Molecular cloning, characterization and expression of a serine protease with clip-domain homologue from scallop Chlamys farreri

Serine proteases play critical roles in a variety of invertebrate immune defense responses, including hemolymph coagulation, antimicrobial peptide synthesis, and melanization. The first mollusk serine protease with clip-domain (designated CFSP1) cDNA was obtained from the scallop Chlamys farreri challenged with Vibrio anguillarum by randomly sequencing a whole tissue cDNA library and rapid amplification of cDNA ends (RACE). The full-length cDNA of the C. farreri serine protease was 1211 bp, consisting of a 5-terminal untranslated region (UTR) of 72 bp, a 3'-terminal UTR of 77 bp with a canonical polyadenylation signal sequence AATAAA and a poly (A) tail, and an open reading frame of 1062 bp. The CFSP1 cDNA encoded a polypeptide of 354 amino acids with a putative signal peptide of 19 amino acids and a mature protein of 335 amino acids. The deduced amino acid sequence of CFSP1 contained an amino-terminal clip domain, a low complexity region, and a carboxyl-terminal serine protease domain. CFSP1 mRNA was mainly expressed constitutively in the hemocytes and was up-regulated and increased 2.9- and 1.9-fold at 16 h after injury and injection of bacteria. (c) 2006 Elsevier Ltd. All rights reserved.

Identification and characterization of a Cystatin gene from Chinese mitten crab Eriocheir sinensis

Cystatins are a superfamily of proteins as reversible inhibitor of cysteine proteinases which play essential roles in a spectrum of physiological and immunological processes In this study, a novel member of Cystatin superfamily was identified from Chinese mitten crab Enocheir sinensis (designated EsCystain) by expressed sequence tag (EST) analysis and rapid amplification of cDNA ends (RACE) approaches The full-length cDNA of EsCystatin was of 1486 bp, consisting of a 5'-terminal untranslated region (UTR) of 92 bp, a 3' UTR of 1034 bp with a polyadenylation signal sequence AATAAA and a polyA tail, and an open reading frame (ORF) of 360 bp encoded a polypeptide of 120 amino acids with the theoretical isoelectric point of 548 and the predicted molecular weight of 13 39 kDa. A signal Cystatin-like domain (Gly(25) to Lys(112)) was found in the putative amino acid sequences of EsCystatin Similar to other Cystatins, the conserved central Q(70)VVSG(74) motif was located in the Cystatin-like domain of EsCystatin But EsCystatin lacked of signal peptide and disulphide bond. The EsCystatin exhibited homology with the other known Cystatins from invertebrates and higher vertebrates, and it was clustered into Cystatin family 1 in the phylogenetic tree. The mRNA transcripts of EsCystain were mainly expressed in hemolymph, gill, hepatopancreas, gonad and muscle, and also marginally detectable in heart After Listonella anguillarum challenge, the relative expression level of EsCystatin in hemolymph was down-regulated to 0 6-fold (P < 0.05) at 3 h post-challenge. Subsequently, it was up-regulated to 3.0-fold (P < 0.01)at 24 h Afterwards. EsCystatin mRNA transcripts suddenly decreased to original level. After Pichia pastoris GS115 challenge, its mRNA expression level in hemolymph was up-regulated to the peak at 3 h (2 8-fold of that in blank (P < 0 01)) The cDNA fragment encoding the mature peptide of EsCystatin was recombined and expressed in Escherichia coli Rosetta-gami (DE3). The recombinant EsCystatin displayed a promoter inhibitory activity against papain When the concentration of EsCystatin protein was of 300 mu g mL(-1), almost 89% of papain activity could be inhibited. These results collectively suggested that EsCystatin was a novel member of protein in Cystatin family, was a potent inhibitor of papain and involved in immune response versus invading microorganisms. (C) 2010 Elsevier Ltd All rights reserved.

AiC1qDC-1, a novel gC1q-domain-containing protein from bay scallop Argopecten irradians with fungi agglutinating activity

