Molecular cloning and characterization of proliferating cell nuclear antigen (PCNA) from Chinese shrimp Fenneropenaeus chinensis

The proliferating cell nuclear antigen gene was cloned from Fenneropenaeus chinensis (FcPCNA). The full-length cDNA sequence of FcPCNA encodes 260 amino acids showing high identity with PCNAs reported in other species. FcPCNA expressed especially high in proliferating tissues of shrimp such as haematopoietic tissue (HPT) and ovary. In order to understand the response of HPT to bacteria and virus challenge, mRNA level of FcPCNA in HPT was analyzed after shrimp were challenged by Vibrio anguillarum and white spot syndrome virus (WSSV). FcPCNA expression in HPT of shrimp was responsive to WSSV and Vibrio challenge, but different expression profiles were obtained after challenge by these two pathogens. The data provide additional information to understand the defense mechanisms of shrimp against virus and bacteria. (c) 2008 Elsevier Inc. All rights reserved.

A toll receptor from Chinese shrimp Fenneropenaeus chinensis is responsive to Vibrio anguillarum infection

Toll-like receptors (TLRs) are an evolutionarily ancient family of pattern recognition receptors (PRRs), playing a crucial role in innate immune responses. Here we present a Toll homolog from Chinese shrimp Fenneropenaeus chinensis, designated FcToll. The full-length cDNA of FcToll is 4115 bp including a poly A-tail of 16 bp, encoding a putative protein of 931 amino acids. The predicted protein consists of an extracellular domain with a potential signal peptide, 16 leucine-rich repeats (LRR), two LRR-C-terminal (LRR-CT) motifs, and two LRR-N-terminal (LRR-NT) motifs, followed by a transmembrane segment of 23 amino acids, and a cytoplasmic Toll/Interteukin-IR (TIR) domain of 139 residues. Genomic structure of FcToll gene contains five exons and four introns. Phylogenetic analysis revealed that it belongs to insect-type invertebrate Toll family. Transcripts of FcToll gene were constitutively expressed in various tissues, with predominant level in lymphoid organ. Real-time PCR assays demonstrated that expression patterns of FcToll were distinctly modulated after bacterial or viral stimulation, with significant enhancement after 5 h post-Vibrio anguillorum challenge but markedly reduced levels immediately after white spot syndrome virus (WSSV) exposure. These results suggest that FcToll might be involved in innate host defense, especially against the pathogen V. anguillarum. (c) 2008 Elsevier Ltd. All rights reserved.

A genetic linkage map of Pacific white shrimp (Litopenaeus vannamei): sex-linked microsatellite markers and high recombination rates

Pacific white shrimp (Litopenaeus vannamei) is the leading species farmed in the Western Hemisphere and an economically important aquaculture species in China. In this project, a genetic linkage map was constructed using amplified fragment length polymorphism (AFLP) and microsatellite markers. One hundred and eight select AFLP primer combinations and 30 polymorphic microsatellite markers produced 2071 markers that were polymorphic in either of the parents and segregated in the progeny. Of these segregating markers, 319 were mapped to 45 linkage groups of the female framework map, covering a total of 4134.4 cM; and 267 markers were assigned to 45 linkage groups of the male map, covering a total of 3220.9 cM. High recombination rates were found in both parental maps. A sex-linked microsatellite marker was mapped on the female map with 6.6 cM to sex and a LOD of 17.8, two other microsatellite markers were also linked with both 8.6 cM to sex and LOD score of 14.3 and 16.4. The genetic maps presented here will serve as a basis for the construction of a high-resolution genetic map, quantitative trait loci (QTLs) detection, marker-assisted selection (MAS) and comparative genome mapping.

cDNA cloning, characterization and expression analysis of the antioxidant enzyme gene, catalase, of Chinese shrimp Fenneropenaeus chinensis

Catalase is an important antioxidant protein that protects organisms against various oxidative stresses by eliminating hydrogen peroxide. The full-length catalase cDNA of Chinese shrimp Fenneropenaeus chinensis was cloned from the hepatopancreas using degenerate primers by the method of 3' and 5' rapid amplification of cDNA ends PCR. The cDNA sequence consists of 1892 bp with a 1560 bp open reading frame, encoding 520 amino acids with high identity to invertebrate, vertebrate and even bacterial catalases. The sequence includes the catalytic residues His71, Asn144, and Tyr354. The molecular mass of the predicted protein is 58824.04 Da with an estimated pl of 6.63. Sequence comparison showed that the deduced amino acid sequence of F. chinensis catalase shares 96%, 73%, 71% and 70% identity with that of Pacific white shrimp Litopenaeus vannamei, Abalone Haliotis discus hannai, Zhikong scallop Chlamys farreri and Human Homo sapiens, respectively. Catalase transcripts were detected in hepatopancreas, hemocytes, lymphoid organ, intestine, ovary, muscle and gill. by real-time PCR. The variation of catalase mRNA transcripts in hemocytes and hepatopancreas was also quantified by real-time PCR and the result indicated that the catalase showed up-regulated expression trends in hemocytes at 14 h and in hepatopancreas at 37 h after injection with white spot syndrome virus (WSSV). (c) 2008 Elsevier Ltd. All rights reserved.

