MOLECULAR CLONING, CHARACTERIZATION, AND EXPRESSION ANALYSIS OF TWO ISOFORMS OF ANTI-LIPOPOLYSACCHARIDE FACTOR FROM THE ORIENTAL RIVER PRAWN, MACROBRACHIUM NIPPONENSE

WANG Ying-Hui, XIU Yun-Ji, GU Wei, MENG Qing-Guoand WANG Wen

(1. Jiangsu Key Laboratory for Biodiversity & Biotechnology and Jiangsu Key Laboratory for Aquatic Crustacean Diseases, College of Life Sciences, Nanjing Normal University, Nanjing 210023, China; 2. Key Laboratory of Sustainable Development of Marine Fisheries, Ministry of Agriculture, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China; 3. Function Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266200, China)

MOLECULAR CLONING, CHARACTERIZATION, AND EXPRESSION ANALYSIS OF TWO ISOFORMS OF ANTI-LIPOPOLYSACCHARIDE FACTOR FROM THE ORIENTAL RIVER PRAWN, MACROBRACHIUM NIPPONENSE

WANG Ying-Hui1, XIU Yun-Ji2,3, GU Wei1, MENG Qing-Guo1and WANG Wen1

(1. Jiangsu Key Laboratory for Biodiversity & Biotechnology and Jiangsu Key Laboratory for Aquatic Crustacean Diseases, College of Life Sciences, Nanjing Normal University, Nanjing 210023, China; 2. Key Laboratory of Sustainable Development of Marine Fisheries, Ministry of Agriculture, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China; 3. Function Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266200, China)

Anti-lipopolysaccharide factors (ALFs), a type of the potent antimicrobial peptide, can bind and neutralize lipopolysaccharide (LPS) and exhibit broad spectrum antimicrobial activities. In order to study the function of ALFs in congenital immunization of Macrobrachium nipponense, two isoforms of the ALF homologues (MnALF1 and MnALF2) were cloned and characterized from the oriental river prawn M. nipponense. The full-length cDNA sequences of MnALF1 and MnALF2 were 1008 and 836 bp, encoding 121 and 124 amino acids, respectively. All of these sequences contained one signal peptide and an LPS-binding domain with two conserved cysteine residues at both ends of the domain. The deduced peptide of MnALF1 and MnALF2 was highly similar to previously identified ALFs in crustaceans. qRT-PCR showed that MnALFs were expressed in all detected tissues. MnALF1 transcript was predominantly detected in heart and intestine and MnALF2 transcript was predominantly detected in hemocytes and hepatopancreas. After challenge with Aeromonas hydrophila, two MnALF transcripts in heart, intestine, hemocytes and hepatopancreas showed a clear time-dependent response expression pattern (the expression levels of MnALF1 present an trend of downregulated first and then increasing, which reached to the highest level at 24h, 12h, 36h, 24h in heart, intestine, hemocytes and hepatopancreas respectively. However, for MnALF2, the expression level present an downregulated trend and then increased in heart, intestine; In hemocytes and hepatopancreas, the expression level present an increasing trend and then down-regulated. MnALF2 transcripts reached to the top at 24h post-challenge). These results suggest that two MnALF isoforms have different tissue specificity and might provide multiple protective functions in immune defense against invading bacteria.

Macrobrachium nipponense; Anti-lipopolysaccharide factor; Expression analysis; Aeromonas hydrophila

The oriental river prawn Macrobrachium nipponense is a freshwater or brackish prawn and it is one of the important economic aquaculture and commercial species in China, Japan and Vietnam[1]. M. nipponense is an indigenous species and the frequency of disease outbreaks has been very low in thepast. However, with rapid development of large-scale culture, various diseases caused by genetic retrogression and viruses, bacteria has become increasingly higher[2]. It is necessary to understand the immune defense mechanisms of M. nipponense to provide new insights into disease prevention in prawn aquaculture.