The globular C1q-domain-containing (C1qDC) proteins are a family of versatile pattern recognition receptors via their globular C1q (gC1q) domain to bind various ligands including several PAMPs on pathogens. In this study, a new gC1q-domain-containing protein (AiC1qDC-1) gene was cloned from Argopecten irradians by rapid amplification of cDNA ends (RACE) approaches and expressed sequence tag (EST) analysis. The full-length cDNA of AiC1qDC-1 was composed of 733 bp, encoding a signal peptide of 19 residues and a typical gC1q domain of 137 residues containing all eight invariant amino acids in human C1qDC proteins and seven aromatic residues essential for effective packing of the hydrophobic core of AiC1qDC-1. The gC1q domain of AiC1qDC-1, which possessed the typical 10-stranded beta-sandwich fold with a jelly-roll topology common to all C1q family members, showed high homology not only to those of Cl qDC proteins in mollusk but also to those of C1qDC proteins in human. The AiC1qDC-1 transcripts were mainly detected in the tissue of hepatopancreas and also marginally detectable in adductor, heart, mantle, gill and hemocytes by fluorescent quantitative real-time PCR. In the microbial challenge experiment, there was a significant up-regulation in the relative expression level of AiC1qDC-1 in hepatopancreas and hemocytes of the scallops challenged by fungi Pichia pastoris GS115, Gram-positive bacteria Micrococcus luteus and Gram-negative bacteria Listonella anguillarum. The recombinant AiC1qDC-1 (rAiC1qDC-1) protein displayed no obvious agglutination against M. luteus and L. anguillarum, but it aggregated P. pastoris remarkably. This agglutination could be inhibited by D-mannose and PGN but not by LPS, glucan or D-galactose. These results indicated that AiC1qDC-1 functioned as a pattern recognition receptor in the immune defense of scallops against pathogens and provided clues for illuminating the evolution of the complement classical pathway. (C) 2010 Elsevier Ltd. All rights reserved.

The cDNA cloning and mRNA expression of heat shock protein 70 gene in the haemocytes of bay scallop (Argopecten irradians, Lamarck 1819) responding to bacteria challenge and naphthalin stress

Heat shock protein 70 (HSP70) is an important member of the heat shock protein superfamily, and it plays a key role in the process of protecting cells, facilitating the folding of nascent peptides and responding to stress. The cDNA of bay scallop Argopecten irradians HSP70 (designated AIHSP70) was cloned by the techniques of homological cloning and rapid amplification of cDNA end (RACE). The full length of AIHSP70 cDNA was 2651 bp in length, having a 5' untranslated region (UTR) of 96 bp, a 3' UTR of 575 bp, and an open reading frame (ORF) of 1980 bp encoding a polypeptide of 659 amino acids with an estimated molecular mass of 71.80 kDa and an estimated isoelectric point of 5.26. BLAST analysis revealed that the AIHSP70 gene shared high identity with other known HSP70 genes. Three classical HSP signature motifs were detected in AIHSP70 by InterPro, analysis. 3-D structural prediction of AIHSP70 showed that its N terminal ATPase activity domain and,C terminal substrate-binding domain shared high similarity with that in human heat shock protein 70. The results indicated that the AIHSP70 was a member of the heat shock protein 70 family. A semi-quantitive RT-PCR method was used to analyse the expression of AIHSP70 gene after the treatment of naphthalin which is one kind of polycyclic aromatic hydrocarbon (PAH) and the challenge of bacteria. mRNA expression of AIHSP70 in scallop was up-regulated significantly after the stimulation of naphthalin and increased with increasing naphthalin concentration. A clearly time-dependent expression pattern of AIHSP70 was observed after the scallops were infected by Vibrio anguillarum, and the mRNA expression reached a maximum level at 8 h and lasted to 16 h, and then dropped progressively. The results indicated that AIHSP70 could play an important role in mediating the environmental stress and immune response in scallop. (c) 2006 Elsevier Ltd. All rights reserved.

The genetic analysis and germplasm identification of the gametophytes of Undaria pinnatifida (Phaeophyceae) with RAPD method

Eleven pairs of Undaria pinnatifida (Harv.) Suringar gametophytes were identified with random amplified polymorphic DNA (RAPD) technique. After screening 100 primers, 20 ten-base primers were determined for the RAPD analysis. A total of 312 polymorphic loci were obtained, of which 97.7% were polymorphic. The primer S198 was found to distinguish all the selected Undaria pinnatifida gametophytes. The genetic distances between each two of the twenty-two U. pinnatifida gametophytes ranged from 0.080 to 0.428, while the distances to the Laminaria was 0.497 on average. After reexamination, two sequences characterized amplification region (SCAR) markers were successfully converted, which could be applied to U. pinnatifida germplasm identification. All these results demonstrated the feasibility of applying RAPD markers to germplasm characterization and identification of U. pinnatifida gametophytes, and to provide a molecular basis for Undaria breeding.

