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A ferritin gene from Procambarus clarkii, molecular characterization and in response to heavy metal stress and lipopolysaccharide challenge
Accepted Manuscript A ferritin gene from Procambarus clarkii, molecular characterization and in response to heavy metal stress and lipopolysaccharide challenge Qiu-Ning Liu, Zhao-Zhe Xin, Yu Liu, Zheng-Fei Wang, Yi-Jing Chen, Dai-Zhen Zhang, Sen-Hao Jiang, Xin-Yue Chai, Chun-Lin Zhou, Bo-Ping Tang PII:
S1050-4648(17)30098-0
DOI:
10.1016/j.fsi.2017.02.025
Reference:
YFSIM 4447
To appear in:
Fish and Shellfish Immunology
Received Date: 13 December 2016 Revised Date:
11 February 2017
Accepted Date: 17 February 2017
Please cite this article as: Liu Q-N, Xin Z-Z, Liu Y, Wang Z-F, Chen Y-J, Zhang D-Z, Jiang S-H, Chai X-Y, Zhou C-L, Tang B-P, A ferritin gene from Procambarus clarkii, molecular characterization and in response to heavy metal stress and lipopolysaccharide challenge, Fish and Shellfish Immunology (2017), doi: 10.1016/j.fsi.2017.02.025. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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A ferritin gene from Procambarus clarkii, molecular characterization
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and in response to heavy metal stress and lipopolysaccharide
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challenge
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Qiu-Ning Liu, Zhao-Zhe Xin, Yu Liu, Zheng-Fei Wang*, Yi-Jing Chen, Dai-Zhen
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Zhang, Sen-Hao Jiang, Xin-Yue Chai, Chun-Lin Zhou, Bo-Ping Tang*
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Jiangsu Key Laboratory for Bioresources of Saline Soils, Jiangsu Synthetic
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Innovation Center for Coastal Bio-agriculture, Jiangsu Provincial Key Laboratory of
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Coastal Wetland Bioresources and Environmental Protection, School of Ocean and
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Biological Engineering, Yancheng Teachers University, Yancheng 224001, Jiangsu
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Province, People's Republic of China.
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* Corresponding author: Zheng-Fei Wang and Bo-Ping Tang
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E-mail: [email protected] (Zheng-Fei Wang) and [email protected] (Bo-Ping Tang)
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Address: Jiangsu Key Laboratory for Bioresources of Saline Soils, Jiangsu Provincial
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Key
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Protection, Jiangsu Synthetic Innovation Center for Coastal Bio-agriculture, School of
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Ocean and Biological Engineering, Yancheng Teachers University, Yancheng 224051,
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Jiangsu Province, People's Republic of China.
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Coastal
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ACCEPTED MANUSCRIPT ABSTRACT
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Ferritin plays important roles in iron storage, detoxification, and immune response.
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Here, a ferritin gene (PcFer) was identified in Procambarus clarkii, an economically
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important freshwater crayfish. Full-length PcFer cDNA was 1022-bp, including a
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135-bp 5ʹ-untranslated region (UTR) with a typical iron responsive element, a 374-bp
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3ʹ-UTR, and a 513-bp open reading frame encoding a polypeptide of 170 amino acids
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which contained the Ferritin domain. PcFer has ion binding sites, a ferrihydrite
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nucleation center, and an iron ion channel. PcFer is phylogenetically closely-related to
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Pacifastacus leniusculus and Eriocheir sinensis ferritins. Real-time quantitative
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reverse-transcription PCR analysis showed that PcFer was expressed in all tested P.
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clarkii tissues, and expressed most in hepatopancreas. After challenge with various
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heavy metals and lipopolysaccharide, respectively, the hepatopancreatic expression
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levels of PcFer were markedly upregulated. These results suggest that expression of
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PcFer might be involved in immune defense and protection of P. clarkii against
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heavy metal stress.
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Keywords: Expression analysis; Ferritin; Heavy metal stress; Immune response;
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Procambarus clarkii.
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1. Introduction
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Ferritin is a primary iron storage protein of most living organisms that
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participates in many biological processes, including antioxidation, angiogenesis, cell
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activation, elimination of the toxicity of some heavy metals and other toxins, immune
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response, and regulation of iron metabolic balance [1,2]. The ferritin proteins,
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consisting of 24 subunits arranged to form a hollow shell with high iron-binding
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capacity (4500 Fe atoms), have similar structures, including a Ferritin domain of
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approximately 140 residues [3]. Ferritin was first characterized and crystallized by
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Laufberger in 1937 from the spleen and liver of horse [4].
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Ferritins are highly conserved proteins and most likely present in all eukaryotes. Ferritins have been widely studied in bacteria, fungi, plants, and animals [5]. The
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ferritin mRNA sequences contain an iron response element (IRE) in their 5ʹ-UTR,
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which regulates the translation of ferritin by interacting with the iron responsive
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element binding protein (IRP)-1 or IRP-2 [6]. Ferritin has been cloned and
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characterized from crustaceans including P. leniusculus [7], Artemia franciscana [8],
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Fenneropenaeus chinensis [9], Litopenaeus vannamei [10], Marsupenaeus japonicas
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[11], Macrobrachium rosenbergii [12], Macrobrachium nipponense [13], Eriocheir
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sinensis [14], and others. Transcriptional expression levels of ferritin can be
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upregulated upon various challenges, for example, high heavy metal concentration,
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pathogens, pH stress, oxidative stress, and thermal stress [15-23]. However, little
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information is available on the regulation and genomic structure of the ferritin in the
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red swamp crayfish Procambarus clarkii.
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P. clarkii is a freshwater crayfish widespread in lakes and rivers, that has been
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widely cultured in Asian countries, especially China, owing to its delicious meat, high
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market value, high migration ability, resistance to environmental changes, and high
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tolerance to low water quality [24]. Infectious diseases and water pollution result in
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large economic losses and food safety problems in farmed P. clarkii; however, little is
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known about the molecular responses of the crayfish. In this paper, we cloned the
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full-length cDNA of ferritin from P. clarkii by RACE-PCR, and investigated its
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expression patterns in different tissues, and in immune response and under heavy
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metal stress, respectively. The data provide novel insight into the physiological role of
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ferritin in P. clarkii.
