in pufferfish Fugu rubripes and zebrafish Danio rerio (Oshiumi et al., 2003; Jault et al., 2004; Meijer et al., 2004). Recent studies demonstrated that zebrafish TLR21 might recognize bacterial genomic DNA or synthetic CpG DNA,functioning as mammalian TLR9 (Yeh et al., 2013). Additionally, up-regulation of TLR21 transcript abundance was detected in orange-spotted grouper Epinephelus coioides, olive flounder Paralichthys olivaceus, and rock bream Oplegnathus fasciatus after bacterial or viral infection and immune stimulation (Li et al., 2012; Gao et al.,2013; Priyathilaka et al., 2014). These findings indicated that TLR21 might be very important in fish immune response.

经过统计测算，优化后的配送线路从原先的120条降低至62条，几乎减少了一半。进行配送路线优化后，百安居也重新寻找了进行城市配送的承运商，并进行运费核算获得一个更好的运费价格。核算时还是以2016年9、10月份从上海物流中心进行配送的数据为基础数据，核算后的汇总分析如表2所示：

Large yellow croaker, Larimichthys crocea, one of the economically important maricultured fish species in Southeast China, has suffered serious diseases including bacterial, viral and other pathogenic infections in recent years, which resulted in huge economic losses (Wang et al.,2001; Zhou et al., 2010). Our previous studies demon-strated that TLR signaling pathway played crucial roles in immune response of L. crocea (Yao et al., 2008, 2009,2012; Huang et al., 2011; Fan et al., 2015). However, as one of the most important members of TLR family, the molecular and expression characteristics of TLR21 gene in large yellow croaker are still poorly understood. In this study, the molecular structure, subcellular localization and immune characterization of TLR21 in large yellow croaker were investigated to better understand its potential role in fish immune responses.

2 Materials and Methods

2.1 Fish Collection and Immune Challenge

The healthy juvenile large yellow croaker (total length 13 ± 1.5 cm, weight 76 ± 25 g) were purchased from a local fish farm of Ningde, Fujian Province, China. Before the experiment, fish was temporarily cultured in 4 m3 tanks at salinity 28, 18–20℃ and a density similar to that of the culture net cages from which the samples were obtained.The fish were cultured for 1 week to adapt to the new environment as described previously (Huang et al., 2011).Then 1.5 mL blood was collected from eugenol anaesthetized fish by cutting the tail and mixed with 3 mL anticoagulant solution (0.48% citric acid, 1.32% sodium citrate and 1.47% glucose). The blood cells were separated by centrifugation at 800 g and 4℃ for 5 min and stored in RNA fixer (Bioteke, Beijing) immediately. Simultaneously, other tissues including head-kidney, kidney, intestine, spleen, liver, gill, skin, brain, heart, muscle, and stomach were dissected from un-stimulated fish and stored in RNA fixer at −20℃ for subsequent RNA extraction.

研究人员发现，近一半的安全座椅与汽车座位并不合适。近日，美国俄亥俄州立大学医学院的研究人员对50辆家用汽车的座位及60个安全座椅（共3600个组合）进行了安全测试，结果发现，座位角度与弹性头枕的位置导致42%的安全座椅与汽车座椅无法完全兼容。

Total RNA was extracted from tissues each (50 mg)using TRIzol reagent (Invitrogen, USA) according to the standard protocol. Before reverse transcription, the RNA was subjected to RNase-free DNase I digestion (Promega,USA) in order to eliminate possible genomic DNA. First strand cDNA was synthesized from total RNA by MMLV reverse transcriptase (Fermentas, China) following the manufacturer’s protocol with Oligo d (T) 18 as primer.

2.2 RNA Extraction and cDNA Synthesis

Fish immune stimulation was performed by intraperitoneal injection of 250 μL polyI:C (27472901, GE, 1 mg mL−1) in phosphate buffered saline (PBS, pH 7.4), 250 μL LPS (L2880, Sigma, 1 mg mL−1) in PBS, or suspension formalin-inactivated Gram-negative bacterium V. parahaemolyticus (108 CFU mL−1) in PBS, respectively (Huang et al., 2011). Fish injected with 250 μL PBS were used as control. Six fish individuals were randomly sampled at each time point from each group. Spleen, head-kidney and liver of each group were collected at 3, 6, 12, 24, 48 and 72 h after injection and preserved for RT-PCR analysis (n = 6).

