cDNA cloning, characterization and mRNA expression of a pacifastin light chain gene from the Chinese mitten crab Eriocheir sinensis

The pacifastin family, characterized by several conserved arrays of six cysteine residues, is a newly identified serine protease inhibitor (SPI) family discovered uniquely in arthropods and plays important roles in multiple biological processes. In the present study, the full-length cDNA of a pacifastin light chain (designated ESPLC) was cloned from the Chinese mitten crab Eriocheir sinensis by expressed sequence tags (ESTs) and PCR techniques. The 1036 bp ESPLC cDNA contained an 831 bp open reading frame (ORF) encoding a putative pacifastin-related peptide of 276 amino acids, a 5'-untranslated region (UTR) of 67 bp, and a 3'-UTR of 138 bp. Six putative conserved domains sharing a characteristic cysteine array (Cys-Xaa(9-12)-Cys-Asn-Xaa-Cys-Xaa-Cys-Xaa(2-3)-Gly-Xaa(3-4)-Cys-Thr-Xaa(3)-Cys) were identified in the deduced amino acid sequence of ESPLC. The conservation of these PLDs (pacifastin light chain domains) and the relative higher similarity of ESPLC to other pacifastin-related precursors suggested that ESPLC was a member of pacifastin family. The mRNA transcripts of ESPLC were found to be higher expressed in hepatopancreas, gill and haemolymph than in gonad, muscle and heart, with the highest expression level in hepatopancreas. The ESPLC mRNA expression in haemolymph of Chinese mitten crab was up-regulated at 2 h and 12 h after challenged with Listonella anguillarum. The tissue distribution and temporal characteristics of ESPLC mRNA expression, similar to that of prophenoloxidase gene in E. sinensis, suggested that ESPLC was potentially involved in the response against invading bacteria, with the possibility that it functioned in the prophenoloxidase system in E sinensis. (C) 2008 Elsevier Ltd. All rights reserved.

cDNA cloning and mRNA expression of heat shock protein 90 gene in the haemocytes of Zhikong scallop Chlamys farreri

Heat shock protein 90 (HSP90) is a highly conserved molecular chaperone contributing to the folding, maintenance of structural integrity and proper regulation of a subset of cytosolic proteins. The full-length cDNA of Zhikong scallop Chlamysfarreri HSP90 (designated CfHSP90) was cloned by EST and rapid RACE techniques. It was of 2710 bp, including an open reading frame (ORF) of 2181 bp encoding a polypeptide of 726 amino acids with all the five HSP90 family signatures. BLAST analysis revealed that the CfHSP90 gene shared high similarity with other known HSP90 genes. Fluorescent real-time quantitative RT-PCR was used to examine the expression pattern of CfHSP90 mRNA in haemocytes of scallops exposed to Cd2+, Pb2+ and Cu2+ for 10 and 20 days, respectively. All the three heavy metals could induce CfHSP90 expression. There was a clear dose-dependent expression pattern of CfHSP90 after heavy metals exposure for 10 days or 20 days. Different concentrations of the same metal resulted in different effects on CfHSP90 expression. The results indicated that CfHSP90 responded to various heavy metal stresses with a dose-dependent expression pattern as well as exposure time effect, and could be used as a molecular biomarker in a heavy metal polluted environment. (c) 2007 Elsevier Inc. All rights reserved.

Sequence analysis of Arthrospira maxima based on fosmid library

Arthrospira (Spirulina) (Setchell& Gardner) is an important cyanobacterium not only in its nutritional potential but in its special biological characteristics. An unbiased fosmid library of Arthrospira maxima FACHB438 that contains 4300 clones was constructed. The size distribution of insert fragments is from 15.5 to 48.9 kb and the average size is 37.6 kb. The recombination frequency is 100%. Therefore the library is 29.9 equivalents to the Arthrospira genome size of 5.4 Mb. A total of 719 sample clones were randomly chosen from the library and 602 available sequences, which consisted of 307,547 bases, covering 5.70% of the whole genome. The codon usage of A. maxima was not strongly biased. GC content at the first position of codons (46.9%) was higher than the second (39.8%) and the third (45.5%) positions. GC content of the genome was 43.6%. Of these sequences, 287 (47.7%) showed high similarities to known genes, 63 (10.5%) to hypothetical genes and the remaining 252 (41.8%) had no significant similarities. The assigned genes were classified into 22 categories with respect to different biological roles. Remarkably, the high presence of 25 sequences (4.2%) encoding reverse transcriptase indicates the RT gene may have multiple copies in the A. maxima genome and might play an important role in the evolutionary history and metabolic regulation. In addition, the sequences encoding the ATP-binding cassette transport system and the two-component signal transduction system were the second and third most frequent genes, respectively. These genomic features provide some clues as to the mechanisms by which this organism adapts to the high concentration of bicarbonate and to the high pH environment.