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2. Materials and Methods
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2.1. Experimental crayfish, heavy metal injections, and lipopolysaccharide (LPS)
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challenge
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Crayfish of both sexes, each about 10 g, were purchased from an aquatic products market in Yancheng, Jiangsu Province, China, in May 2015, and were cultured at
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room temperature before commencing the experiment. Eight tissues were dissected:
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gill, heart, hepatopancreas, hemocytes, intestine, muscle, ovary, and testis. For heavy
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metal injections, 60 crayfish were injected in the abdominal muscle with 10 µL sterile
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solution containing ZnSO4 (12 mg/L), CuSO4 (2 mg/L), Fe2(SO4)3 (6 mg/L), or
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K2Cr2O7 (4 mg/L), respectively, while 10 µL sterile water was injected into the
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crayfish in the negative control group. Hepatopancreases of crayfish from each group
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were collected 0, 3, 6, 9, and 12 h after heavy metal injection. To determine immune
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response, 40 crayfish were randomly divided into two groups: one group was
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challenged by injecting 10 µL LPS (1 µg/µL) into the abdominal muscle, the control
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group was injected with 10 µL PBS. After treatment, hepatopancreases were collected
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at 3, 6, 12, 24, and 36 h. All samples were immediately frozen in liquid nitrogen and
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stored at −80 °C until RNA extraction.
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2.2. RNA extraction and cDNA synthesis
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Total RNA was extracted using TRIzol reagent (Aidlab, China) per the
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manufacturer’s instructions. RNase-free DNase I (Promega, USA) was used to remove contaminating genomic DNA. RNA integrity and DNA contamination were
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assessed on a 1% formaldehyde gel. The concentration of RNA in the samples was
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measured using a NanoDrop 2000c spectrophotometer. Only samples with 260
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nm/280 nm absorbance ratios ranging from 1.8 to 2.0 were used. First strand cDNA
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was generated by using 1 µg hepatopancreas of total RNA per sample with a
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TRUEscript cDNA Synthesis Kit (Aidlab). For rapid amplification of the cDNA ends
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(RACE), single-stranded cDNAs were synthesized using the SMART™ RACE
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cDNA Amplification Kit (Clontech, USA).
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2.3. Cloning of PcFer gene
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The full-length cDNA of PcFer was obtained by using reverse transcription PCR
ACCEPTED MANUSCRIPT and RACE-PCR. Oligonucleotide primers (Table 1) were designed using Primer
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Premier 5.0 software. The primers RC3 and RC5 were used for RACE-PCR; the
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thermal program consisted of 5 min at 94 °C, followed by 5 cycles of 94 °C for 1 min
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and 65 °C for 2 min, and then 35 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C
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for 40 s. The PCR products were analyzed on 1% agarose gels (Aidlab), then purified
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PCR products were ligated into the T-vector (Sangon, China) and sequenced
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(Sunbiotech, China).
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2.4. Sequence analysis of PcFer
BLAST searches were performed at http://www.ncbi.nlm.nih.gov/blast.cgi.
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Translation of the cDNA was performed using the Expert Protein Analysis System
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(ExPAsy; http://au.expasy.org/), and the translated sequence was analyzed using
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DNASTAR software (Madison, USA). The isoelectric point (pI) and molecular
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weight (MW) of the deduced amino acid sequence were predicted using the Compute
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pI/MW Tool at ExPAsy (http://web.expasy.org/compute_pi/). Putative signal peptide
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prediction was performed using the SignalP 4.0 Server
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(http://www.cbs.dtu.dk/services/SignalP/). A motif scan was performed at
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http://hits.isb-sib.ch/cgi-bin/motif_scan. The functional domain was predicted by
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using SMART software (http://smart.embl-heidelberg.de/). The transmembrane
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protein topological structure was analyzed with the TMHMM server on-line tool
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(http://www.cbs.dtu.dk/services/TMHMM/).
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2.5. Homologous alignment and phylogenetic analysis
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Homology searches were performed using BLASTn and BLASTp at the National
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Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). The amino
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acid sequences of ferritin from different organisms used for phylogenetic analysis
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were downloaded from GenBank. Multiple sequence alignments were carried out
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using Clustal X software [25]. A phylogenetic tree was constructed using Molecular
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Evolutionary Genetics Analysis (MEGA) version 6.0 [26]. The data were analyzed
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using Poisson correction, and gaps were removed by complete deletion. The
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topological stability of the neighbor-joining trees was evaluated by 1000 bootstrap
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replications.
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2.6. qPCR analysis of expression patterns of PcFer qPCR was performed to determine mRNA expression levels of the PcFer gene in
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several tissues and following heavy metal injection and LPS challenge. The 18S
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rRNA gene (GenBank: AF436001) was selected as the internal reference for
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normalization. Table 1 lists the primers used in qPCR. The qPCR was performed on a
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Mastercycler ep realplex (Eppendorf, Germany) using the 2× SYBR Green qPCR Mix
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Kit (Aidlab). Reaction mixtures (20 µL) contained 10 µL 2× SYBR® Premix Ex
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Taq™ buffer, 1 µL forward and reverse primers, 1 µL cDNA, and 7 µL RNase-free
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H2O. The PCR procedure was: 95 °C for 10 s, followed by 40 cycles of 95 °C for 15 s,
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60 °C for 15 s and 72 °C for 30 s. At the end of the reaction, a melting curve was
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produced by monitoring the fluorescence continuously while slowly heating the
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sample from 60 to 95 °C. Each independent experiment was conducted in triplicate
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and the relative expression level of the gene was determined using a previously
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reported method [27].
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2.7. Data analysis
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Data are presented as the mean ± standard error of the mean (SEM). The data
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were subjected to one-way analysis of variance, followed by Duncan’s multiple range
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test. Statistical analysis was performed using SPSS 16.0 software (SPSS, USA) Data
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were considered statistically significant when the P-value was <0.05 and highly
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significant for P < 0.01.