“都市时间”栏目组接电话的值班记者是一女孩，她一脸茫然，因为对方的手机突然断了，没有了任何声音。她自言自语道：“怎么挂机了？莫名其妙。”她这话被从值班室走向里间的栏目组曾真听到了，她停住，问：“什么情况？”值班记者说：“有个人打来电话，要我们去胜利大厦给他来一场电视直播。”“电视直播？直播什么？”“没听清，电话断了。根据以往的经验，十有八九是

2.3 Cloning and Sequencing of LcTLR21 cDNA

Based on a known partial cDNA sequence of TLR21 gene from our EST database, the 3’ and 5’ ends were obtained by rapid amplification of cDNA ends (RACE) approaches with muscle cDNA as template. The 3’end PCR of LcTLR21 gene was performed using the gene specific primers TLR21-3F1/F2 and adaptor primers AOLP/AP(Table 1) by nest PCR at annealing temperature of 50℃and 57.5℃, respectively. To clone the 5’ end of LcTLR21 gene, the muscle mRNA was transcribed by M-MLV reverse transcriptase with Oligo d (T) 18 as primer, followed by purifying the cDNA with a DNA purification kit(Omega, Bio-Tek) and tailed with poly(C) at the 3’ end by terminal deoxynucleotidyl transferrase (TdT) (Fermentas,China). The first round PCR was performed at annealing temperature of 50℃ with primer TLR21-5R1 and adaptor primer AAP using the muscle cDNA as template, and then nested PCR was carried out with a specific primer TLR21-5R2 and adapter primer AP at 53℃.

PCR product was purified with Gel Extraction Kit(Omega, Bio-Tek). The purified PCR product was ligated into pMD19-T vector (Takara, China) and transformed into the competent E. coli TOP10 cells. Positive clones containing the expected-size inserts were screened by colony PCR and then sequenced by Sangon Corp (Shanghai, China). All primers were listed in Table 1.

Table 1 Primers used for LcTLR21 gene cloning and expression analysis

Primers Sequence (5’-3’) Purpose TLR21-3F1 TTGCCTACATCACAGGGACT TLR21-3F2 CGGGATGTTCTCCTGCTCG 3’ RACE method TLR21-5R1 CAGGTCGTTTACAATAGCC TLR21-5R2 CAAGATTGGGAACAGTAAG 5’ RACE method AOLP GGCCACGCGTCGACTAGTAC(T)16 AAP GGCCACGCGTCGACTAGTAC(G)10 AP GGCCACGCGTCGACTAGTAC General primers for RACE TLR21-F AGCTGGGCCGTGATATTGTG TLR21-R TTTCCAGCCTACTCCCCGT β-actin-RT-F TTATGAAGGCTATGCCCTGCC β-actin-RT-R TGAAGGAGTAGCCACGCTCTGT mRNA expression pTurbo-TLR21-F CCGCTCGAGATGGCAAGTCTTATTCATCA pTurbo-TLR21-R CGGGGTACCGTAGGTTGCAAGTAAAAGTTTTC Subcellular localization

2.4 Sequence Analysis and Phylogenetic Tree Construction

To investigate the subcellular localization of LcTLR21,HEK293T cells seeded in 6-well plates were cultured overnight. Transfections were carried out with 2.5 μg purified plasmids pTurbo-GFP-TLR21 and pTurbo-GFP-N(vector control) using lipofetamine 3000 (Invitrogen, CA)according to the manufacturers’ instructions, respectively.At 24 h post transfection, cells were washed and cultured with fresh DMEM and stained with Hoechst 33342 (Sigma-Aldrich, MO) at a final concentration of 1 μg mL−1 for 20 min. Subsequently, the transfected cells were directly observed under a fluorescence microscope (Leica, German) with excitation at 488 nm and emission at 511 nm.