Invertebrates, which lack adaptive immune systems, depend completely on the innate immune system that is composed of humoral and cellular responses[3]. Antimicrobial peptides (AMPs), components of the humoral immune responses, play essential functions in the innate immune system[4,5]. They distributed broadly in the whole living kingdom with killing activities against bacteria, fungi or viral pathogens[6].

ALF is one of the group of AMPs, exhibiting binding and neutralizing activities to lipopolysaccharides[7]. The first ALF was found in Limulus polyphemus (LpALF)[8]and showed strong antimicrobial effect on Gram-negative R-type bacteria[9]. From crystal structure analysis, the LPS binding domain of LpALF contains two highly conserved-cysteine residues[10,11]. The LpALF derived peptide modulates cytokine gene expression and promotes resolution of bacterial acute infection in mice[12]. More recently, a number of crustacean ALFs with similar functions have been reported in penaeid shrimps[13,14], freshwater prawns[15—17], crayfish[18], lobster[19], and crabs[20,21]. Antibacterial activity against both Gram-negative and Gram-positive bacteria which was confirmed by the in vitro assays in decapods[22,23]. Many investigations have revealed that ALFs presented multiple biological activities against fungi and virus[24,25].

Although AMPs have been widely investigated in invertebrates, ALF has not been isolated from M. nipponense. In the current study, two ALFs from M. nipponense were identified and characterized. Here, the full length cDNAs of MnALF1 and MnALF2 were cloned. The deduced amino acid sequences were compared with other known crustacean ALFs. The expressions of MnALF1 and MnALF2 in different tissues were investigated, and their expression profiles in heart, intestine, hemocytes and hepatopancreas were studied after challenge with Aeromonas hydrophila.

1 Materials and methods

1.1 Animal and RNA extraction

The oriental river prawns, M. nipponense (2—3 g per prawn) were purchased from an aquaculture market in Nanjing, Jiangsu Province, China. They were cultured in tanks at 20℃ with freshwater and an aeration system. Prawn health status was assessed daily during acclimation by monitoring both general activity and food intake. Total RNA from different tissues was extracted by using TRIzol Reagent following the manufacturer’s instructions. RNA quality was assessed by electrophoresis on 1.2% agarose gel and the RNA concentration was measured by the absorbance at 260 nm on a spectrophotometer.

1.2 cDNA library construction and EST analysis

A cDNA library was constructed from the hemocytes of M. nipponense (Shanghai Hanyu Bio-Tech). Random sequencing of the library using illumina Hiseq 2000 genome analyzer yielded 43289 ESTs. BLASTx analysis revealed that two ESTs were homologous to ALF in Macrobrachium rosenbergii (ADI80708 and ACG60660). These two ESTs were selected for further cloning of MnALFs.

1.3 Cloning the full-length cDNA of MnALFs

SMARTerTMRACE cDNA Amplification Kit (Takara, Japan) was used for Rapid amplification of cDNA ends (RACE). Two pairs of gene specific primers were designed based on the corresponding EST sequences. For the 5′-RACE, The PCR reactions were performed with MnALF1-R1 or MnALF2-R1 and Universal Primer A Mix (UPM). For the 3′-RACE, the PCR reactions were performed with MnALF1-F1 or MnALF2-F1 and UPM. The polymerase chain reaction was performed as follows: 1 cycle at 94℃ for 2min; 30 cycles at 94℃ for 30s, 68℃ for 30s, 72℃ for 3min, and 1 cycle at 72℃ for 10min. The PCR fragments were cloned into pMD19-T vector and sequenced by Springen (Nanjing) Biotechnology Company.

