Isolation and characterization of a virulent Vibrio sp. bacterium from clams (Meretrix meretrix) with mass mortality

MM5 was a bacterial strain isolated from moribund clam (Meretrix meretrix) collected from a farm with mass mortality outbreak. Primary genotypic and phenotypic identification including 16S rDNA sequence analysis, multilocus sequence analysis (MLSA) of four housekeeping genes (gapA, ftsZ, mreB and topA) and biochemical tests suggested that strain MM5 was a Vibrio species closest to but different from Vibrio furnissii. Our previous study indicated that MM5 could induce a high mortality of M. meretrix (Yue et al., 2010). Quantitative challenge test was performed in this study to further evaluate the pathogenic potential of MM5, which showed that at 84 h post-inoculation, the cumulative mortalities of the MM5-injected group were significantly higher than those of control groups (P < 0.05). Cytopathological and histopathological features of the clam infected by MM5 were carried out by transmission electron microscopy (TEM) and Hematoxylin and Eosin (H&E) staining, respectively. Cytopathologically, foci of MM5 were found in hepatocytes of the clam infected by MM5. In addition, cytopathological lesion was detected in foot of infected clam. Histopathologically, MM5 was detected in different tissues of infected clam, including hepatopancreas, mantle and gill. Challenge test combined with pathological features indicated that MM5 was virulent to M. meretrix.     

Cloning and transcriptional analysis of two sialic acid-binding lectins (SABLs) from razor clam Solen grandis

Sialic acid-binding lectin (SABL) plays crucial role in both innate and adaptive immune responses benefiting from its predominant affinity toward glycan. In the present study, two SABLs from razor clam Solen grandis (designated as SgSABL-1 and SgSABL-2) were identified, and their expression patterns, both in tissues and towards microorganism glycan stimulation, were then characterized. The cDNA of SgSABL-1 and SgSABL-2 was 988 and 1281 bp, containing an open reading frame (ORF) of 744 and 570 bp, respectively, and deduced amino acid sequences showed high similarity to other invertebrates SABLs. Both SgSABL-1 and SgSABL-2 encoded a C1q domain. SgSABL-1 and SgSABL-2 were found to be constitutively expressed in a wide range of tissues with different levels, including mantle, gill, gonad, hemocyte, muscle, and hepatopancreas, and both of them were highly expressed in hepatopancreas. SgSABL-1 and SgSABL-2 could be significantly induced after razor clams were stimulated by acetylated subunits-containing glycan LPS and PGN, suggesting the two SgSABLs might perform potential function of glycan recognition. In addition, SgSABL-2 could also be induced by β-1,3-glucan. All these results indicated that SgSABL-1 and SgSABL-2 might be involved in the immune response against microbe infection and contributed to the pathogens recognition.     

泥蚶谷胱甘肽过氧化物酶基因的全长克隆与表达分析

为了探讨谷胱甘肽过氧化物酶（Glutathione peroxidase,GPx）基因在泥蚶应激反应中的作用,研究采用RACE技术克隆了泥蚶GPx基因（TgGPx）cDNA全长,其cDNA全长1195 bp,包含45 bp 5’-UTR,639 bp开放阅读框（ORF）和511 bp 3’-UTR。ORF编码212个氨基酸残基,预测蛋白分子量为24.3 kD,理论等电点为8.33,其中,第53个氨基酸U是由密码子202UGA204编码的硒代半胱氨酸（Se-Cys）。在3’-UTR上存在一段序列,形成一种独特的茎环结构,即SECIS元件。SECIS元件在密码子UGA翻译为Se-Cys的过程中起决定性作用。通过序列比对与系统进化分析,发现软体动物中也存在不同种类的GPx基因,TgGPx与GPx1和GPx2的亲缘关系较近。利用qRT-PCR技术对TgGPx在泥蚶的不同组织以及重金属刺激后的表达量进行分析,结果表明,TgGPx在泥蚶的5个组织中都有表达,但存在组织特异性,在外套膜中的表达量最高,在血细胞中的表达量最低。用重金属铅、铜、镉刺激后,TgGPx在肝胰脏中的表达量显著升高,表明TgGPx在维护机体正常功能方面及泥蚶抵御外界刺激的应激反应中发挥作用。     

Cloning and characterization of a hsp70 gene from Asiatic hard clam Meretrix meretrix which is involved in the immune response against bacterial infection

In the present study, a 71.43 kDa heat shock protein cDNA was cloned from Asiatic hard clam Meretrix meretrix. The cDNA was 2292 bp, containing an open reading frame (ORF) of 1959 bp, which encodes a protein of 652 amino acids with a theoretical molecular weight of 71.43 kDa and an isoelectric point of 5.32. Based on the amino acid sequence analysis and phylogenetic analysis, this hsp70 cDNA is a member of cytoplasmic hsc70 (constitutive genes) subfamily in the hsp70 family, and is designated as MmeHsc71. Quantitative RT-PCR was carried out to compare the spatial and temporal expression patterns of MmeHsc71 in the mRNA level between control clams and Vibrio parahaemolyticus-infected clams. Spatially, MmeHsc71 mRNA was found in all tested tissues, including foot, hepatopancreas, mantle and gill. MmeHsc71 mRNA expression level in hepatopancreas and gill displayed a significant increase in vibrio-challenged clams at 24h post-infection compared to control clams (P < 0.05). Temporally, there was a significant increase of MmeHsc71 mRNA level in hepathopancreas of vibrio-challenged clams compared to control clams at 6, 12, and 24h post-challenge, respectively. The result of quantitative immunofluorescence also indicated that there was obvious increase of MmeHsc71 in hepatopancreas of vibrio-challenged clams compared to control clams in protein level at 24h post-infection. The results suggested that MmeHsc71 may play an important role in mediating the immune responses of M. meretrix to bacterial challenge.     