2004年，高志明的妻子因病去世。之后不断有人给他介绍对象，高志明的脑子里却总有夏小凡的影子。也不是没试过主动去找她，却发现夏小凡虽然一直没有再婚，身边却总是有人。她长袖善舞，真情假意，她要创一个自己的世界，就得学会应酬各式各样的男人。

解析：制取NH3需要NH4Cl与Ca(OH)2反应,若只加热NH4Cl生成NH3和HCl,在试管口遇冷又生成固体NH4Cl,可能堵塞导管,A项错误;制取NaHCO3时,向NH3和食盐的饱和溶液中通入CO2的导气管应长进短出,B项错误;NaHCO3在水中的溶解度小,CO2通入氨化的饱和食盐水生成NaHCO3晶体析出(NH3+NaCl+CO2+H2O==NaHCO3↓+NH4Cl),可用过滤法分离,C项正确;加热NaHCO3会分解,无法得到干燥的NaHCO3,并且加热时装置中烧杯应垫上石棉网,D项错误。

Table 2 The putative TLR21 protein sequences used for multiple alignment and phylogenetic analysis

2.5 Semi-Quantitative RT-PCR Analysis of LcTLR21 mRNA Expression

Expression level of LcTLR21 gene in different tissues of non-stimulated and challenged large yellow croaker was measured by semi-quantitative RT-PCR. The house keeping β-actin gene transcripts were utilized as the internal control. Gene specific primers TLR21-F and TLR21-R for LcTLR21, β-actin-F and β-actin-R for β-actin (Table 1) were used to amplify specific fragments with the annealing temperature of 54℃ and 32 cycles, 48℃ and 24 cycles, respectively. The PCR product was sequenced to verify the specificity of above genes. After the PCR program, the PCR product was analyzed by electrophoresis in 1.5% agarose gel containing ethidium bromide(EtBr) with the electrophoresis photograph analyzed with Image J software. All data were given in terms of relative mRNA expression as means ± SE (Standard Error). The data obtained from three independent biological replications were subjected to t-test and the difference was significant if P < 0.05.

2.6 Cell Culture, Plasmid Construction and Subcellular Localization of LcTLR21

HEK-293T cells were seeded in Dulbecco’s Modified Eagle Medium (DMEM, Invitrogen-Gibco) supplemented with 10% fetal bovine serum (FBS, Invitrogen-Gibco),1% antibiotics (100 μg penicillin mL−1, 100 U streptomycinn mL−1). Cells were cultured at 37℃ and in 5% CO2 in 25 cm2 culture flasks (NUNC) for further analysis.

To construct the expression plasmid for the TLR21-GFP fusion protein, the ORF of LcTLR21 gene was amplified with primer pTurbo-TLR21-F and pTurbo-TLR21-R (Table 1). The PCR product was purified and inserted into the PMD19-T vector (Promega, USA) for sequencing and then was cleaved with endonuclease Xho I and Kpn I. The DNA fragment was then inserted into pTurbo-GFP-N vector (Invitrogen, CA) for construction pTurbo-GFP-TLR21 which was then confirmed by sequencing.

随着数据积累和医院持续改进，李红和林茜开始把医院管理经验转化为数据详实的论文，将科研与管理很好地结合起来。而且，这篇论文还被国际期刊收录。

The sequence homology analysis and the deduced amino acid sequence comparison were carried out by BLAST program (http://blast.ncbi.nlm.nih.gov/Blast.cgi)and the Expert Protein Analysis System (EXPASY)(http://www.expasy.org/), independently. The protein domain features were predicted by Simple Modular Architecture Reach Tool (SMART, http://smart.embl-Heidelberg.de/). Multiple alignment of putative amino acid sequence of LcTLR21 with TLR21 from other species (Table 2) was performed using Clustal W (http://www.ebi.ac.uk/clustalw/). Phylogenetic tree was constructed by MEGA 5.05 (http://www.megasoftware.Net) with Neighbor-Joining (NJ) method (Saitou and Nei, 1987).

2.7 Statistical Analysis

The data were subjected to the analysis of t-test with SPSS 19.0 for windows (SPSS Inc.), and the difference was considered statistically significant if P < 0.05 and extremely significant if P < 0.01. One-way ANOVA was employed for the analysis and the results were plotted to figures by Origin 8.0 software (Origin Lab Corporation,MS, USA). Asterisks denote statistically significant differences between experimental treatments and the control.