Cflec-5, a pattern recognition receptor in scallop Chlamys farreri agglutinating yeast Pichia pastoris

C-type lectins are a superfamily of carbohydrate-recognition proteins which play crucial roles as pattern recognition receptors (PRRs) in the innate immunity. In this study, the full-length cDNA of a C-type lectin was cloned from scallop Chlamys farreri (designated as Cflec-5) by expression sequence tag (EST) analysis and rapid amplification of cDNA ends (RACE) approach The full-length cDNA of Cflec-5 was of 1412 bp. The open reading frame encoded a polypeptide of 153 amino acids, including a signal sequence and a conserved carbohydrate-recognition domain with the EPN motif determining the mannose-binding specificity The deduced amino acid sequence of Cflec-5 showed high similarity to members of C-type lectin superfamily. The quantitative real-time PCR was performed to investigate the tissue distribution of Cflec-5 mRNA and its temporal expression profiles in hemocytes post pathogen-associated molecular patterns (PAMPs) stimulation. In healthy scallops, the Cflec-5 mRNA was mainly detected in gill and mantle, and marginally in other tissues The mRNA expression of Cflec-5 could be significantly induced by lipopolysaccharide (LPS) and glucan stimulation and reached the maximum level at 6 h and 12 h, respectively But its expression level did not change significantly during peptidoglycan (PGN) stimulation The function of Cflec-5 was investigated by recombination and expression of the cDNA fragment encoding its mature peptide in Escherichia coli Rosetta Gami (DE3) The recombinant Cflec-5 agglutinated Pichia pastoris in a calcium-independent way The agglutinating activity could be inhibited by D-mannose. LPS and glucan, but not by D-galactose or PGN. These results collectively suggested that Cflec-5 was involved in the innate Immune response of scallops and might contribute to nonself-recognition through its interaction with various PAMPs (C) 2010 Elsevier Ltd All rights reserved

Molecular cloning, characterization and expression of a novel serine proteinase inhibitor gene in bay scallops (Argopecten irradians, Lamarck 1819)

Serine protease inhibitors, critical regulators of endogenous proteases, are found in all multicellular organisms and play crucial roles in host physiological and immunological effector mechanisms. The first mollusk serine proteinase inhibitor (designated AISPI) cDNA was obtained from the bay scallop Argopecten irradians by randomly sequencing a whole tissue cDNA library and rapid amplification of cDNA ends (RACE). The full-length cDNA of the scallop serine protease inhibitor was 1020 bp, consisting of a 5'-terminal untranslated region (UTR) of 39 bp, a 3'-terminal UTR of 147 bp with a canonical polyadenylation signal sequence AATAAA and a poly(A) tail, and an open reading frame of 834 bp. The AISPI cDNA encoded a polypeptide of 278 amino acids with a putative signal peptide of 22 amino acids and a mature protein of 256 amino acids. The deduced amino-acid sequence of AISPI contained six tandem and homologous domains similar to that of Kazal-type serine protease inhibitors, including the conserved sequence C-X(7)-C-X(6)-Y-X(3)-C-X(2,3)-C and six cysteine residues responsible for the formation of disulfide bridges, indicating that the AISPI protein from bay scallop should be a member of the Kazal-type serine protease inhibitor family. The temporal expression of AISPI was measured by semi-quantitative RT-PCR after injury or bacterial challenge. After the adductor muscle was wounded or injected with Vibrio anguillarum, the expression of AISPI mRNA in hemolymph was up-regulated and reached the maximum level at 8 and 16 h, respectively, and then progressively dropped back to the original level. The results indicated that AISPI could play an important role in injury healing and immune response in mollusks as it could be induced by injury and bacterial challenge. (c) 2005 Elsevier Ltd. All rights reserved.

Characterization and mapping of expressed sequence tags isolated from a subtracted cDNA library of Litopenaeus vannamei injected with white spot syndrome virus

A large number of polymorphic simple sequence repeats (SSRs) or microsatellites are needed to develop a genetic map for shrimp. However, developing an SSR map is very time-consuming, expensive, and most SSRs are not specifically linked to gene loci of immediate interest. We report here on our strategy to develop polymorphic markers using expressed sequence tags (ESTs) by designing primers flanking single or multiple SSRs with three or more repeats. A subtracted cDNA library was prepared using RNA from specific pathogen-free (SPF) Litopenaeus vannamei juveniles (similar to 1 g) collected before (0) and after (48 h) inoculation with the China isolate of white spot syndrome virus (WSSV). A total of 224 clones were sequenced, 194 of which were useful for homology comparisons against annotated genes in NCBI nonredundant (nr) and protein databases, providing 179 sequences encoded by nuclear DNA, 4 mitochondrial DNA, and 11 were similar to portions of WSSV genome. The nuclear sequences clustered in 43 groups, 11 of which were homologous to various ESTs of unknown function, 4 had no homology to any sequence, and 28 showed similarities to known genes of invertebrates and vertebrates, representatives of cellular metabolic processes such as calcium ion balance, cytoskeleton mRNAs, and protein synthesis. A few sequences were homologous to immune system-related (allergens) genes and two were similar to motifs of the sex-lethal gene of Drosophila. A large number of EST sequences were similar to domains of the EF-hand superfamily (Ca2+ binding motif and FRQ protein domain of myosin light chains). Single or multiple SSRs with three or more repeats were found in approximately 61 % of the 179 nuclear sequences. Primer sets were designed from 28 sequences representing 19 known or putative genes and tested for polymorphism (EST-SSR marker) in a small test panel containing 16 individuals. Ten (53%) of the 19 putative or unknown function genes were polymorphic, 4 monomorphic, and 3 either failed to satisfactorily amplify genomic DNA or the allele amplification conditions need to be further optimized. Five polymorphic ESTs were genotyped with the entire reference mapping family, two of them (actin, accession #CX535973 and shrimp allergen arginine kinase, accession #CX535999) did not amplify with all offspring of the IRMF panel suggesting presence of null alleles, and three of them amplified in most of the IRM F offspring and were used for linkage analysis. EF-hand motif of myosin light chain (accession #CX535935) was placed in ShrimpMap's linkage group 7, whereas ribosomal protein S5 (accession #CX535957) and troponin I (accession #CX535976) remained unassigned. Results indicate that (a) a large number of ESTs isolated from this cDNA library are similar to cytoskeleton mRNAs and may reflect a normal pathway of the cellular response after im infection with WSSV, and (b) primers flanking single or multiple SSRs with three or more repeats from shrimp ESTs could be an efficient approach to develop polymorphic markers useful for linkage mapping. Work is underway to map additional SSR-containing ESTs from this and other cDNA libraries as a plausible strategy to increase marker density in ShrimpMap.