A Dicer-1 gene from white shrimp Litopenaeus vannamei: Expression pattern in the processes of immune response and larval development

Dicer is a member of the RNAase III family which catalyzes the cleavage of double-stranded RNA to small interfering RNAs and micro RNAs, and then directs sequence-specific gene silencing. In this paper, the full-length cDNA of Dicer-1 was cloned from white shrimp Litopenaeus vannamei (designated as LvDcr1). It was of 7636 bp, including a poly A tail, a 5' UTR of 136 bp, a 3' UTR of 78 bp, and an open reading frame (ORF) of 7422 bp encoding a putative protein of 2473 amino acids. The predicted amino acid sequence comprised all recognized functional domains found in other Dicer-1 homologues and showed the highest (97.7%) similarity to the Dicer-1 from tiger shrimp Penaeus mondon. Quantitative real-time PCR was employed to investigate the tissue distribution of LvDcr1 mRNA, and its expression in shrimps under virus challenge and larvae at different developmental stages. The LvDcr1 mRNA could be detected in all examined tissues with the highest expression level in hemocyte, and was up-regulated in hemocytes and gills after virus injection. These results indicated that LvDcr1 was involved in antiviral defense in adult shrimp. During the developmental stages from fertilized egg to postlarva VII, LvDcr1 was constitutively expressed at all examined development stages, but the expression level varied significantly. The highest expression level was observed in fertilized eggs and followed a decrease from fertilized egg to nauplius I stage. Then, the higher levels of expression were detected at nauplius V and postlarva stages. LvDcr1 expression regularly increased at the upper phase of nauplius, zoea and mysis stages than their prophase. The different expression of LvDcr1 in the larval stages could provide clues for understanding the early innate immunity in the process of shrimp larval development. (C) 2010 Elsevier Ltd. All rights reserved.

Cloning, expression and identification of ferritin from Chinese shrimp, Fenneropenaeus chinensis

Ferritin, the iron storage protein, plays a key role in iron metabolism. A cDNA encoding ferritin (FcFer) was cloned from hepatopancreas of Chinese shrimp, Fenneropenaeus chinensis. The predicted protein contains 170 amino acid residues with a predicted molecular weight (MW) about 19, 422.89 Da and theoretical isoelectric point (PI) of 4.73. Amino acid alignment of FcFer revealed 97% homology with Litopenaeus vannamei ferritin. Results of the RT-PCR showed that the expression of FcFer mRNA was up-regulated after shrimp was challenged with either white spot syndrome virus (WSSV) or heavy metal ions (Zn2+ and Cu2+) in the laboratory. A fusion protein containing FcFer was produced and the purified recombinant protein exhibited similar function of iron uptake in vitro. The result of in-gel digestion and identification using LC-ESI-MS showed that two peptide fragments (-DDVALPGFAK- and -LLEDEYLEEQVDS1KK-) of the recombinant protein were identical to the corresponding sequence of L. vannamei ferritin. The recombinant FcFer protein will be proved useful for study on the structure and function of ferritin in F chinensis. (c) 2006 Elsevier B.V. All rights reserved.

主要环境因子对对虾感染白斑综合症的影响研究

白斑综合症病毒（whitespotsyndromevirus,WSSV）是近来危害对虾养殖业的主要病原之一,自出现以来给对虾养殖业带来巨大经济损失.研究表明,对虾病害的发生是对虾自身的健康状况、病原微生物、以及环境条件等各种因素相互作用的结果,因此对虾病毒问题的研究必须从多角度综合考虑,其中研究环境胁迫因子与养殖动物抗病力和病原生物致病能力的相互作用关系是解决病害问题的关键.该文在国家重点基础研究发展规划项目“环境胁迫对对虾抗病力的影响及其人工调控”的支持下,利用从暴发白斑综合症的中国对虾中分离出的白斑综合症病毒（WSSV）源,通过对虾血细胞计数（totalhaemocytecount,THC）、对虾血清酚氧化酶活性（phenoloxidaseactivity,PO）、对虾累计死亡率及平均死亡时间等方面研究,探讨了感染方式、赤潮异弯藻及盐度变化对对虾感染白斑综合症病毒的影响.