A transglutaminase from Chinese shrimp (Fenneropenaeus chinensis), full-length cDNA cloning, tissue localization and expression profile after challenge

Transglutaminase can catalyze the cross-linking reaction between soluble clotting protein molecules from the plasma for prevention of excess blood loss from a wound and obstructing micro-organisms from invading the wound in crustaceans. A novel transglutaminase (FcTG) gene was cloned from hemocytes of Chinese shrimp Fenneropenaeus chinensis by 3' and 5' rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA consists of 2972 bp, encoding 757 amino acids with a calculated molecular mass of 84.96 kDa and a theoretical isoelectric point of 5.61. FcTG contains a typical transglutaminase-like homologue (TGc domain: E-value = 1.94e-38). Three catalytic sites (Cys-324, His-391 and Asp-414) are present in this domain. The deduced amino acid sequence of FcTG showed high identity with black tiger shrimp TG, kuruma shrimp TG and crayfish TG. Transcripts of FcTG mRNA were mainly detected in gill, lymphoid organ and hemocytes by RT-PCR. RNA in situ hybridization further confirmed that FcTG was constitutively expressed in hemocytes both in the circulatory system and lymphoid organ. The variation of mRNA transcription level in hemocytes and lymphoid organ following injection of killed bacteria or infection with white spot syndrome virus (WSSV) was quantified by RT-PCR. The up-regulated expression of FcTG in shrimp lymphoid organ following injection of bacteria indicates that it is inducible and might be associated with bacterial challenge. (c) 2006 Elsevier Ltd. All rights reserved.

The cDNA cloning and mRNA expression of cytoplasmic Cu, Zn superoxide dismutase (SOD) gene in scallop Chlamys farreri

Cu, Zn superoxide dismutases (SODs) are rnetalloenzymes that represent one important line of defence against reactive oxygen species (ROS). A cytoplasmic Cu. Zn SOD cDNA sequence was cloned from scallop Chlamys farreri by the homology-based cloning technique. The full-length cDNA of scallop cytoplasmic Cu, Zn SOD (designated CfSOD) was 1022 bp with a 459 bp open reading frame encoding a polypeptide of 153 amino acids. The predicted amino acid sequence of CfSOD shared high identity with cytoplasmic Cu. Zn SOD in molluscs, insects, mammals and other animals, such as cytoplasmic Cu, Zn SOD in oyster Crassostrea sostrea gigas (CAD42722), mosquito Aedes aegypti (ABF18094), and cow Bos taurus (XP_584414). A quantitative reverse transcriptase real-time PCR (qRT-PCR) assay was developed to assess the mRNA expression of CfSOD in different tissues and the temporal expression of CfSOD in scallop challenged with Listonella anguillarum, Micrococcus luteus and Candida lipolytica respectively. Higher-level mRNA expression of CfSOD was detected in the tissues of haemocytes, gill filaments and kidney. The expression of CfSOD dropped in the first 8-16 h and then recovered after challenge with L. anguillarum and M. litteus, but no change was induced by the C. lipolytica challenge. The results indicated that CfSOD was a constitutive and inducible acute-phase protein, and could play an important role in the immune responses against L. anguillarum and M. luteus infection. (C) 2007 Elsevier Ltd. All rights reserved.