1.4 Sequence analysis

The homology searches for nucleotide and amino acid sequence similarities were conducted with BLAST programs (http://blast.ncbi.nlm.nih.gov/ Blast.cgi). The deduced amino acid sequence was analyzed with the Expert Protein Analysis System (http://www.expasy.org/). SignalP 4.1 program was utilized to predict the presence and location of signal peptide (http://www.cbs.dtu.dk/services/SignalP/). Multiple sequences alignment was performed using the ClustalW2 (http://www.ebi.ac.uk/Tools/msa/ clustalw2/). A cladogram was constructed based on the amino sequences alignment by the neighbor-joining (NJ) algorithm embedded in MEGA 5 program.

1.5 Bacterial challenge and sample preparation

During the experiment, the prawns were fed once daily with commercial feed. The prawns were randomly divided into two groups and 50 prawns injected individually with 50 μL live A. hydrophila sus-pension (104cells/mL) which used as a challenge group. For the control group, 50 prawns were injected with 50 μL saline (0.85% NaCl) (pH=7.0). Every three individuals were randomly sampled to eliminate individual differences at 0, 1h, 12h, 24h and 36h post challenge. Heart, intestine, hemocytes and hepatopancreas were collected, respectively, and all of samples extracted at different times were stored at–80℃ for subsequent total RNA extraction.

1.6 Expression analysis of MnALFs transcript

The mRNA expressions of MnALFs transcript in different tissues, including hemocytes, heart, hepatopancreas, gill, intestine, nerve and muscle of untreated prawns, and the temporal expressions of MnALFs in heart, intestine, hemocytes and hepatopancreas of prawns challenged with A. hydrophila were determined by quantitative real-time RT-PCR.

The first-strand cDNA was synthesized by using PrimeScriptTM1st Strand cDNA Synthesis Kit (TaKaRa) with 1 μg of total RNA. Two pairs of gene-specific primer (MnALF1-RT-F, MnALF1-RT-R and MnALF2-RT-F, MnALF2-RT-R) (Tab. 1) were used to amplify MnALF1 and MnALF2 genes, respectively. The primers Mnβ-actinF and Mnβ-actinR (Tab. 1) were used to amplify β-actin for internal standardization[26,27].

qRT-PCR was carried out in a total volume of 20 μL (10 μL of 2×SYBR Premix Ex Taq, 1 μL cDNA mix, 0.5 μL of each primer (10 μmol/L), and 8 μL of sterile distilled H2O) . The PCR program was 95℃for 30s, followed by 40 cycles of 95℃ for 5s and 60℃ for 30s. All samples were run thrice. All datawere given in terms of relative mRNA expression as mean±S.E. Statistical significance was determined by one-way ANOVA and post-hoc Duncan multiple range tests. Significance was set at P＜0.05.

Tab. 1 Primers used in the present study

2 Results

2.1 cDNA cloning and sequence analysis of MnALF1 and MnALF2

In this research, two different forms of ALF cDNA were identified from the oriental river prawn, M. nipponense. These ALF genes were designated as MnALF1 and MnALF2. The complete cDNA sequence of MnALFs were obtained by overlapping the corresponding EST with the amplified fragments. The complete cDNA sequence of MnALF1 is 1008 bp, including a 146 bp 5′ UTR, a 496 bp 3′ UTR, and a 366 bp ORF encoding a protein of 121 amino acids. The poly (A) tail was found in MnALF1, while no poly (A) signal (AATAAA) was detected. MnALF1 protein contains a signal peptide of 20 residues and an LPS-binding domain of 22 residues with 2 conserved cysteines. The theoretical pI of MnALF1 is 6.29 and molecular weight (Mw) is 13869.14 Da. The theoretical pI of LPS-binding domain is 9.14. The sequence data was deposited in GenBank with accession number KF696705.

The complete sequence of MnALF2 cDNA is 836 bp in length consisting of a 120 bp 5′ UTR, a 341 bp 3′ UTR, and a 375 bp ORF encoding a protein of 124 amino acids. A canonical polyadenylation signalsequence AATAAA was present at 15 nucleotides upstream of the poly (A) tail. A signal peptide of 25 amino acids and an LPS-binding domain were also detected in MnALF2. MnALF2 has a pI of 9.12 and an Mw of 13868.11 Da. The LPS-binding domain of MnALF2 has a pI of 9.14. The sequence data was deposited in GenBank with accession number KF696706.