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3. Results and discussion
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3.1. Sequence analysis of the PcFer gene
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The PcFer gene was isolated from hepatopancreas of red swamp crayfish. Figure
ACCEPTED MANUSCRIPT 1 shows the nucleotide and deduced amino acid sequences of PcFer. The full-length
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cDNA of PcFer was obtained by RT-PCR and RACE-PCR. It contains a 135-bp
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5′-untranslated region (UTR), a putative ORF of 513 bp encoding a polypeptide of
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170 amino acids which contains the Ferritin domain, a 374-bp 3′-UTR with a
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polyadenylation signal sequence AATAAA at position 675–680, and a 29-bp poly (A)
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tail. Similar results were found for F. chinensis [9] and L. vannamei [10], suggesting
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that the ferritin genes have similar functions. Based on the entire amino acid sequence,
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the predicted molecular weight and isoelectric point of PcFer were 19.39 kDa and
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5.11, respectively. No signal peptide was predicted by the SignalP 4.1 Server,
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suggesting that PcFer is not a secretory protein. A putative IRE was observed in the
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5ʹ-UTR with a well-conserved 5ʹ-CAGUGU-3ʹ sequence [28]. Transmembrane
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topological structure was found in PcFer protein. The 3D structure of PcFer protein
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predicted by the SWISS-MODEL protein fold server contains an N-terminus,
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C-terminus, four long α-helices, and one short helix, similar to that of ferritin from
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other animals [29]. Putative ion binding sites (red) at position 24, 31, 58, 59, 62, 104,
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and 138, putative ferrihydrite nucleation center (blue) at position 54-61, and putative
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iron ion channel (yellow) at position 115, 128, and 131, respectively (Fig. 2).
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Conserved domain prediction using SMART software showed that the PcFer protein
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contains the Ferritin domain; this binds a mineral core of hydrated ferric oxide, and
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forms a multi-subunit protein shell that encloses the former and assures its solubility
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in an aqueous environment [30]. The P. clarkii ferritin cDNA sequence has been
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deposited in NCBI with GenBank accession number KM283424.
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3.2. Homologous alignment and phylogenetic analysis
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Figure 2 shows the deduced amino acid sequence of PcFer aligned with several
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other known and predicted ferritin peptides by Clustal X software to assess the
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relatedness of the ferritins. The PcFer protein sequence has 86%, 61% and 58%
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identity with those from P. leniusculus, Danio rerio, and Homo sapiens, respectively.
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Sequence alignment and prediction of functional domains by BLASTp revealed the
ACCEPTED MANUSCRIPT amino acid sequences of conserved features of PcFer, which were identical to those of
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known ferritins; for example, seven amino acid residues (Glu24, Try31, Glu58, Glu59,
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His62, Glu104, Gln138) form the ferroxidase diiron center involved in ion binding
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[31], three residues (His115, Lys128, Glu131) constitute the iron ion channel [31,32],
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and four residues (Glu54, Asp57, Glu58, Glu61) form the ferrihydrite nucleation
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center, which plays a role in iron nucleation [33].
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To classify and analyze the molecular evolution of ferritins, 24 representative
ferritin sequences were used to reconstruct their phylogenetic relationships based on
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amino acid sequences. The evolutionary history was inferred using the
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neighbor-joining method, with complete deletion of gaps and 1000 bootstrap
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replications. The sequences could be classified into two main clades with high
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bootstrap support, corresponding to invertebrates and vertebrates (Fig. 3). Invertebrate
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ferritins clustered into two main groups. PcFer was closely related to the ferritin of P.
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leniusculus, and then clustered with E. sinensis. Overall, the phylogenetic tree was
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consistent with relationships between species. The results suggest that ferritin proteins
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are highly conserved in animals [34].
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3.3. Tissue distribution of the PcFer gene
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to determine PcFer mRNA transcripts. The expression level in each of the tissues
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examined was normalized relative to that of the 18S rRNA gene and compared with
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expression in hemocytes (i.e. hemocyte expression was defined as 1). The PcFer gene
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was expressed in all tested tissues; the highest expression level was in hepatopancreas
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which is thought to be an important metabolizing organ [35], followed by intestine,
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muscle and gill; the lowest expression was in hemocytes (Fig. 4). Similar results were
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found in other animals: a study of M. nipponense showed that ferritin was expressed
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most in the hepatopancreas and least in the heart and hemocytes [13]; the expression
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of ferritin in M. rosenbergii and Scylla paramamosain was high in the hepatopancreas,
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but the gene was not expressed in hemocytes [12,36]. However, in contrast to our
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ACCEPTED MANUSCRIPT results, the highest expression levels of ferritin in M. japonicus and L. vannamei were
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detected in hemocytes [36]. Both hemocytes and the hepatopancreas are major
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metabolic centers for the production of reactive oxygen species in crustaceans [37].
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The different expression patterns across different species suggest that ferritin might
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play different roles in different species. Further studies are needed to examine the
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different functions of ferritin in different tissues and species.
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3.4. Quantitative analysis of PcFer mRNA after heavy metal challenge
To determine the transcriptional expression profile of PcFer in response to heavy
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metal challenge, crayfish were injected with Fe3+, Cu2+, Zn2+ or Cr6+ solution. qPCR
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was employed to examine the PcFer transcription levels in the hepatopancreas at
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different time points. Compared with the control, the mRNA expression level of
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PcFer in the hepatopancreas was significantly increased at 6 h after injection with
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Zn2+ solution (Fig. 5a). The expression of PcFer was significantly upregulated at 3, 9
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and 12 h in response to Cr6+ stress (Fig. 5b). Cu2+ treatment significantly induced
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PcFer mRNA expression levels compared to the control group from 3–12 h (Fig. 5c).