中华绒螯蟹（Eriocheir sinensis）cDNA文库的构建、EST分析及其酚氧化酶系统关键基因的研究

中华绒螯蟹（Eriocheir sinensis）是我国的特色物种，具有重要的经济和科研价值。酚氧化酶系统作为节肢动物特有的免疫机制，在中华绒螯蟹的免疫反应中发挥重要作用。本研究构建了一个中华绒螯蟹的cDNA文库，利用表达序列标签 (Expressed Sequence Tag，EST) 技术，对中华绒螯蟹表达序列进行了大规模测序分析，并利用cDNA末端快速扩增（rapid amplification of cDNA ends，RACE）、实时定量PCR、原核重组和RNAi等技术研究了其酚氧化酶免疫系统的分子基础及其相应功能。 用鳗弧菌和金黄色葡萄球菌同时感染中华绒螯蟹，提取血细胞的RNA构建了一个库容为3.3×106 克隆cDNA文库。随机测序后获得7535条高质量的EST序列，其中在GenBank数据库中未发现同源序列的为4593 条，而具有较高同源性2942条可以分为20个功能类别，参与了23个生物学反应。 进一步分析发现，969 条（32.9% ）EST与免疫相关，可拼接成221个免疫基因。这个比例高于其它任何一个已公布的甲壳动物cDNA文库。在免疫相关EST中，抗菌肽比例最高，约占总数的20.1%（195条EST）。免疫基因的高比例和抗菌肽的高表达，证明细菌刺激是提高cDNA 文库中免疫基因丰度的有效方法。 EST序列的获得和免疫基因的富集，丰富了中华绒螯蟹的基因组信息，初步了解了中华绒螯蟹固有免疫系统的概况, 为进一步克隆和研究中华绒螯蟹免疫防御功能基因提供了序列基础。 本研究在EST分析的基础上，克隆获得了中华绒螯蟹酚氧化酶系统10个基因的cDNA全长序列, 它们分别是前酚氧化酶（EsproPO），丝氨酸蛋白酶同源物（EsSPH), 丝氨酸蛋白酶抑制剂pacifastin, serpin, PAPII (EsPLC, Es serpin, EsPAPII), 模式识别丝氨酸蛋白酶（EsPRSP），peroxinectin (Esperoxinectin)和3个前酚氧化酶激活酶 (EsPAP1, 2, 3)。它们与相近物种的酚氧化酶系统相应基因均具有较高同源性，并含有胰酶催化结构域，CLIP结构域，PLD结构域，KAZAL结构域，Serpin结构域以及酚氧化酶结构域等酚氧化酶系统相应基因典型的特征结构域。分析发现，PAPs的CLIP结构域和PRSP，Pacifastin，Proxinectin，proPO基因是节肢动物特有的，是酚氧化酶系统作为节肢动物特有免疫机制的分子基础。本研究从多个基因的3′UTR区发现了调控元件，如15-LOX-DICE，K-box和 Brd-Box。在所推断的蛋白中，EsPAP3和EsPAPII的等电点呈碱性，Esperoxinetin的为中性，而EsPRSP，EsSPH，EsproPO, EsPAPII, Esserpin，EsPAP1的等电点在酸性区间。健康中华绒螯蟹 EsPAP1，EsPAP2，EsPAPII基因在肌肉中的表达量最高，而在血细胞中的表达量相对较低；EsPAP3，EsproPO，EsPLC基因在血细胞中表达量较高，在肌肉中的表达量最低。其中，EsPAP3在血细胞中的表达量是其在肌肉组织中表达量的526.35倍。调控元件和多种激活酶与抑制剂的存在、组织分布和等电点的差异，说明中华绒螯蟹酚氧化酶系统在转录、翻译、激活等多个层次上受到了调控。在中华绒螯蟹受到鳗弧菌刺激后，EsPAP1，EsPAP2，EsPAP3，EsPLC和EsPAPII基因的表达量呈上升或下降的趋势，但表达量的极限值均出现在2小时和12小时，这一规律与EsproPO应激后的mRNA表达和酶比活力的变化特点相吻合，说明中华绒螯蟹酚氧化酶系统各因子相互协调共同参与中华绒螯蟹对入侵细菌的防御反应。同时EsPAP2，EsPAP3，EsproPO，EsPAPII，EsPLC在中华绒螯蟹受到鳗弧菌刺激后的表达呈现反复多次上升，表明酚氧化酶系统可能参与了多种免疫反应。研究还发现EsPAP1参与中华绒螯蟹血液凝集过程，而EsPAP3是蟹血细胞中的有效的前酚氧化酶激活因子。研究结果初步揭示了中华绒螯蟹酚氧化酶系统的分子基础、对微生物的响应机制及其调控机制和演化趋势，为节肢动物固有免疫系统研究奠定了良好基础。