3 Results

3.1 Characterization of the Full-Length cDNA of LcTLR21

The expression level of LcTLR21 in liver after immune-stimulation is shown in Fig.6C. After LPS stimulation, LcTLR21 transcripts increased significantly at 3 h(P < 0.05). Then, after a short recovery at 6 h, it increased significantly from 12 h to 48 h post-injection (P < 0.05),with the highest value being 24 times higher than that of control (at 24 h) (P < 0.05). Then it returned to the control level at 72 h. LcTLR21 gene expression level showed a significant increase from 6 h to 48 h post-injection with poly I:C (P < 0.05), with the peak (31 folds of control found at 24 h (P < 0.05). Then it recovered to the control level at 72 h. LcTLR21 transcripts increased significantly from 12 to 48 h after V. parahemolyticus stimulation, with the highest (14-fold of control) reached at 12 h after injection (P < 0.05). Then it recovered to the control level at 72 h.

PCS7是一种基于现场总线的过程控制系统，它兼具可编程控制器与分散控制系统的优点[9]。汽包水位控制系统在SMPT-1000设备上的实现，以PCS7为控制器，并进行SMPT-1000与PCS7的通信连接，运行系统，测试其控制效果。

Fig.1 Nucleotide and deduced amino acid sequence of LcTLR21. The start codon (ATG) is boxed and the stop codon(TGA) is marked with an asterisk. The signal peptide is represented with dot underline (1-23 aa); LRR domains are shown with underscore (81–104 aa, 105–128 aa, 129–152 aa, 179–202 aa, 428–451 aa, 452–475 aa, 476–499 aa, 500–526 aa, 580–603 aa, 604–629 aa, 630–653 aa, 654–677 aa); transmembrane domain is shown with double underscore (754-767 aa); and the TIR domain is marked by light grey (795–944 aa). In the 3’-UTR, the polyadeylation signal (AATAA) is in bold and mRNA instability motif (ATTTA) is underlined and italicized.

中俄界江地区的生态旅游资源不但种类丰富多样、地域特征突出、颇具观赏特性、开发价值极大，辐射范围极广等基本表征，同时生态旅游潜力的构成要素完备（见图1），有极大的开发价值。

Fig.2 The conservative structures of LcTLR21. Predicted protein domain of LcTLR21 determined by SMART analysis.Signal peptide in red, TM domain in dark blue, LRR and TIR domain are labeled accordingly; Conserved D442 and F444 residues were predicted in LRR domains of TLR21. Conserved Box 1–3 were predicted in TIR domain. Accession numbers of the sequences are given in Table 2. (For interpretation of the references to in this figure legend, the reader is referred to the web version of this article.)

Phylogenetic tree analysis revealed that the putative amino acid sequence of LcTLR21 was clustered into the subgroup TLR21 from other teleosts, with the closest relationship with TLR21 from rock bream (Fig.3). The observed relationships within this cluster reflected the taxonomic position of the species.

Prediction of domain structure by SMART revealed that LcTLR21 contained a signal peptide (1–23 aa) at the N-terminal, followed by 12 LRRs domain at the position of 81 to 677 aa, one transmembrane domain at position of 745 to 767 aa and one TIR domain at the C-terminal(795–944 aa) (Fig.1). Multiple alignments of LcTLR21 amino acids shared high level of sequence identity with TLR21 from rock bream, yellowtail Seriola lalandi and channel catfish (75%, 72% and 71%, respectively). The TIR domain identities of them were 93%, 89% and 91%,respectively. The conserved amino acid residues (D442 and F444) (Fig.2) which participate in recognition and interaction with CpG DNA, three highly conserved active motifs BOX 1 (YDAFXXXX), BOX 2 (XCLXXRDXXXG) and BOX 3 (FWXXX) in the TIR domain (Fig.2) were also identified in LcTLR21.

Fig.3 Phylogenetic tree of LcTLR21 sequence. Complete amino acids sequences were aligned by using CLUSTAL W, and the tree was constructed with NJ method in MEGA 5.05 and a bootstrap analysis was performed using 1000 replicates to test the relative support for particular clades. GenBank accession numbers of these genes are listed in Table 2.