2.2 Similarity and phylogenetic analyses

BLAST search indicated that two forms exhi bited similarity with other crustacean ALFs. MnALF1 showed the highest similarity with ALF2 and ALF3 from M. rosenbergii (81% identity). MnALF2 has 90% identity with ALF4 from M. rosenbergii and 86% identity with ALF from Macrobrachium olfersii, respectively.

Multiple sequence alignment of MnALF1 and MnALF2 with ALFs in crustaceans revealed that all these ALFs were conserved; they manifested a signal peptide and an LPS-binding domain, especially the two conserved cysteine residues at both ends of the domain.

A phylogenetic tree was constructed with ALF homologs from crustaceans to identify their evolutionary relationships (Fig. 1). Based on the tree topo-logy, ALF homologs could be classified into three main clades: A, B, and C. MnALF1 together with MrAlf3, FcAlf5 and PtAlf4 forming a small group belonged to Clade C. MnALF2 has a closer evolutionary relationship with MrAlf4 grouped into Clade A.

2.3 Tissue distributions of MnALF1 and MnALF2

qRT-PCR was employed to quantify mRNA expressions of MnALF1 and MnALF2 in the tissues of healthy prawns, including hemocytes, heart, hepatopancreas, gill, intestine, nerve and muscle (Fig. 2). The mRNA transcripts of MnALF1 and MnALF2 were detected in all the selected tissues. Fig. 2A showed that MnALF1 was mainly expressed in heart and intestine. The transcript of MnALF2 was detected mainly in the hemocytes and hepatopancreas (Fig. 2B).

Fig. 1 Neighbor-joining phylogenetic tree of crustaceans ALF genes. The reliability of the branching was tested by bootstrap resampling (1000 pseudoreplicates)

2.4 Expression profiles of MnALF1 and MnALF2 after A. hydrophila challenge

The temporal mRNA expressions of MnALF1 and MnALF2 transcript in heart, intestine, hemocytes and hepatopancreas post A. hydrophila challenge are shown in Fig. 3.

Fig. 2 Tissue distributions of MnALF1 (A) and MnALF2 (B) transcripts. Vertical bars represent the mean±S.E.

Fig. 3 After challenge with A. hydrophila, the time-course expression levels of MnALF1 and MnALF2 in heart (A), intestine (B), hemocytes (C) and hepatopancreas (D). Vertical bars represent the mean±S.E. (n=3). β-actin was used as internal control

In heart, MnALF1 was down-regulated signifi-cantly at 1h post-challenge (26%). As time progressed, the subsequent expression was up-regulated and returned to the normal level at 24h post-challenge. For MnALF2, its expression level decreased during the first 12h after A. hydrophila challenge, and the lowest level occurred at 12h (49%). MnALF2 increased from 12h to 24h and reached the highest level at 24h, which was a 2.91-fold increase compared to that in the internal group.

In intestine, both MnALF1 and MnALF2 decreased initially at 1h post challenge. Afterward, MnALF1 transcript expression increased to the peak at 12h post injection (3.14-fold). The peak of MnALF2 occurred at 24h, which was a 8.15-fold increase compared to that in the control group.

In hemocytes, the temporal increase was detected for MnALF2 at 1h post challenge. Then, MnALF1 and MnALF2 transcript expressions decreased significantly at 12h. As time progressed, the expression of MnALF2 reached the peak at 24h.

In hepatopancreas, two MnALFs transcript expression fluctuated slightly during the first 12h. At 24h, both expression levels of MnALF1 and MnALF2 increased significantly and reached to their highest levels, which were 3.62-fold and 4.29-fold higher than that in the control group, respectively.