SNAP-23蛋白在鲈鱼巨噬细胞白细胞介素1β分泌过程中的功能

SNARE蛋白家族是所有真核细胞胞吐及分泌作用中的关键因子，由其介导的运输囊泡膜与靶膜的锚靠、融合为胞内蛋白的运出提供了一条重要途径。体外试验表明，Syntaxin6-Syntaxin7-Vti1b，SNAP-23-Syntaxin4等SNARE核心蛋白之间精确的相互作用是哺乳动物巨噬细胞肿瘤坏死因子α (TNF-α)运输和分泌的必备条件，在机体非特异性免疫应答反应过程中起重要作用。 本研究受上述启示，旨在揭示SNARE蛋白在海洋鱼类免疫细胞内重要细胞因子白细胞介素1β (IL-1β)的分泌过程中的作用。参照Percoll密度梯度离心技术，从鲈鱼头肾组织分离纯化巨噬细胞进行稳定培养；利用RT-PCR方法克隆出鲈鱼t-SNARE蛋白SNAP-23和Syntaxin3的部分cDNA序列，再结合先前克隆的VAMP2和已知的鲈鱼IL-1β，TNF-α和IL-8的基因序列，设计特异性引物。利用Real-time PCR技术在mRNA水平上精确测定鲈鱼巨噬细胞中上述6种基因在革兰氏阴性菌脂多糖(LPS)分子刺激下的表达变化，发现SNAP-23基因与三种细胞因子基因的表达正相关；通过免疫印迹检测SNAP-23蛋白表达变化，利用酶联免疫吸附试验(ELISA)检测IL-1β的分泌水平，在蛋白水平上验证了SNAP-23表达与IL-1β分泌的正相关性；利用5`RACE和3`RACE技术克隆出鲈鱼SNAP-23全长基因，结合定点突变策略和靶向PCR克隆手段，构建鲈鱼SNAP-23野生型融合质粒pEGFP-SNAP-23wt，Cys缺失突变融合质粒pEGFP-SNAP-23ΔCys和模拟E型肉毒神经毒素(BoNT/E)切割突变融合质粒pEGFP-SNAP-23ΔBoNT/E，以及鲈鱼IL-1β野生型融合表达质粒IL-1β-pEGFP和IL-1β-pEYFP。所有融合蛋白均在鲈鱼巨噬细胞内成功表达，结合ELISA实验结果发现，SNAP-23野生型的表达对IL-1β的分泌有促进作用，而Cys缺失突变体的表达则抑制IL-1β向胞外分泌。首次证实了鱼类巨噬细胞内SNAP-23蛋白在IL-1β分泌过程中的重要作用。此外通过与GFP共表达，定位了IL-1β分子在巨噬细胞内的分布，发现新合成的IL-1β分子很可能像TNFα一样经“内质网－胞质－伪足－胞外” 的分泌路径运出胞外。