环境因子对养殖生物白斑综合症传播和分子生物标志物系统的影响研究

本文研究了海洋微藻在白斑综合症(white spot syndrome)暴发中的可能作用，以及阴离子表面活性剂十二烷基硫酸钠(SDS)和十二烷基苯磺酸钠(SDBS)长期暴露对紫贻贝(Mytilus galloprovincialis)生物标志物系统的影响(72 d)。 1．海洋微藻在养殖对虾白斑综合症传播中的作用研究 为了证实海洋微藻是否是养殖对虾白斑综合症的传播途径，我们首先将六种海洋微藻：球定边金藻(Isochrysis galbana)、中肋骨条藻(Skeletonema costatum)、小球藻(Chlorella sp. )、赤潮异湾藻(Heterosigma akashiwo)、锥状斯氏藻(Scrippsiella trochoidea)和盐藻(Dunaliella salina)，与人工注射感染白斑病毒(white spot syndrome virus)的成体日本囊对虾共同培养，用套氏PCR方法检测共培养的微藻能否携带白斑病毒。在此基础上，进一步研究了共培养后的海洋微藻是否能感染幼体日本囊对虾。研究结果表明，除了H. akashiwo，实验海洋微藻均可携带白斑病毒，但它们携带病毒的能力有明显差异，Chlorella sp.和S. trochoidea携带白斑病毒的能力较强；但是，与白斑病毒的其他携带者（如桡足类等）不同，携带病毒的海洋微藻10天后病毒检测结果均呈阴性。共培养后小球藻组可感染幼体日本囊对虾，但幼体携带病毒的量只能通过二步PCR方法才能检测到。上述结果表明，海洋微藻在WSSV的水平传播途径中具有一定的作用。 2．表面活性剂对紫贻贝生物标志物系统的影响研究 以青岛胶州湾现场调查数据为依据，选择阴离子表面活性剂十二烷基硫酸钠(SDS)和十二烷基苯磺酸钠(SDBS)作为污染物、以近海底栖生物紫贻贝为受试生物，研究了长期暴露后紫贻贝生化指标(SOD, CAT, GSH, GPx, GST, iNOS, AKP)和遗传毒理指标(AFLP指纹图谱)的变化。实验结果发现: 经过72d不同浓度暴露后，SDBS实验组紫贻贝体内的SOD、CAT和iNOS活性均有显著下降（除CAT 0.1mg/L组外），GSH、GST和GPx在3.0mg /L SDS、SDBS组较各自对照组均有显著升高。SDBS对紫贻贝生化指标影响的显著性水平大于SDS。统计分析显示，SDBS暴露组下GST与GPx呈显著正相关关系，iNOS与SOD也表现出一定正相关,但GSH与CAT、GSH与SOD呈现显著负相关关系。SDS浓度与GST呈显著正相关，而SDBS浓度与CAT呈显著负相关。另外，实验结果发现后闭壳肌中iNOS是一个具有应用前景的阴离子表面活性剂暴露生物标志物。AFLP标记结果统计显示，在实验给定的污染物浓度下，SDBS基因毒性要大于SDS；不同的DNA指纹图谱以及遗传距离图显示不同的污染物造成的DNA损伤是不同的。结果表明，在长期暴露条件(72 d)下，一定浓度的阴离子表面活性剂可以对岗哨生物紫贻贝的SOD, CAT, GSH, GPx, GST, iNOS和AFLP指纹图谱一组指标产生显著影响。

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 complete mitochondrial genome of the ridgetail white prawn Exopalaemon carinicauda Holthuis, 1950 (Crustacean: Decapoda: Palaemonidae) revealed a novel rearrangement of tRNA genes

The complete mitochondrial (mt) DNA sequence was determined for a ridgetail white prawn, Exopalaemon carinicauda Holthuis, 1950 (Crustacea: Decopoda: Palaemonidae). The mt genome is 15,730 bp in length, encoding a standard set of 13 protein-coding genes, 2 ribosomal RNA genes, and 22 transfer RNA genes, which is typical for metazoans. The majority-strand consists of 33.6% A, 23.0% C, 13.4% G, and 30.0% T bases (AT skew = 0.057: GC skew = -0.264). A total of 1045 bp of non-coding nucleotides were observed in 16 intergenic regions,,including a major A+ T rich (79.7%) noncoding region (886 bp). A novel translocation of tRNA(Pro) and tRNA(Thr) was found when comparing this genome with the pancrustacean ground pattern indicating that gene order is not conserved among caridean mitochondria. Furthermore, the rate of Ka/Ks in 13 protein-coding genes between three caridean species is Much less than 1, which indicates a strong Purifying selection within this group. To investigate the phylogenetic relationship within Malacostraca, phylogenetic trees based oil Currently available malacostracan complete mitochondrial sequences were built with the maximum likelihood and Bayesian models. All analyses based oil nucleotide and amino acid data strongly support the monophyly of Decapoda. The Penaeidae, Reptantia, Caridea, and Meiura clades were also recovered as monophyletic groups with Strong Statistical Support. However, the phylogenetic relationships within Pleocyemata are unstable, as represented by the inclusion or exclusion of Caridea. (C) 2009 Elsevier B.V. All rights reserved.