3 Discussion

ALFs play important roles in the innate immune system of crustaceans. Previous studies reported that the potent antimicrobial activity against both bacterial and viral made ALFs becoming potential therapeutic agents[24,28]. In this study, two isoforms of ALF were identified and characterized from the oriental river prawn, M. nipponense. The deduced protein sequences of MnALF1 and MnALF2 revealed a conserved structure composed of a signal peptide, an LPS-binding domain and two conserved cysteine residues. The two cysteine residues formed a disulfide loop, within which the positively charged amino acid residues were mainly clustered. All data demonstrate that the LPS-binding domain and the positively charged amino acids are essential for ALFs biological activities[10,29,30].

Together with other ALFs, the sequences of the MnALFs were used to construct a neighbor-joining phylogenetic tree to reveal the relationships of crustacean ALFs. On the tree topology, the MnALF1 and MnALF2 cluster together with MrALF3 and MrALF4, respectively. Based on the tree topology, crustacean ALFs could be classified into three main clades: A, B, and C. MnALF1 was classified within Clade C and MnALF2 was grouped into Clade A (Fig. 1). Meanwhile, three isoforms of M. rosenbergii ALFs were classified within two Clades. In previous research, ALFs from P. monodon were also divided into two different groups according to their genomic loci[15]. It suggests that the common ancestor of decapods proba bly had two ALF homologues.

The hypothetical LPS-binding domain is stabi lized by a disulfide bond. For MnALF1, this binding site consists of six positively charged amino acid residues (three Lys, two Arg and one His). For MnALF2, this binding site consists of five positively charged amino acid residues (four Lys and one Arg). An amphipathic loop, which possesses an alternating series of hydrophilic and hydrophobic residues, is generated by the disulfide bond. It was predicted that the hydrophobic residues forming one hydrophobic side can enter into the lipid bilayer, leaving the positively charged residues on the other side pointing out to bind to the hydrophilic and phosphate groups of lipid A on the bacterial membrane[20,30].

MnALF1 was mainly expressed in the heart and intestine and MnALF2 was mainly expressed in the hemocytes and hepatopancreas. Previous studies showed that most ALF transcripts were mainly situated in hemocytes. However, inconsistent results were obtained in some crustaceans, where ALF transcripts were prominently expressed in hepatopancreas, intestines, gills, or other tissues[24]. Previous researches have demonstrated that hemocytes are the major sites of AMP production, and the intestine or gonad is the secondary expression site for AMPs[31]. In this study, the results showed that the heart and hepatopancreas are also important production sites for MnALFs.

In this study, the expression patterns of MnALF1 and MnALF2 under bacterial challenge were investigated. The expression transcripts of MnALFs mostly decreased during the initial hours of A. hydrophila challenge, which is similar to the pattern observed in PtALF5[32]and PtALF7[33]. ALF isoforms exhibit clear time-dependent expression patterns[34], but the activation times are different. For MnALF1, its expression level was down-regulated after A. hydrophila stimulation and then increased and reached to the highest level at 24h, 12h, 36h, 24h in heart, intestine, hemocytes and hepatopancreas respectively. Moreover, in heart and intestine MnALF2 was down-regulated significantly at 1h post-challenge and reached the highest level at 24h. However, in hemocytes and hepatopancreas, the expression of MnALF2 start to increase after A. hydrophila stimulation and then downregulated significantly and reached their highest levelat 24h. In short, the expression level of MnALF1 and MnALF2 transcripts reached the top at 24h post-challenge, which is the same as observed in PtALF5 and PtALF7. This result is consistent with the conclusion that ALFs possess potent antimicrobial activity against Gram-negative bacteria.

In conclusion, we cloned and characterized two isoforms of the ALF homologues (MnALF1, MnALF2) from M. nipponense. Both of them contained one signal peptide and an LPS-binding domain with two conserved cysteine residues at both ends of the domain. MnALF1 and MnALF2 had different tissue distribution and after challenge with Aeromonas hydrophila, the two MnALF transcripts in heart, intestine, hemocytes and hepatopancreas showed a clear time-dependent response expression pattern. The present results might provide multiple protective functions in immune defense against invading bacteria.