3.2 Tissue Distribution of LcTLR21

Expression level of LcTLR21 gene in head-kidney after stimulation with LPS, poly I:C and V. parahemolyticus is shown in Fig.6B. After LPS challenge, the expression of LcTLR21 increased significantly from 6 h to 48 h (P <0.05), with the peak value (7.5-fold of control) reached at 12 h (P < 0.05). After injection with poly I:C, LcTLR21 transcripts increased significantly from 3 h to 48 h (P <0.05), and the highest (5-fold of control) was found at 12 h (P < 0.05), then it recovered to that of control at 72 h.After V. parahemolyticus stimulation, LcTLR21 transcripts showed a significant increase from 3 to 72 h (P < 0.05),with the greatest (4.7-fold of control) appeared at 24 h after stimulation (P < 0.05).

Fig.4 Relative expression of LcTLR21 gene in different tissues of large yellow croaker, including heart, liver, blood, intestine, kidney, muscle, gill, skin, brain, spleen, head-kidney and stomach, respectively (n = 6).

3.3 Subcellular Distribution of LcTLR21-GFP Fusion Protein

In order to probe the intracellular localization of LcTLR21 protein, the plasmid expressing GFP fused with LcTLR21 (LcTLR21-GFP) was constructed. The control plasmid pTurbo-GFP-N was randomly distributed both in the cytoplasm and nucleus (as shown in Fig.5). However,transfection of LcTLR21-GFP plasmid, which drives the expression of a fusion protein consisting of full-length LcTLR21 and GFP, were constitutively expressed in the cytoplasm.

Fig.5 The subcellular localization of LcTLR21 and GFP (control) in HEK-293T cell line. The left panels are LcTLR21-GFP fusion protein and GFP expression profile under fluorescence, the middle panels are the cell nucleus stained with Hoechst 33342 while the right panels are the combined images of LcTLR21-GFP fusion protein and GFP with cell nucleus. Bar = 20 μm.

3.4 Expression Profile of LcTLR21 After Immune Challenge

The expression profile of LcTLR21 in spleen after immune stimulation is shown in Fig.6A. Significant upregulation of LcTLR21 gene expression was detected from 3 h to 48 h after LPS injection, with the peak (8.6-fold of control) found at 24 h (P < 0.05). LcTLR21 tran-scripts showed significant change from 3 to 48 h after poly I:C challenge, with the highest (12 times of control)reached at 48 h (P < 0.05). After stimulation with V. parahemolyticus, the LcTLR21 expression increased significantly at 3 h (P < 0.05). Then it returned to that of control from 6 to 12 h post-injection. However, significant upregulation of expression was detected again from 24 to 72 h, peaking (approximately 6.5 folds of control) at 72 h (P< 0.05).

The constitutive expression of LcTLR21 gene in tissues revealed that the transcripts of TLR21 gene were determined in all examined tissues, with the most predominant expression in spleen, followed by head-kidney, liver and stomach and the weakest expression was detected in brain(Fig.4).

Based on the 2400 bp sequence of LcTLR21 gene in the RNAseq database of large yellow croaker, a 800 bp fragment containing poly(A) tail was obtained by 3’-RACE PCR and a fragment of 300 bp containing the initial start codon ATG was amplified by 5’-RACE PCR,respectively. As a result, the 3365 bp full-length cDNA sequence of LcTLR21 gene (GenBank accession number:KX928778) was obtained, which included an ORF of 2937 bp encoding a polypeptide of 978 amino acid residues, a 5’-untranslated region (UTR) of 93 bp and a 3’-UTR of 331 bp (Fig.1). The calculated isoelectric point(pI) of LcTLR21 was 8.35, and the theoretical molecular weight (Mw) was 113.3 kDa.

Fig.6 Analysis of LcTLR21 gene expression relative to that of control in spleen (A), head-kidney (B) and liver (C) after LPS, poly I:C and V. parahaemolyticus challenges. The data are shown as means ± SE. Asterisk indicate significant difference at P < 0.05 between the experimental group and the control at the corresponding points (n =6).

4 Discussion

In the present study, the full-length LcTLR21 cDNA was cloned, which contained typical conservative structure of the TLR family. The theoretical ectodomain of LcTLR21 contained 12 LRRs, including 10 conserved LRRs and 2 typical LRRs (LRRTYP), which is similar with TLR21 from olive flounder (18 LRRs), yellowtail(17 LRRs), orange-spotted grouper (16 LRRs) and grass carp (17 LRRs) (Li et al., 2012; Gao et al., 2013; Reyes-Becerril et al., 2016; Wang et al., 2013). In addition, two conserved amino acid residues (D442 and F444) participating in CpG DNA recognition and interaction (Reyes-Becerril et al., 2016) were identified in the LRR domain of LcTLR21.This is similar with TLR21 from rock bream(D443 and F445) and yellowtail (D420 and F422) (Priyathilaka et al., 2014; Reyes-Becerril et al., 2016), suggesting that LcTLR21 might have the similar recognition ability.However, TLR21 from zebrafish and grass carp contained the conserved D and another amino acid residue Y, which replaced conserved F (Fig.2), indicating the two typical sites are not very conservative in teleosts. However, the impact of this replacement on ligand-bind- ing function of TLR21 is still unknown.