中华绒螯蟹（Eriocheir sinensis）Peroxiredoxin 6和Thioredoxin1基因的克隆及表达

中华绒螯蟹是我国重要的水产经济动物，近年来养殖规模不断扩大，产量持续增加。但是，伴随着养殖规模的扩大，养殖环境也日益恶化并导致了大量疾病的发生，严重制约了中华绒螯蟹养殖业的健康发展。因此，疾病预防和控制对中华绒螯蟹养殖业的可持续发展具有举足轻重的作用。与其他无脊椎动物一样，中华绒螯蟹的免疫系统没有免疫球蛋白和淋巴细胞，而是依靠由细胞免疫和体液免疫构成的固有免疫系统来对病原进行识别和清除。中华绒螯蟹的固有免疫机制的研究有助于推动中华绒螯蟹病害防治工作的开展。 本研究采用大规模EST测序方法，结合末端快速扩增（rapid amplification of cDNA ends，RACE）技术从中华绒螯蟹血淋巴中克隆到了过氧化物还原酶（peroxiredoxin，EsPrx6）和硫氧还蛋白（thioredoxin，EsTrx1）基因的cDNA 全长序列；采用实时荧光定量PCR 技术检测了这两个基因在健康个体中表达的组织分布情况以及鳗弧菌刺激后血淋巴细胞中的时序表达规律；同时，将这两个基因的编码区克隆到pET 系列载体，并在大肠杆菌中实现了重组表达，并进行了体外活性检测。 过氧化物还原酶是一个抗氧化蛋白超家族，在保护机体免受活性氧（reactive oxygen species，ROS）的伤害中发挥着重要作用。中华绒螯蟹Prx6（EsPrx6） 基因的cDNA 全长为1076 bp，5` UTR（untranslated region，UTR） 为69 bp，3` UTR 为347 bp，开放阅读框（open reading frame，ORF）为660 bp，编码219 个氨基酸的蛋白。mRNA 3`-端具有多聚腺苷酸加尾信号（polyadenylation signal）AATAAA 和polyA 尾巴。EsPrx6 的预测分子量为 24 kDa，理论等电点为6.21，具有一个保守的Prx 结构域、一个AhpC 结构域和过氧化物酶催化活性中心PVCTTE，表明EsPrx6 属于1-Cys 型Prx。在所检测的组织中均有EsPrx6 的表达，其中以肝胰腺表达量最高，为血淋巴细胞中表达量的17.4 倍。鳗弧菌刺激后，血淋巴细胞中EsPrx6 的表达下降，到12 h 时，实验组显著低于对照组（P<0.05）；随时间推移，表达水平逐渐回升，但在整个实验期间，都没有恢复到起始水平。将EsPrx6 进行体外重组并在大肠杆菌E. coli BL21（DE3）中实现表达，重组EsPrx6 具有预期的抗氧化活性和过氧化物酶活性，其中抗氧化活力为14.69 U/mg 蛋白，高于相同条件下GSH 的抗氧化力（P<0.05），过氧化物酶活力为23.46 U/mg 蛋白。结果表明，EsPrx6 作为一种重要的抗氧化剂，在中华绒螯蟹抵御ROS 可能引起的氧化损伤方面具有重要作用。 硫氧还蛋白是广泛存在于生物体内的一种具有硫醇依赖性的具有还原活性的蛋白。中华绒螯蟹Trx1（EsTrx1）基因的cDNA 全长为641 bp，5` UTR 为17 bp，3` UTR 为306 bp，开放阅读框为318 bp，编码105 个氨基酸。EsTrx1 的预测分子量为12.2 kDa，理论等电点为4.8。EsTrx1 不含信号肽，其氨基酸序列与其他动物的Trx1s 具有高度相似性，如与地中海黄蝎的Trx1 相似度达到73%；而与其他物种Trx2 的同源性很低，相似度仅为14.3-22.8%，表明EsTrx1 属于Trx1 亚族。实时荧光定量PCR 检测发现，EsTrx1 在鳃、性腺、肝胰腺、肌肉、心脏和血淋巴细胞中都有表达。血淋巴细胞中EsTrx1 mRNA 的表达量在菌刺激后上升，刺激后6 h，实验组表达量显著高于对照组和空白组（P<0.05），然后逐渐恢复到刺激前水平。为进一步探讨其生物学功能，将EsTrx1 进行体外重组并在大肠杆菌E. coli BL21（DE3）得到表达，重组EsTrx1 具有预期的氧化还原调节活性，抗氧化活力为3.06 U/mg，且抗氧化活力高于GSH（P<0.05）。rEsTrx1 的二硫键还原活力为5.03，低于凡纳滨对虾的二硫键还原活力（10.44），接近于大肠杆菌（4.93），小牛胸腺（6.50）和小牛肝脏（5.09），而高于鲍鱼Trx2（1.83）活力。结果表明，EsTrx1 在生理条件下能够作为一种重要的抗氧化剂，参与对细菌感染的免疫应答反应。