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高速的退場，已能看出相關(guān)邏輯所在。那么為何選擇西王？為何將打造了多年的“準(zhǔn)冠軍”球隊(duì)轉(zhuǎn)讓給一家民營企業(yè)——西王集團(tuán)？

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日本沼蝦兩種抗脂多糖因子的特性研究

王英慧1修云吉2,3顧 偉1孟慶國1王 文1

(1. 南京師范大學(xué)生命科學(xué)學(xué)院, 江蘇省生物多樣性與生物技術(shù)重點(diǎn)實(shí)驗(yàn)室, 江蘇省水生甲殼動(dòng)物病害重點(diǎn)實(shí)驗(yàn)室, 南京210023; 2. 中國水產(chǎn)科學(xué)研究院黃海水產(chǎn)研究所, 農(nóng)業(yè)部海洋漁業(yè)可持續(xù)發(fā)展重點(diǎn)實(shí)驗(yàn)室, 青島 266071; 3. 海洋科學(xué)與技術(shù)國家實(shí)驗(yàn)室，海洋漁業(yè)科學(xué)與食物產(chǎn)出過程功能實(shí)驗(yàn)室, 青島 266200)

為了研究抗脂多糖因子ALFs在日本沼蝦先天性免疫中的功能作用, 研究從日本沼蝦中克隆了2種抗脂多糖因子MnALF1、MnALF2。MnALF1 cDNA 全長1008 bp, 編碼121個(gè)氨基酸; MnALF2 cDNA 全長836 bp,編碼124個(gè)氨基酸。這2種氨基酸均包含有一個(gè)信號(hào)肽序列和一個(gè)LPS結(jié)合位點(diǎn), 并且在結(jié)合位點(diǎn)的兩端(N-端和C-端)都有2個(gè)保守的半胱氨酸殘基。這2種MnALFs與之前發(fā)現(xiàn)的甲殼動(dòng)物的ALFs是非常相似的。qRTPCR結(jié)果顯示MnALFs在所有被檢測的組織中均有表達(dá)。其中MnALF1主要在心臟和小腸內(nèi)表達(dá), 而MnALF2則主要在血細(xì)胞和肝胰臟中表達(dá)。在用嗜水氣單胞菌刺激之后發(fā)現(xiàn)2種MnALFs在心臟、小腸、血細(xì)胞、肝胰臟中都呈現(xiàn)出明顯的時(shí)間依賴表達(dá)模式(MnALF1在刺激之后呈現(xiàn)出先減少后增加的趨勢, 之后分別在不同組織的不同時(shí)間點(diǎn)達(dá)到最大值; 然而, 對于MnALF2, 在心臟和小腸中先減少后增加, 在血細(xì)胞和肝胰臟中呈現(xiàn)出先增加后減少, 最后都在24h達(dá)到最大值)。結(jié)果提示這2種MnALF具有不同的組織特異性, 并且在細(xì)菌侵染的免疫防御中起著重要的保護(hù)作用。

日本沼蝦; 抗脂多糖因子; 表達(dá)分析; 嗜水氣單胞菌

Q344+.1

A

1000-3207(2017)05-0977-07

10.7541/2017.122

date： 2016-06-08; Accepted date： 2016-11-12

Supported by the National Natural Science Foundation of China (31570176, 31602198); the Natural Science Foundation of Jiangsu Province (BK20151545); Project for Aquaculture in Jiangsu Province (D2015-13, Y2016-28)

Brief introduction of author： WANG Ying-Hui (1990—), Female, Heze, Shandong; Master; Research field: Disease of aquatic organism. E-mail: 1223775449@qq.com

WANG Wen (1957—), Female, Shihezi, Xinjiang; Doctor; Research field: Disease of aquatic organism. E-mail: wenwang@njnu.edu.cn