Identification and characterization of a myeloid differentiation factor 88 (MyD88) cDNA from Zhikong scallop Chlamys farreri

Myeloid differentiation factor 88 (MyD88) is a universal and essential adapter for the TLR/IL-1R family. In this report, the first mollusk Myd88 ortholog (named as CfMyd88) was cloned from Zhikong scallop (Chlamys farreri). The full-length cDNA of CfMyd88 was of 1554 bp, including a 5 '-terminal untranslated region (UTR) of 427 bp, a polyA tail, and an open reading frame (ORF) of 1104 bp encoding a polypeptide of 367 amino acids containing the typical TLR and IL-1R-related (TIR) domain and death domain (DD). Homology analysis revealed that the predicted amino acid sequence of CfMyd88 was homologous to a variety of previously identified Myd88s with more than 30% identity. The temporal expressions of CfMyd88 mRNA in the mixed primary cultured haemocytes stimulated by lipopolysaccharide (LPS) and peptidoglycans (PGN) were measured by real-time RT-PCR system. The mRNA expression of CfMyd88 decreased after stimulation with both LPS and PGN, and the lowest level was about 1/3 times (at 6 h) and 1/10 times (at 9 h) to that in the control group, respectively. The expression then recovered and was upregulated to two-fold at 9 h after LPS stimulation or to the original level at 12 It after PGN stimulation. The results suggest that the MyD88-dependent signaling pathway exists in scallop and was involved in the defense system. (c) 2007 Elsevier Ltd. All rights reserved.

Molecular cloning and characterization of a short type peptidoglycan recognition protein (CfPGRP-S1) cDNA from Zhikong scallop Chlamys farreri

Peptidoglycan recognition protein (PGRP) specifically binds to peptidoglycan and plays a crucial role in the innate immune responses as a pattern recognition receptor (PRR). The cDNA of a short type PGRP was cloned from scallop Chlamys farreri (named CfPGRP-SI) by homology cloning with degenerate primers, and confirmed by virtual Northern blots. The full length of CfPGRP-SI cDNA was 1073 bp in length, including a 5 ' untranslated region (UTR) of 59 bp, a 3 ' UTR of 255 bp, and an open reading frame (ORF) of 759 bp encoding a polypeptide of 252 amino acids with an estimated molecular mass of 27.88 kDa and a predicted isoelectric point of 8.69. BLAST analysis revealed that CfPGRP-S1 shared high identities with other known PGRPs. A conserved PGRP domain and three zinc-binding sites were present at its C-terminus. The temporal expression of QPGRP-S1 gene in healthy, Vibrio anguillarum-challenged and Micrococcus lysodeikticus-challenged scallops was measured by RT-PCR analysis. The expression of CfPGRP-S1 was upregulated initially in the first 12 h or 24 h either by M. lysodeikticus or V. anguillarum challenge and reached the maximum level at 24 h or 36 h, then dropped progressively, and recovered to the original level as the stimulation decreased at 72 h. There was no significant difference between V. anguillarum and M. lysodeikticus challenge. The results indicated that the CfPGRP-S1 was a constitutive and inducible acute-phase protein which was involved in the immune response against bacterial infection. (c) 2007 Elsevier Ltd. All rights reserved.

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.

Construction and characterization of two bacterial artificial chromosome libraries of Zhikong scallop, Chlamys farreri Jones et Preston, and identification of BAC clones containing the genes involved in its innate immune system

Large-insert bacterial artificial chromosome (BAC) libraries are necessary for advanced genetics and genomics research. To facilitate gene cloning and characterization, genome analysis, and physical mapping of scallop, two BAC libraries were constructed from nuclear DNA of Zhikong scallop, Chlamys farreri Jones et Preston. The libraries were constructed in the BamHI and MboI sites of the vector pECBAC1, respectively. The BamHI library consists of 73,728 clones, and approximately 99% of the clones contain scallop nuclear DNA inserts with an average size of 110 kb, covering 8.0x haploid genome equivalents. Similarly, the MboI library consists of 7680 clones, with an average insert of 145 kb and no insert-empty clones, thus providing a genome coverage of 1.1x. The combined libraries collectively contain a total of 81,408 BAC clones arrayed in 212 384-well microtiter plates, representing 9.1x haploid genome equivalents and having a probability of greater than 99% of discovering at least one positive clone with a single-copy sequence. High-density clone filters prepared from a subset of the two libraries were screened with nine pairs of Overgos designed from the cDNA or DNA sequences of six genes involved in the innate immune system of mollusks. Positive clones were identified for every gene, with an average of 5.3 BAC clones per gene probe. These results suggest that the two scallop BAC libraries provide useful tools for gene cloning, genome physical mapping, and large-scale sequencing in the species.