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After Fe3+ challenge, the expression of PcFer reached its peak at 9 h in
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hepatopancreas with the highest relative expression level we observed in these
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experiments (Fig. 5d). It has been reported that ferritin expression levels can be
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upregulated by iron and other heavy metals, for example, the expressions of ferritin in
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F. chinensis and Exopalaemon carinicauda were significantly upregulated after the
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shrimp were exposed to Fe3+, Cu2+ or Cd2+ [9, 38]. However, there were no significant
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differences in the amount of ferritin mRNA in the hepatopancreas and other tissues
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showing a high expression level in iron-injected and non-injected P. leniusculus and
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M. rosenbergii specimens [7,12], These results suggest that ferritin might play a role
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in tissue-specific transcriptional regulation upon heavy metal challenge.
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3.5. Expression of PcFer in response to LPS challenge
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ACCEPTED MANUSCRIPT qPCR was used to investigate the role of ferritin in P. clarkii immunity. The
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expression levels of PcFer were identified in hepatopancreas at different time points
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in response to LPS challenge. After LPS challenge, the expression of PcFer was
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upregulated significantly compared to the controls (Fig. 6). The peak of PcFer mRNA
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transcripts was observed 24 h post-injection. Ferritin is considered to play key roles in
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host defense responses. The expression of F. chinensis ferritin was upregulated in
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response to white spot syndrome virus (WSSV) challenge [9]. In the hepatopancreas
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of M. japonicus, the expression level of the ferritin gene reached a peak 24 h after
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injection of WSSV [11]. The mRNA levels of M. nipponense ferritin were
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significantly increased in hemocytes upon on Aeromonas hydrophila injection [13].
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The mRNA transcript levels of ferritin from the postlarvae of Fenneropenaeus indicus
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were upregulated in the head kidney and spleen after challenge with Vibrio harveyi
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[39]. Similar results were also found for Scophthalmus maximus, where the expression
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levels of ferritin were induced in liver after stimulation by LPS and
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polyinosinic:polycytidylic acid [40]. All these results imply that ferritin plays an
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important role in defense against pathogens.
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4. Conclusion
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In this study, a cDNA containing a ferritin gene was isolated from P. clarkii and characterized. We analyzed its phylogenetic relationships and expression pattern
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following different challenges. The results suggest that PcFer might play an important
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role in regulating heavy metal stress and immune responses. Further studies are
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required to uncover the detailed physiological functions and structure-function
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relationships of ferritins.
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Acknowledgements
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The authors declare no competing interests. This work was supported by the
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Natural Science Foundation of Jiangsu Province (BK20160444), the National Natural
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Laboratory for Bioresources of Saline Soils (JKLBS2014013 and JKLBS2015004),
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the Natural Science Research General Program of Jiangsu Provincial Higher
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Education Institutions (15KJB240002 and 12KJA180009), the Special Guide Fund
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Project of Agricultural Science and Technology Innovation of Yancheng city
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(YKN2014022), the Jiangsu Provincial Key Laboratory of Coastal Wetland
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Bioresources and Environmental Protection (JLCBE14006).
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[38] J. Zhang, T. Gui, J. Wang, J. Xiang, The ferritin gene in ridgetail white prawn
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Exopalaemon carinicauda: Cloning, expression and function. Int. J. Biol.
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Macromol. 72 (2015) 320-325. [39] S. Nayak, K.M. Ajay, N. Ramaiah, R.M. Meena, R.A. Sreepada, Profiling of a
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[40] W.J. Zheng, Y.H. Hu, Z.Z. Xiao, L. Sun, Cloning and analysis of a ferritin subunit from turbot (Scophthalmus maximus). Fish. Shellfish Immunol. 28 (2010)
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829-836.
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Fig. 1 Complete nucleotide and deduced amino acid sequence of the Procambarus
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clarkii ferritin (PcFer) gene. The amino acid residues are represented by one-letter
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codes. The open reading frame, from the initiation codon ATG to the termination
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codon TAA, is in uppercase. The ferritin domain is highlighted in purple, and the iron
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responsive element in the 5ʹ-untranslated region (UTR) of the gene is in grey; the
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polyadenylation sequence in the 3ʹ-UTR is underlined.
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Fig. 2 Sequence alignment of the PcFer protein with its homologs from other animals.
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Identical amino acids are highlighted in black, similar amino acids are highlighted in
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gray. Key conserved domains, motifs and residues are indicated. Putative ion binding
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sites (red) at position 24, 31, 58, 59, 62, 104, and 138, putative ferrihydrite nucleation
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center (blue) at position 54-61, and putative iron ion channel (yellow) at position 115,
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128, and 131, respectively.
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Fig. 3 Phylogenetic tree based on ferritin amino acid sequences constructed using
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MEGA version 6.06 and the neighbor-joining algorithm. Bootstrap percentages (1000
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repetitions) of the branches are indicated.
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Fig. 4 Expression analysis of PcFer mRNA in various tissues. Relative expression
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levels were analyzed by qPCR and the 18S gene was used as an internal standard.
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Gene expression level in the control group was set to 1.0. The data were expressed as
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the mean fold change (means ± SE, n = 3) relative to the untreated group.
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Fig. 5 Relative mRNA expression levels of PcFer in response to heavy metals stress
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including Zn (a), Cr (b), Cu (c), Fe (d), respectively. The 18S gene was used as an
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were expressed as the mean fold change (means ± SE, n = 3) relative to the untreated
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group. The values were significantly different to the control at the same time point
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when marked with asterisks (*P < 0.05).
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Fig. 6 Relative mRNA expression levels of PcFer in response to lipopolysaccharide
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(LPS) challenge. The 18S gene was used as an internal standard. Gene expression
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level in the control group was set to 1.0. The data were expressed as the mean fold
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change (means ± SE, n = 3) relative to the untreated group. The values were
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significantly different to the control at the same time point when marked with
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asterisks (*P < 0.05).