中国明对虾免疫系统中抗氧化相关基因的克隆与表达分析

对虾病害在世界范围内的广泛传播，给水产养殖和沿海农村经济造成了重大损失。深入开展对虾免疫机制研究并在此基础上寻找对虾疾病防治的有效方法已成为当务之急。研究表明，当对虾等甲壳动物受到外界病原刺激时，其体内的吞噬细胞在吞噬活动中会激活磷酸己糖支路的代谢，引起呼吸爆发，产生多种活性氧分子。另外，受到病原侵染的对虾还会产生其他多种免疫反应，这些免疫反应将消耗大量的能量（ATP），产能的呼吸链会加速运转，由此也会引发大量活性氧的产生。这些活性氧分子可以杀灭入侵的病原微生物，但同时由于活性氧分子反应的非特异性，它们也会对宿主的细胞、组织和器官造成严重伤害，进而导致对虾生理机能的损伤和免疫系统的破坏。所以，消除对虾体内因过度免疫反应产生的过量氧自由基将能够增强其抵御病原侵染的能力，提高免疫力。本论文从中国明对虾体内克隆了线粒体型超氧化物歧化酶（mMnSOD）、胞质型超氧化物歧化酶（cMnSOD）、过氧化氢酶（Catalase）和过氧化物还原酶（Peroxiredoxin）等四种与免疫系统相关的抗氧化酶基因，分析了它们的分子结构特征，组织分布及应答不同病原刺激的表达变化模式，并对其中的mMnSOD基因和Peroxiredoxin基因进行了体外重组表达、分离纯化和酶活性分析。 采用RACE技术从中国明对虾血细胞中克隆了两个超氧化物歧化酶（SOD）基因，通过序列比对分析发现，其中一个为mMnSOD基因，另一个为cMnSOD基因。mMnSOD基因的cDNA全长为1185个碱基，其中开放阅读框为660个碱基，编码220个氨基酸，其中推测的信号肽为20个氨基酸。多序列比对结果显示中国明对虾mMnSOD基因的推导氨基酸序列与罗氏沼虾、蓝蟹的推导氨基酸序列同源性分别为88%和82%。Northern blot结果表明，该基因在对虾的肝胰脏、血细胞、淋巴器官、肠、卵巢、肌肉和鳃等组织中均有表达。半定量RT-PCR结果显示，对虾感染病毒3 h时，该基因在血细胞和肝胰脏中的转录水平显著升高。此外，通过构建原核表达载体，本研究对该基因进行了体外重组表达，并对纯化的重组蛋白进行了质谱鉴定和酶活分析。cMnSOD基因的cDNA全长为1284个碱基，其中开放阅读框为861个碱基，编码287个氨基酸。多序列比对结果显示中国明对虾cMnSOD基因的推导氨基酸序列与斑节对虾和凡纳滨对虾的同源性高达98%和94%。组织半定量结果显示，cMnSOD基因在对虾被检测的各个组织中均有表达。 另外，半定量RT-PCR结果表明，对虾感染病毒23h时，该基因在肝胰脏中的转录上升到正常水平的3.5倍；而感染后59 h时，该基因在血细胞中的转录上升到正常水平的2.5倍。 利用根据其他生物过氧化氢酶保守氨基酸序列设计的简并引物，结合RACE技术，从中国明对虾肝胰脏中克隆到了过氧化氢酶基因的部分片段，片段长1725个碱基。多序列比对结果发现目前所得中国明对虾Catalase基因部分片段的推导氨基酸序列与罗氏沼虾和皱纹盘鲍Catalase氨基酸序列的同源性分别达到95%和73%。通过实时荧光定量PCR技术对中国明对虾Catalase基因在各个组织中的分布情况及病毒感染后该基因在血细胞和肝胰脏中的转录变化进行了研究。结果发现，该基因在肝胰脏、鳃、肠和血细胞中表达水平较高，在卵巢、淋巴器官和肌肉中的表达水平相对较弱；感染病毒23 h和37 h时，对虾血细胞和肝胰脏中该基因mRNA的表达量分别出现显著性上升。 依据中国明对虾头胸部cDNA文库提供的部分片段信息，结合SMART-RACE技术，从中国明对虾肝胰脏中克隆到了过氧化物还原酶基因（Peroxiredoxin）， 该基因的cDNA全长为942个碱基，其中开放阅读框为594个碱基，编码198个氨基酸。中国明对虾Peroxiredoxin基因的推断氨基酸序列与伊蚊、文昌鱼和果蝇等Peroxiredoxin基因的推断氨基酸序列同源性分别为77%、76%和73%。其蛋白理论分子量为22041.17 Da，pI为5.17。Northern blot结果表明，Peroxiredoxin基因在对虾的肝胰脏、血细胞、淋巴器官、肠、卵巢、肌肉和鳃等组织中均有表达。实时荧光定量PCR结果显示，弧菌感染后，该基因在对虾血细胞和肝胰脏中的转录水平都有明显变化并且表达模式不同。另外，对该基因进行了体外重组表达，并对纯化的重组蛋白进行了质谱鉴定和酶活性分析。酶活性分析表明，复性后的重组蛋白能在DTT存在的条件下还原H2O2。