cDNA cloning and mRNA expression of a selenium-dependent glutathione peroxidase from Zhikong scallop Chlamys farreri

The glutathione peroxidases are essential enzymes of the cellular antioxidant defence system. In the present study, the full-length cDNA sequence encoding an extracellular glutathione peroxidase (designated CfGPx3) was isolated from Zhikong scallop Chlamys farreri. The complete cDNA was of 1194 bp, containing a 5' untranslated region (UTR) of 50 bp, a 3' UTR of 490 bp and an open reading frame (ORF) of 654 bp encoding a polypeptide of 217 amino acids. CfGPx3 possessed all the conserved features critical for the fundamental structure and function of glutathione peroxidase, such as the selenocysteine encoded by stop codon UGA, the GPx signature motif ((96)LGVPCNQFI(103)) and the active site motif ((WNFEKF184)-W-179). The high similarity of CfGPx3 with GPx from other organisms indicated that CfGPx3 should be a new member of the glutathione peroxidase family. By fluorescent quantitative real-time PCR, the CfGPx3 mRNA was universally detected in the tissues of haemocytes, gill, gonad, muscle and hepatopancreas with the highest expression in hepatopancreas. After scallops were challenged by Listonella anguillarum, the expression level of CfGPx3 transcript in haemocytes was significantly up-regulated (P<0.05) at 8 h post challenge. These results suggested that CfGPx3 was potentially involved in the immune response of scallops and perhaps contributed to the protective effects against oxidative stress. (C) 2010 Elsevier Inc. All rights reserved.

Identification and molecular characterization of a peritrophin-like protein from fleshy prawn (Fenneropenaeus chinensis)

Peritrophin, one of the components of the peritrophic matrix, was first isolated from the intestine of insects. It is thought to protect insects from invasion of microorganisms and to stimulate digestion of food. Peritrophin-like proteins have also been found in crustaceans, as a component of the egg layer. In this study, one fragment of the peritrophin-like gene was obtained from fleshy prawn (Chinese shrimp) (Fenneropenaeus chinensis) by panning the T7 phage display library constructed with the shrimp hemocyte cDNA. The total sequence of the peritrophin cDNA was cloned by modified SMART cDNA and LD-PCR methods. The full cDNA is 1048 bp and the deduced protein is composed of 274 amino acids, including 21 amino acid signal peptide, and four peritrophin A domains and the latter three forming three chitin-binding domains. Similarity analysis results showed that the peritrophin-like protein from F chinensis has significant similarities with peritrophin-like and cortical rod proteins from other shrimp. It was inducing expression in hemocytes, heart, stomach, gut, and gills of the infected shrimp, and constitutive expression in the ovaries. No expression signal was detected in the hepatopancreas of either infected or noninfected shrimp. The recombinant peritrophin-like protein has the activity of binding Gram-negative bacteria and strong binding activity to chitin. Therefore, the bacteria and chitin binding activities of the peritrophin-like protein suggest that it may plays a role in immune defense and other physiological resposes. (c) 2005 Elsevier Ltd. All rights reserved.

The cDNA cloning and mRNA expression of a potential selenium-binding protein gene in the scallop Chlamys farreri