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Table 1 The primers used in this study
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Primer
Primer sequence (5ʹ-3ʹ)
Purpose
TCAMAYCCKCSAGAMCDACCA
RACE-PCR
RC-R
TKAAGCMCAAWGCKTCHTCCC
RACE-PCR
RC5
ACAGTTCCAAGTTGATCTGCT
RC3
ACCATGAGGACTGCGAAGCA
F1
GCACCAACAGTGCAAGAATGG
qPCR
R1
CTCGATGGACTCCACCTGCTC
qPCR
18S-F
CTGTGATGCCCTTAGATGTT
qPCR
18S-R
GCGAGGGGTAGAACATCCAA
qPCR
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RC-F
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RACE-PCR
RACE-PCR
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ACCEPTED MANUSCRIPT Highlights A ferritin gene from Procambarus clarkii was identified and characterized. The expression profiles of P. clarkii ferritin were investigated in various tissues.
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Ferritin transcripts were measured in response to LPS and heavy metal challenge.
S1050-4648(17)30098-0
DOI:
10.1016/j.fsi.2017.02.025
Reference:
YFSIM 4447
To appear in:
Fish and Shellfish Immunology
Received Date: 13 December 2016 Revised Date:
11 February 2017
Accepted Date: 17 February 2017
Please cite this article as: Liu Q-N, Xin Z-Z, Liu Y, Wang Z-F, Chen Y-J, Zhang D-Z, Jiang S-H, Chai X-Y, Zhou C-L, Tang B-P, A ferritin gene from Procambarus clarkii, molecular characterization and in response to heavy metal stress and lipopolysaccharide challenge, Fish and Shellfish Immunology (2017), doi: 10.1016/j.fsi.2017.02.025. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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A ferritin gene from Procambarus clarkii, molecular characterization
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and in response to heavy metal stress and lipopolysaccharide
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challenge
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Qiu-Ning Liu, Zhao-Zhe Xin, Yu Liu, Zheng-Fei Wang*, Yi-Jing Chen, Dai-Zhen
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Zhang, Sen-Hao Jiang, Xin-Yue Chai, Chun-Lin Zhou, Bo-Ping Tang*
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Jiangsu Key Laboratory for Bioresources of Saline Soils, Jiangsu Synthetic
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Innovation Center for Coastal Bio-agriculture, Jiangsu Provincial Key Laboratory of
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Coastal Wetland Bioresources and Environmental Protection, School of Ocean and
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Biological Engineering, Yancheng Teachers University, Yancheng 224001, Jiangsu
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Province, People's Republic of China.
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* Corresponding author: Zheng-Fei Wang and Bo-Ping Tang
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E-mail: [email protected] (Zheng-Fei Wang) and [email protected] (Bo-Ping Tang)
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Address: Jiangsu Key Laboratory for Bioresources of Saline Soils, Jiangsu Provincial
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Key
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Protection, Jiangsu Synthetic Innovation Center for Coastal Bio-agriculture, School of
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Ocean and Biological Engineering, Yancheng Teachers University, Yancheng 224051,
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Jiangsu Province, People's Republic of China.
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Coastal
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ACCEPTED MANUSCRIPT ABSTRACT
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Ferritin plays important roles in iron storage, detoxification, and immune response.
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Here, a ferritin gene (PcFer) was identified in Procambarus clarkii, an economically
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important freshwater crayfish. Full-length PcFer cDNA was 1022-bp, including a
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135-bp 5ʹ-untranslated region (UTR) with a typical iron responsive element, a 374-bp
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3ʹ-UTR, and a 513-bp open reading frame encoding a polypeptide of 170 amino acids
36
which contained the Ferritin domain. PcFer has ion binding sites, a ferrihydrite
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nucleation center, and an iron ion channel. PcFer is phylogenetically closely-related to
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Pacifastacus leniusculus and Eriocheir sinensis ferritins. Real-time quantitative
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reverse-transcription PCR analysis showed that PcFer was expressed in all tested P.
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clarkii tissues, and expressed most in hepatopancreas. After challenge with various
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heavy metals and lipopolysaccharide, respectively, the hepatopancreatic expression
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levels of PcFer were markedly upregulated. These results suggest that expression of
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PcFer might be involved in immune defense and protection of P. clarkii against
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heavy metal stress.
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Keywords: Expression analysis; Ferritin; Heavy metal stress; Immune response;
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Procambarus clarkii.
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1. Introduction
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Ferritin is a primary iron storage protein of most living organisms that
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participates in many biological processes, including antioxidation, angiogenesis, cell
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activation, elimination of the toxicity of some heavy metals and other toxins, immune
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response, and regulation of iron metabolic balance [1,2]. The ferritin proteins,
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consisting of 24 subunits arranged to form a hollow shell with high iron-binding
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capacity (4500 Fe atoms), have similar structures, including a Ferritin domain of
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approximately 140 residues [3]. Ferritin was first characterized and crystallized by
56
Laufberger in 1937 from the spleen and liver of horse [4].
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Ferritins are highly conserved proteins and most likely present in all eukaryotes. Ferritins have been widely studied in bacteria, fungi, plants, and animals [5]. The
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ferritin mRNA sequences contain an iron response element (IRE) in their 5ʹ-UTR,
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which regulates the translation of ferritin by interacting with the iron responsive
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element binding protein (IRP)-1 or IRP-2 [6]. Ferritin has been cloned and
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characterized from crustaceans including P. leniusculus [7], Artemia franciscana [8],
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Fenneropenaeus chinensis [9], Litopenaeus vannamei [10], Marsupenaeus japonicas
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[11], Macrobrachium rosenbergii [12], Macrobrachium nipponense [13], Eriocheir
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sinensis [14], and others. Transcriptional expression levels of ferritin can be
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upregulated upon various challenges, for example, high heavy metal concentration,
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pathogens, pH stress, oxidative stress, and thermal stress [15-23]. However, little
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information is available on the regulation and genomic structure of the ferritin in the
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red swamp crayfish Procambarus clarkii.