三疣梭子蟹HMGBa基因克隆及其应答不同病原入侵的表达特征

为研究三疣梭子蟹(Portunus trituberculatus)高迁移率族蛋白B (High-mobility group box protein, HMGB)在其先天免疫中发挥的功能, 利用RACE技术首次克隆得到了三疣梭子蟹HMGBa基因, 命名为PtHMGBa。其cDNA序列全长1030 bp, 其中5′端非编码区(UTR)为94 bp, 3′端非编码区(UTR)为255 bp, 开放阅读框(ORF)为681 bp, 编码一个含有227个氨基酸, 分子量25.82 kD, 理论等电点为5.94的蛋白质。PtHMGBa蛋白包含2个HMG盒结构域和一个酸性尾部结构域。分析表明, 三疣梭子蟹HMGBa氨基酸序列与凡纳滨对虾(Litopenaeus vannamei) HMGBa相似度最高。实时荧光定量PCR结果显示, PtHMGBa基因在血细胞和肝胰腺的表达量最高, 在眼柄中表达量最低。在副溶血弧菌和WSSV感染过程中, PtHMGBa基因在肝胰腺和血细胞中均出现了表达上调。其中, 经副溶血弧菌感染后, 该基因在上述2种组织中分别于48h和6h达到表达量的峰值; 经WSSV感染后, 该基因在2种组织中均在12h达到表达量的峰值。结果表明PtHMGBa基因参与了三疣梭子蟹抵御外来病原的免疫响应, 研究为深入开展三疣梭子蟹和其他甲壳动物的免疫调控机理提供了科学依据。     

Molecular properties and immune defense of two ferritin subunits from freshwater pearl mussel,Hyriopsis schlegelii

Ferritin is a conserved iron-binding protein involved in cellular iron metabolism and host defense. In the present study, two distinct cDNAs for ferritins in the freshwater pearl mussel Hyriopsis schlegelii were identified (designated as HsFer-1 and HsFer-2) by SMART RACE approach and expressed sequence tag (EST) analysis. The full-length cDNAs of HsFer-1 and HsFer-2 were of 760 and 877 bp, respectively. Both of the two cDNAs contained an open reading frame (ORF) of 522 bp encoding for 174 amino acid residues. Sequence characterization and homology alignment indicated that HsFer-1 and HsFer-2 had higher similarity to H-type subunit of vertebrate ferritins than L-type subunit. Analysis of the HsFer-1 and HsFer-2 untranslated regions (UTR) showed that both of them had an iron response element (IRE) in the 5′-UTR, which was considered to be the binding site for iron regulatory protein (IRP). Quantitative real-time PCR (qPCR) assays were employed to examine the mRNA expression profiles. Under normal physiological conditions, the expression level of both HsFer-1 and HsFer-2 mRNA were the highest in hepatopancreas, moderate in gonad, axe foot, intestine, kidney, heart, gill, adductor muscle and mantle, the lowest in hemocytes. After stimulation with bacteria Aeromonas hydrophila, HsFer-1 mRNA experienced a different degree of increase in the tissues of hepatopancreas, gonad and hemocytes, the peak level was 2.47-fold, 9.59-fold and 1.37-fold, respectively. Comparatively, HsFer-2 showed up-regulation in gonad but down-regulation in hepatopancreas and hemocytes. Varying expression patterns indicate that two types of ferritins in H. schlegelii might play different roles in response to bacterial challenge. Further bacteriostatic analysis showed that both the purified recombinant ferritins inhibited the growth of A. hydrophila to a certain degree. Collectively, our results suggest that HsFer-1 and HsFer-2 are likely to be functional proteins involved in immune defense against bacterial infection.     

Selenium-dependent glutathione peroxidase gene expression during gonad development and its response to LPS and H(2)O(2) challenge in Scylla paramamosain

A selenium-dependent glutathione peroxidase cDNA was obtained from green mud crab Scylla paramamosain (SpGPx) by homology PCR technique and rapid amplification of cDNA ends (RACE) methods. The 1135?bp full-length cDNA contains a 9?bp 5'-untranslated region (UTR), an open reading frame (ORF) of 564 bp encoded a deduced protein of 187 amino acids (aa), and a 562?bp 3'-UTR with a 100 bp conserved eukaryotic selenocysteine insertion sequence (SECIS). It involves a putative selenocysteine (Sec(40), or U(40)) residue which is encoded by an opal codon, (127)TGA(129), and forms an active site with residues Q(74) and W(142). Sequence characterization revealed that SpGPx contain a characteristic GPx signature motif 2 ((64)LAFPCNQF(71)), an active site motif ((152)WNFEKF(157)), a potential N-glycosylation site ((76)NTT(78)), and two residues (R(90) and R(168)) which contribute to the electrostatic architecture by directing the glutathione donor substrate. Multiple sequence alignment and phylogenetic analysis showed that SpGPx share a high level of identities and closer relationship with other selected invertebrate GPxs and vertebrate GPx1 and GPx2. Molecular modelling analysis results also supported these observations. Real time quantitative PCR analysis revealed that SpGPx was constitutively expressed in 10 selected tissues, and its expression level in gill and testis was higher than that in the other tissues (p?相似文献