Selenium binding proteins (SeBP) represent a family of proteins that are believed to be involved in controlling the oxidation/reduction in many physiological processes. The cDNA of Zhikong Scallop Chlamys farreri selenium binding protein (zSeBP) was cloned by expressed sequence tag (EST) and RACE techniques. The high similarity of zSeBP deduced amino acid sequence with the SeBP in other organisms, such as bird, fish, frog, mosquito, fruit fly, mammalian, and even nematode and microorganism indicated that zSeBP should be a member of SeBP family. The temporal expression of zSeBP in the hemocytes was measured by semi-quantitative RT-PCR after scallops were stimulated by either oxidative stress or microbial challenge. The expression of zSeBP was up-regulated progressively after stimulation, and then dropped gradually to the original level. Meanwhile, malondialdehyde (MDA) measured by the colorimetric method in the microbial challenged scallops increased immediately after scallops was challenged by microbes, and was significantly higher than that in the control scallops. Results indicated that the microbial infection could incense the disorder of oxidation/reduction and may result in high MDA production. The negative correlation between the expression level of zSeBP and the MDA content suggested that zSeBP could play an important role in mediating the anti-oxidation mechanisms and immune response in marine invertebrates. (c) 2005 Published by Elsevier Ltd.

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.

Fine-scale vertical distribution of bacteria in the East Pacific deep-sea sediments determined via 16S rRNA gene T-RFLP and clone library analyses

Although the deep-sea sediments harbor diverse and novel bacteria with important ecological and environmental functions, a comprehensive view of their community characteristics is still lacking, considering the vast area and volume of the deep-sea sedimentary environments. Sediment bacteria vertical distribution and community structure were studied of the E272 site in the East Pacific Ocean with the molecular methods of 16S rRNA gene T-RFLP (terminal restriction fragment length polymorphism) and clone library analyses. Layered distribution of the bacterial assemblages was detected by both methods, indicating that the shallow sediments (40 cm in depth) harbored a diverse and distinct bacterial composition with fine-scale spatial heterogeneity. Substantial bacterial diversity was detected and nine major bacterial lineages were obtained, including Acidobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Nitrospirae, Planctomycetes, Proteobacteria, and the candidate divisions OP8 and TM6. Three subdivisions of the Proteobacteria presented in our libraries, including the alpha-, gamma- and delta-Proteobacteria. Most of our sequences have low similarity with known bacterial 16S rRNA genes, indicating that these sequences may represent as-yet-uncultivated novel bacteria. Most of our sequences were related to the GenBank nearest neighboring sequences retrieved from marine sediments, especially from deep-sea methane seep, gas hydrate or mud volcano environments. Several sequences were related to the sequences recovered from the deep-sea hydrothermal vent or basalt glasses-bearing sediments, indicating that our deep-sea sampling site might be influenced to certain degree by the nearby hydrothermal field of the East Pacific Rise at 13A degrees N.

编码四种甲壳动物蜕皮抑制激素的部分cDNA的克隆和序列分析

本文分别提取了中国对虾、中华绒螯蟹、鹰爪虾和蓝对虾的眼柄总RNA。用无DNA污染的眼柄总RNA为模板，根据日本对虾的MIH设计兼并引物P1和P2，进行RT－PCR扩增，在适宜的反应条件下，均分别得到一特异性的产物。以蓝对虾基因组DNA为模板的PCR扩增也能得到一与特异性cDNA片段大小相似的特异性的DNA片段。分别将这些片段亚克隆到载体中进行测序，测得中国对虾特异性cDNA片段由203个碱基组成，中华绒螯蟹、鹰爪虾、蓝对虾的特异性cDNA片段及蓝对虾的特异性DNA片段由215个碱基组成，其中蓝对虾特异性cDNA与由蓝对虾的基因组DNA扩增得到的特异性DNA片段的碱基序列几乎完全相同。利用互联网上的在线工具Fasta3查找这些cDNA推定的氨基酸序列的相似序列，可以发现大量的甲壳动物的CHH家族的神经肽与它们相似。在所有的相似序列中，分别由中国对虾、中华绒螯蟹、鹰爪虾和蓝对虾的特异性cDNA片段推定的氨基酸序列与蜕皮抑制激素（MIH）的相似度最高，这一结果提示分别由中国对虾、中华绒螯蟹、鹰爪虾和蓝对虾的眼柄特异性cDNA片段推定的氨基酸序列可能是这四种甲壳动物的MIH片段。根据核苷酸数据和氨基酸数据，比较这四种甲壳动物的MIH以及已发表的六种MIH之间的相似度，同时分析这十种MIH的系统进化关系，结果是蓝对虾、鹰爪虾和中华绒螯蟹的MIH相似性较高，亲缘关系较近，这与物种的系统演化不相符。