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P. clarkii is a freshwater crayfish widespread in lakes and rivers, that has been
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widely cultured in Asian countries, especially China, owing to its delicious meat, high
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market value, high migration ability, resistance to environmental changes, and high
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tolerance to low water quality [24]. Infectious diseases and water pollution result in
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large economic losses and food safety problems in farmed P. clarkii; however, little is
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known about the molecular responses of the crayfish. In this paper, we cloned the
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full-length cDNA of ferritin from P. clarkii by RACE-PCR, and investigated its
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expression patterns in different tissues, and in immune response and under heavy
78
metal stress, respectively. The data provide novel insight into the physiological role of
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ferritin in P. clarkii.
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2. Materials and Methods
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2.1. Experimental crayfish, heavy metal injections, and lipopolysaccharide (LPS)
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challenge
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Crayfish of both sexes, each about 10 g, were purchased from an aquatic products market in Yancheng, Jiangsu Province, China, in May 2015, and were cultured at
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room temperature before commencing the experiment. Eight tissues were dissected:
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gill, heart, hepatopancreas, hemocytes, intestine, muscle, ovary, and testis. For heavy
87
metal injections, 60 crayfish were injected in the abdominal muscle with 10 µL sterile
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solution containing ZnSO4 (12 mg/L), CuSO4 (2 mg/L), Fe2(SO4)3 (6 mg/L), or
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K2Cr2O7 (4 mg/L), respectively, while 10 µL sterile water was injected into the
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crayfish in the negative control group. Hepatopancreases of crayfish from each group
91
were collected 0, 3, 6, 9, and 12 h after heavy metal injection. To determine immune
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response, 40 crayfish were randomly divided into two groups: one group was
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challenged by injecting 10 µL LPS (1 µg/µL) into the abdominal muscle, the control
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group was injected with 10 µL PBS. After treatment, hepatopancreases were collected
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at 3, 6, 12, 24, and 36 h. All samples were immediately frozen in liquid nitrogen and
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stored at −80 °C until RNA extraction.
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2.2. RNA extraction and cDNA synthesis
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Total RNA was extracted using TRIzol reagent (Aidlab, China) per the
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manufacturer’s instructions. RNase-free DNase I (Promega, USA) was used to remove contaminating genomic DNA. RNA integrity and DNA contamination were
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assessed on a 1% formaldehyde gel. The concentration of RNA in the samples was
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measured using a NanoDrop 2000c spectrophotometer. Only samples with 260
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nm/280 nm absorbance ratios ranging from 1.8 to 2.0 were used. First strand cDNA
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was generated by using 1 µg hepatopancreas of total RNA per sample with a
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TRUEscript cDNA Synthesis Kit (Aidlab). For rapid amplification of the cDNA ends
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(RACE), single-stranded cDNAs were synthesized using the SMART™ RACE
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cDNA Amplification Kit (Clontech, USA).
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2.3. Cloning of PcFer gene
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The full-length cDNA of PcFer was obtained by using reverse transcription PCR
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Premier 5.0 software. The primers RC3 and RC5 were used for RACE-PCR; the
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thermal program consisted of 5 min at 94 °C, followed by 5 cycles of 94 °C for 1 min
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and 65 °C for 2 min, and then 35 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C
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for 40 s. The PCR products were analyzed on 1% agarose gels (Aidlab), then purified
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PCR products were ligated into the T-vector (Sangon, China) and sequenced
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(Sunbiotech, China).
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2.4. Sequence analysis of PcFer
BLAST searches were performed at http://www.ncbi.nlm.nih.gov/blast.cgi.
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Translation of the cDNA was performed using the Expert Protein Analysis System
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(ExPAsy; http://au.expasy.org/), and the translated sequence was analyzed using
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DNASTAR software (Madison, USA). The isoelectric point (pI) and molecular
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weight (MW) of the deduced amino acid sequence were predicted using the Compute
123
pI/MW Tool at ExPAsy (http://web.expasy.org/compute_pi/). Putative signal peptide
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prediction was performed using the SignalP 4.0 Server
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(http://www.cbs.dtu.dk/services/SignalP/). A motif scan was performed at
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http://hits.isb-sib.ch/cgi-bin/motif_scan. The functional domain was predicted by
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using SMART software (http://smart.embl-heidelberg.de/). The transmembrane
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protein topological structure was analyzed with the TMHMM server on-line tool
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(http://www.cbs.dtu.dk/services/TMHMM/).
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2.5. Homologous alignment and phylogenetic analysis
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Homology searches were performed using BLASTn and BLASTp at the National
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Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). The amino
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acid sequences of ferritin from different organisms used for phylogenetic analysis
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were downloaded from GenBank. Multiple sequence alignments were carried out
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using Clustal X software [25]. A phylogenetic tree was constructed using Molecular
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Evolutionary Genetics Analysis (MEGA) version 6.0 [26]. The data were analyzed
137
using Poisson correction, and gaps were removed by complete deletion. The
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topological stability of the neighbor-joining trees was evaluated by 1000 bootstrap
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replications.
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2.6. qPCR analysis of expression patterns of PcFer qPCR was performed to determine mRNA expression levels of the PcFer gene in
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several tissues and following heavy metal injection and LPS challenge. The 18S
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rRNA gene (GenBank: AF436001) was selected as the internal reference for
144
normalization. Table 1 lists the primers used in qPCR. The qPCR was performed on a
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Mastercycler ep realplex (Eppendorf, Germany) using the 2× SYBR Green qPCR Mix
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Kit (Aidlab). Reaction mixtures (20 µL) contained 10 µL 2× SYBR® Premix Ex
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Taq™ buffer, 1 µL forward and reverse primers, 1 µL cDNA, and 7 µL RNase-free
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H2O. The PCR procedure was: 95 °C for 10 s, followed by 40 cycles of 95 °C for 15 s,
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60 °C for 15 s and 72 °C for 30 s. At the end of the reaction, a melting curve was
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produced by monitoring the fluorescence continuously while slowly heating the
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sample from 60 to 95 °C. Each independent experiment was conducted in triplicate
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and the relative expression level of the gene was determined using a previously
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reported method [27].
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2.7. Data analysis
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Data are presented as the mean ± standard error of the mean (SEM). The data
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were subjected to one-way analysis of variance, followed by Duncan’s multiple range
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test. Statistical analysis was performed using SPSS 16.0 software (SPSS, USA) Data
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were considered statistically significant when the P-value was <0.05 and highly
159
significant for P < 0.01.