The typical TIR domain, which has a function for TLR signal transduction, shared high identities with TLR21 from other fish species (Table 2). Three conservative motifs BOX, including BOX 1, BOX 2 and BOX 3 were also identified in TIR domain of LcTLR21 (Fig.2). BOX 1 and BOX 2 are mainly involved in regulation of the downstream signal transduction pathway, while Box 3 is primarily involved in directing localization of receptor (Slack et al., 2000). Our findings suggested that LcTLR21 might play a similar intracellular signal transduction function.

Phylogenetic analysis demonstrated that LcTLR21 was clustered in the teleost TLR21 clade, with the closest relationship with the TLR21 isoform from rock bream. The TLR21 from birds was also clustered into their corresponding subgroup, which reflected the traditional classification (Fig.3).

Little is known about the subcellular localization of TLR21 in fish. Our results showed that the LcTLR21-GFP fusion protein mainly distributed in the cytoplasm of HEK-293T cells. Previous studies demonstrated that TLR21 was preferentially localized in the endoplasmic reticulum in zebrafish and chicken (Yeh et al., 2013; Brownlie et al.,2009), indicating that LcTLR21 might have a function in cell signaling transduction.

Tissue expression profiles showed that LcTLR21 gene transcripts were broadly expressed in most examined tissues of large yellow croaker, with the most prominent found in spleen followed by that in head-kidney. Similar expression characterizations were also reported in orange spotted grouper (Li et al., 2012), yellowtail (Reyes-Becerril et al., 2016) and grass carp (Wang et al., 2013). These results suggested that LcTLR21 mainly expressed in the main immunity-related tissues and might play important roles in fish immune response.

In the present study, significant up-regulation of LcTLR21 transcripts was detected in spleen, head-kidney and liver after LPS and V. parahemolyticus injection (P <0.05) (Fig.6), indicating that LcTLR21 could be induced after bacterial infection in immunity-related tissues of large yellow croaker. Our findings are similar with the previous studies in olive flounder, grass carp, rock bream and channel catfish (Gao et al., 2013; Priyathilaka et al.,2014; Wang et al., 2013; Baoprasertkul et al., 2007). After poly I:C challenge, LcTLR21 gene transcripts also increased significantly in all examined tissues (P < 0.05)(Fig.6). Similarly, significant up-regulations of TLR21 were reported in olive flounder and zebrafish larvae after poly I:C stimulation (Gao et al., 2013; Sundaram et al.,2012), indicating that LcTLR21 expression also could be induced by viral infection. Therefore, our findings suggested that LcTLR21 could be induced in fish defense against bacterial and viral infections, which is similar with the previous studies in other fish species (Gao et al.,2013; Priyathilaka et al., 2014; Reyes-Becerril et al., 2016;Wang et al., 2013; Sundaram et al., 2012; Lee et al., 2014).

In summary, the full-length cDNA of TLR21 gene was cloned from large yellow croaker, which showed high identities with other TLRs and contained the conservative structures of TLR21. LcTLR21 transcripts were broadly expressed in most examined tissues with the highest abundance found in spleen and the weakest in brain.Subcellular localization demonstrated that LcTLR21 constitutively existed in the cytoplasm. The expression of LcTLR21 could be induced significantly in spleen, headkidney and liver after stimulated with LPS, poly I:C and V.parahemolyticus. Our findings suggested that LcTLR21 might play an important role in large yellow croaker resistance to bacterial and viral infections.

Acknowledgements

This work was funded by the Qingdao National Laboratory for Marine Science and Technology to Cuiluan Yao, the Natural Science Foundation of Fujian(No. 2018J01454), and the National Natural Science Foundation of China (Nos. 31101882 and 41276178).

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