中国明对虾Toll样受体基因的克隆与表达分析

对虾病害在世界范围内的广泛传播，给水产养殖和沿海农村经济造成了重大损失。自1993 年对虾白斑病暴发以来，中国明对虾的养殖一直一蹶不振。引起对虾大规模死亡的原因是多方面的，其主要原因是养殖环境恶化、对虾种质退化和抗病力下降。因此，深入开展对虾免疫机制研究，并在此基础上寻找对虾疾病防治的有效方法，改良种质和培育抗病品系，已成为对虾养殖业走可持续发展之路的当务之急。 Toll 样受体（Toll-like receptors, TLRs）家族是进化保守的哺乳动物模式识别蛋白（pattern recognition receptors, PRR），在先天免疫系统中起着非常重要的作用。本研究采用同源克隆和RACE（rapid amplification of cDNA ends）技术从中国明对虾中克隆到Toll 样受体同源基因，并将其命名为FcToll。它全长4115 bp，3’UTR 包含16 个poly A 尾巴，开放阅读框编码931 个氨基酸的多肽。预测的该多肽包含典型的Toll 样受体结构，分为胞外区、跨膜区和胞内区。其中胞外区有信号肽，有16 个富含亮氨酸的重复序列eucine-rich repeats, LRR），并含有2个LRR-C 末端基序和2 个LRR-N 末端基序；跨膜区是23 个氨基酸的一次跨膜结构域；胞内区是含有139 个氨基酸的TIR 结构域（Toll/Interleukin-1R）。克隆 发现FcToll 的基因组结构包含5 个外显子和4 个内含子。系统发生分析揭示FcToll归属于“昆虫型”的无脊椎动物Toll 样受体家族。组织分布研究发现FcToll 在中国明对虾中是组成型表达的，在淋巴器官中表达量较显著。分别利用不同病原体刺激健康的中国明对虾，Real-time PCR 发现该基因在刺激后表达水平呈现不同的表达谱：灭活鳗弧菌（Vibrio anguillarum）注射后5 小时，该基因表达显著 上调；而WSSV（white spot syndrome virus）注射后该基因表达则迅速下调，感染后23 小时内其表达水平均低于对应时间点的对照组。这就表明FcToll 可能参与中国明对虾的先天免疫防御，尤其可能参与入侵弧菌的免疫应答。

The responsive expression of heat shock protein 22 gene in zhikong scallop Chlamys farreri against a bacterial challenge

HSP22 is a member of a small HSP subfamily contributing to the growth, transformation and apoptosis of the cell as well as acting as a molecular chaperone. In the present study, CfHSP22 cDNA was cloned from Chlamys farreri by the rapid amplification of cDNA ends technique. The full-length cDNA of CfHSP22 was of 1279 bp, consisting of a 5'-terminal untranslated region (5'UTR) of 122 bp, a 3'UTR of 581 bp with a canonical polyadenylation signal sequence AATAAA and a poly( A) tail, and an open reading frame of 576 bp encoding a polypeptide with a molecular mass of 22.21 kDa and a predicted isoelectric point of 9.69. There was an alpha-crystallin domain, a hallmark of the sHSP subfamily, in the C-terminus, and the deduced amino acid sequence of CfHSP22 showed high similarity to previously identified HSP22s. CfHSP22 was constitutively expressed in the haemocyte, muscle, kidney, gonad, gill, heart and hepatopancreas, and the expression level in the hepatopancreas was higher than that in the other tissues. CfHSP22 transcription was up-regulated and reached a maximal level at 12 h after the bacterial challenge, and then declined progressively to the original level at 48 h. These results suggested that CfHSP22 perhaps play a critical role in response to the bacterial challenge in haemocytes of scallop C. farreri.

Identification of quantitative trait loci for growth-related traits in the Pacific abalone Haliotis discus hannai Ino

The locations and effects of quantitative trait loci (QTL) were estimated for nine characters for growth-related traits in the Pacific abalone (Haliotis discus hannai Ino) using a randomly amplified polymorphic DNA (RAPD), amplification fragment length polymorphism (AFLP) and SSR genetic linkage map. Twenty-eight putatively significant QTLs (LOD > 2.4) were detected for nine traits (shell length, shell width, total weight, shell weight, weight of soft part, muscle weight, gonad and digestive gland weight, mantle weight and gill weight). The percentage of phenotypic variation explained by a single QTL ranged from 8.0% to 35.9%. The significant correlations (P < 0.001) were found among all the growth-related traits, and Pearson's correlation coefficients were more than 0.81. For the female map, the QTL for growth were concentrated on groups 1 and 4 linkage maps. On the male map, the QTL that influenced growth-related traits gathered on the groups 1 and 9 linkage maps. Genetic linkage map construction and QTL analysis for growth-related traits are the basis for the marker-assisted selection and will eventually improve production and quality of the Pacific abalone.