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3. Results and discussion
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3.1. Sequence analysis of the PcFer gene
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The PcFer gene was isolated from hepatopancreas of red swamp crayfish. Figure
ACCEPTED MANUSCRIPT 1 shows the nucleotide and deduced amino acid sequences of PcFer. The full-length
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cDNA of PcFer was obtained by RT-PCR and RACE-PCR. It contains a 135-bp
165
5′-untranslated region (UTR), a putative ORF of 513 bp encoding a polypeptide of
166
170 amino acids which contains the Ferritin domain, a 374-bp 3′-UTR with a
167
polyadenylation signal sequence AATAAA at position 675–680, and a 29-bp poly (A)
168
tail. Similar results were found for F. chinensis [9] and L. vannamei [10], suggesting
169
that the ferritin genes have similar functions. Based on the entire amino acid sequence,
170
the predicted molecular weight and isoelectric point of PcFer were 19.39 kDa and
171
5.11, respectively. No signal peptide was predicted by the SignalP 4.1 Server,
172
suggesting that PcFer is not a secretory protein. A putative IRE was observed in the
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5ʹ-UTR with a well-conserved 5ʹ-CAGUGU-3ʹ sequence [28]. Transmembrane
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topological structure was found in PcFer protein. The 3D structure of PcFer protein
175
predicted by the SWISS-MODEL protein fold server contains an N-terminus,
176
C-terminus, four long α-helices, and one short helix, similar to that of ferritin from
177
other animals [29]. Putative ion binding sites (red) at position 24, 31, 58, 59, 62, 104,
178
and 138, putative ferrihydrite nucleation center (blue) at position 54-61, and putative
179
iron ion channel (yellow) at position 115, 128, and 131, respectively (Fig. 2).
180
Conserved domain prediction using SMART software showed that the PcFer protein
181
contains the Ferritin domain; this binds a mineral core of hydrated ferric oxide, and
182
forms a multi-subunit protein shell that encloses the former and assures its solubility
183
in an aqueous environment [30]. The P. clarkii ferritin cDNA sequence has been
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deposited in NCBI with GenBank accession number KM283424.
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3.2. Homologous alignment and phylogenetic analysis
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Figure 2 shows the deduced amino acid sequence of PcFer aligned with several
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other known and predicted ferritin peptides by Clustal X software to assess the
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relatedness of the ferritins. The PcFer protein sequence has 86%, 61% and 58%
189
identity with those from P. leniusculus, Danio rerio, and Homo sapiens, respectively.
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Sequence alignment and prediction of functional domains by BLASTp revealed the
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known ferritins; for example, seven amino acid residues (Glu24, Try31, Glu58, Glu59,
193
His62, Glu104, Gln138) form the ferroxidase diiron center involved in ion binding
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[31], three residues (His115, Lys128, Glu131) constitute the iron ion channel [31,32],
195
and four residues (Glu54, Asp57, Glu58, Glu61) form the ferrihydrite nucleation
196
center, which plays a role in iron nucleation [33].
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To classify and analyze the molecular evolution of ferritins, 24 representative
ferritin sequences were used to reconstruct their phylogenetic relationships based on
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amino acid sequences. The evolutionary history was inferred using the
200
neighbor-joining method, with complete deletion of gaps and 1000 bootstrap
201
replications. The sequences could be classified into two main clades with high
202
bootstrap support, corresponding to invertebrates and vertebrates (Fig. 3). Invertebrate
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ferritins clustered into two main groups. PcFer was closely related to the ferritin of P.
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leniusculus, and then clustered with E. sinensis. Overall, the phylogenetic tree was
205
consistent with relationships between species. The results suggest that ferritin proteins
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are highly conserved in animals [34].
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3.3. Tissue distribution of the PcFer gene
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to determine PcFer mRNA transcripts. The expression level in each of the tissues
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examined was normalized relative to that of the 18S rRNA gene and compared with
211
expression in hemocytes (i.e. hemocyte expression was defined as 1). The PcFer gene
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was expressed in all tested tissues; the highest expression level was in hepatopancreas
213
which is thought to be an important metabolizing organ [35], followed by intestine,
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muscle and gill; the lowest expression was in hemocytes (Fig. 4). Similar results were
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found in other animals: a study of M. nipponense showed that ferritin was expressed
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most in the hepatopancreas and least in the heart and hemocytes [13]; the expression
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of ferritin in M. rosenbergii and Scylla paramamosain was high in the hepatopancreas,
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but the gene was not expressed in hemocytes [12,36]. However, in contrast to our
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detected in hemocytes [36]. Both hemocytes and the hepatopancreas are major
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metabolic centers for the production of reactive oxygen species in crustaceans [37].
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The different expression patterns across different species suggest that ferritin might
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play different roles in different species. Further studies are needed to examine the
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different functions of ferritin in different tissues and species.
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3.4. Quantitative analysis of PcFer mRNA after heavy metal challenge
To determine the transcriptional expression profile of PcFer in response to heavy
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metal challenge, crayfish were injected with Fe3+, Cu2+, Zn2+ or Cr6+ solution. qPCR
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was employed to examine the PcFer transcription levels in the hepatopancreas at
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different time points. Compared with the control, the mRNA expression level of
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PcFer in the hepatopancreas was significantly increased at 6 h after injection with
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Zn2+ solution (Fig. 5a). The expression of PcFer was significantly upregulated at 3, 9
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and 12 h in response to Cr6+ stress (Fig. 5b). Cu2+ treatment significantly induced
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PcFer mRNA expression levels compared to the control group from 3–12 h (Fig. 5c).
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After Fe3+ challenge, the expression of PcFer reached its peak at 9 h in
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hepatopancreas with the highest relative expression level we observed in these
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experiments (Fig. 5d). It has been reported that ferritin expression levels can be
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upregulated by iron and other heavy metals, for example, the expressions of ferritin in
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F. chinensis and Exopalaemon carinicauda were significantly upregulated after the
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shrimp were exposed to Fe3+, Cu2+ or Cd2+ [9, 38]. However, there were no significant
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differences in the amount of ferritin mRNA in the hepatopancreas and other tissues
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showing a high expression level in iron-injected and non-injected P. leniusculus and
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M. rosenbergii specimens [7,12], These results suggest that ferritin might play a role
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in tissue-specific transcriptional regulation upon heavy metal challenge.
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3.5. Expression of PcFer in response to LPS challenge
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expression levels of PcFer were identified in hepatopancreas at different time points
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in response to LPS challenge. After LPS challenge, the expression of PcFer was
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upregulated significantly compared to the controls (Fig. 6). The peak of PcFer mRNA
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transcripts was observed 24 h post-injection. Ferritin is considered to play key roles in
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host defense responses. The expression of F. chinensis ferritin was upregulated in
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response to white spot syndrome virus (WSSV) challenge [9]. In the hepatopancreas
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of M. japonicus, the expression level of the ferritin gene reached a peak 24 h after
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injection of WSSV [11]. The mRNA levels of M. nipponense ferritin were
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significantly increased in hemocytes upon on Aeromonas hydrophila injection [13].
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The mRNA transcript levels of ferritin from the postlarvae of Fenneropenaeus indicus
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were upregulated in the head kidney and spleen after challenge with Vibrio harveyi
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[39]. Similar results were also found for Scophthalmus maximus, where the expression
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levels of ferritin were induced in liver after stimulation by LPS and
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polyinosinic:polycytidylic acid [40]. All these results imply that ferritin plays an
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important role in defense against pathogens.
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4. Conclusion
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In this study, a cDNA containing a ferritin gene was isolated from P. clarkii and characterized. We analyzed its phylogenetic relationships and expression pattern
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following different challenges. The results suggest that PcFer might play an important
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role in regulating heavy metal stress and immune responses. Further studies are
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required to uncover the detailed physiological functions and structure-function
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relationships of ferritins.
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Acknowledgements
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The authors declare no competing interests. This work was supported by the
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Natural Science Foundation of Jiangsu Province (BK20160444), the National Natural
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Laboratory for Bioresources of Saline Soils (JKLBS2014013 and JKLBS2015004),
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the Natural Science Research General Program of Jiangsu Provincial Higher
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Education Institutions (15KJB240002 and 12KJA180009), the Special Guide Fund
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Project of Agricultural Science and Technology Innovation of Yancheng city
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(YKN2014022), the Jiangsu Provincial Key Laboratory of Coastal Wetland
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Bioresources and Environmental Protection (JLCBE14006).
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ACCEPTED MANUSCRIPT Figure and Table captions
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Fig. 1 Complete nucleotide and deduced amino acid sequence of the Procambarus
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clarkii ferritin (PcFer) gene. The amino acid residues are represented by one-letter
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codes. The open reading frame, from the initiation codon ATG to the termination
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codon TAA, is in uppercase. The ferritin domain is highlighted in purple, and the iron
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responsive element in the 5ʹ-untranslated region (UTR) of the gene is in grey; the
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polyadenylation sequence in the 3ʹ-UTR is underlined.
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Fig. 2 Sequence alignment of the PcFer protein with its homologs from other animals.
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Identical amino acids are highlighted in black, similar amino acids are highlighted in
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gray. Key conserved domains, motifs and residues are indicated. Putative ion binding
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sites (red) at position 24, 31, 58, 59, 62, 104, and 138, putative ferrihydrite nucleation
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center (blue) at position 54-61, and putative iron ion channel (yellow) at position 115,
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128, and 131, respectively.
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Fig. 3 Phylogenetic tree based on ferritin amino acid sequences constructed using
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MEGA version 6.06 and the neighbor-joining algorithm. Bootstrap percentages (1000
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repetitions) of the branches are indicated.
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Fig. 4 Expression analysis of PcFer mRNA in various tissues. Relative expression
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levels were analyzed by qPCR and the 18S gene was used as an internal standard.
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Gene expression level in the control group was set to 1.0. The data were expressed as
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the mean fold change (means ± SE, n = 3) relative to the untreated group.
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Fig. 5 Relative mRNA expression levels of PcFer in response to heavy metals stress
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including Zn (a), Cr (b), Cu (c), Fe (d), respectively. The 18S gene was used as an
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ACCEPTED MANUSCRIPT internal standard. Gene expression level in the control group was set to 1.0. The data
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were expressed as the mean fold change (means ± SE, n = 3) relative to the untreated
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group. The values were significantly different to the control at the same time point
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when marked with asterisks (*P < 0.05).
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Fig. 6 Relative mRNA expression levels of PcFer in response to lipopolysaccharide
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(LPS) challenge. The 18S gene was used as an internal standard. Gene expression
430
level in the control group was set to 1.0. The data were expressed as the mean fold
431
change (means ± SE, n = 3) relative to the untreated group. The values were
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significantly different to the control at the same time point when marked with
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asterisks (*P < 0.05).
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Table 1 The primers used in this study
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Primer
Primer sequence (5ʹ-3ʹ)
Purpose
TCAMAYCCKCSAGAMCDACCA
RACE-PCR
RC-R
TKAAGCMCAAWGCKTCHTCCC
RACE-PCR
RC5
ACAGTTCCAAGTTGATCTGCT
RC3
ACCATGAGGACTGCGAAGCA
F1
GCACCAACAGTGCAAGAATGG
qPCR
R1
CTCGATGGACTCCACCTGCTC
qPCR
18S-F
CTGTGATGCCCTTAGATGTT
qPCR
18S-R
GCGAGGGGTAGAACATCCAA
qPCR
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RC-F
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RACE-PCR
RACE-PCR
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ACCEPTED MANUSCRIPT Highlights A ferritin gene from Procambarus clarkii was identified and characterized. The expression profiles of P. clarkii ferritin were investigated in various tissues.
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Ferritin transcripts were measured in response to LPS and heavy metal challenge.