Cloning and expression analysis of a HSP70 gene from Pacific abalone (Haliotis discus hannai)

Heat shock protein 70 (HSP70), the primary member of HSPs that are responsive of thermal stress, is found in all multicellular organisms and functions mostly as molecular chaperon. The inducible HSP70 cDNA cloned from Pacific abalone (Haliotis discus hannai) using rapid amplification of cDNA ends (RACE), was highly homologous to other HSP70 genes. The full-length cDNA of the Pacific abalone HSP70 was 2631bp, consisting of a 5'-terminal untranslated region (UTR) of 90bp, a 3'-terminal UTR of 573bp with a canonical polyadenylation signal sequence AATAAA and a poly (A) tail, and an open reading frame of 1968bp. The HSP70 cDNA encoded a polypeptide of 655 amino acids with an ATPase domain of 382 amino acids, the substrate peptide binding domain of 161 amino acids and a C-terminus domain of 112 amino acids. The temporal expression of HSP70 was measured by semi-quantitative RT-PCR after heat shock and bacterial challenge. Challenge of Pacific abalone with heat shock or the pathogenic bacteria Vibrio anguillarum resulted in a dramatic increase in the expression of HSP70 mRNA level in muscle, followed by a recovery to normal level after 96h. Unlike the muscle, the levels of HSP70 expression in gills reached the top at 12h and maintained a relatively high level compared with the control after thermal and bacterial challenge. The upregulated mRNA expression of HSP70 in the abalone following heat shock and infection response indicates that the HSP70 gene is inducible and involved in immune response.     

Molecular cloning and immune responsive expression of a novel C-type lectin gene from bay scallop Argopecten irradians

C-type lectins are Ca(2+)-dependent carbohydrate-recognition proteins that play crucial roles in innate immunity. The cDNA of C-type lectin (AiCTL1) in the bay scallop Argopecten irradians was cloned by expressed sequence tag (EST) and RACE techniques. The full-length cDNA of AiCTL1 was 660 bp, consisting of a 5'-terminal untranslated region (UTR) of 30 bp and a 3' UTR of 132 bp with a polyadenylation signal sequence AATAAA and a poly(A) tail. The AiCTL1 cDNA encoded a polypeptide of 166 amino acids with a putative signal peptide of 20 amino acid residues and a mature protein of 146 amino acids. The deduced amino acid sequence of AiCTL1 was highly similar to those of the C-type lectins from other animals and contained a typical carbohydrate-recognition domain (CRD) of 121 residues, which has four conserved disulfide-bonded cysteine residues that define the CRD and two additional cysteine residues at the amino terminus. AiCTL1 mRNA was dominantly expressed in the hemocytes of the bay scallop. The temporal expression of AiCTL1 mRNA in hemocytes was increased by 5.7- and 4.9-fold at 6h after injury and 8h after injection of bacteria, respectively. The structural features, high similarity and expression pattern of AiCTL1 indicate that the gene may be involved in injury healing and the immune response in A. irradians.     

A novel serine protease with clip domain from scallop Chlamys farreri

The serine proteases with clip domain are involved in various innate immune functions in invertebrate such as antimicrobial activity, cell adhesion, pattern recognition and regulation of the prophenoloxidase system. A serine protease with clip-domain cDNA (Cf SP) was obtained by Expressed sequence taggings (ESTs) method and rapid amplification of cDNA ends (RACE). The Cf SP full-length cDNA was of 1,152 bp, including a 5'-terminal untranslated region (UTR) of 63 bp, a 3'-terminal UTR of 81 bp with a canonical polyadenylation signal sequence AATAAA and a poly(A) tail, and an open reading frame of 1,008 bp encoding a polypeptide of 336 amino acids with a putative signal peptide of 19 amino acids. The deduced amino acid sequence of Cf SP contained an amino-terminal clip domain with three disulfide bonds formed six conserved Cys residues, a carboxyl-terminal trypsin-like domain with the conserved His-Asp-Ser catalytic triad, and a low complexity linker sequence. The Cf SP was strongly expressed in hemocytes and the mRNA expression of Cf SP was up-regulated and increased 3.2-fold and 2.6-fold at 16 h after injection of Vibrio anguillarum and Micrococcus luteus. The results suggested that Cf SP gene might be involved in immune response of Gram-negative and Gram-positive microbial infection in scallop.     

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 1211bp, consisting of a 5'-terminal untranslated region (UTR) of 72bp, a 3'-terminal UTR of 77bp with a canonical polyadenylation signal sequence AATAAA and a poly (A) tail, and an open reading frame of 1062bp. 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 16h after injury and injection of bacteria.     

Structure, organization and expression of common carp (Cyprinus carpio L.) SLP-76 gene

SLP-76 is an important member of the SLP-76 family of adapters, and it plays a key role in TCR signaling and T cell function. Partial cDNA sequence of SLP-76 of common carp (Cyprinus carpio L.) was isolated from thymus cDNA library by the method of suppression subtractive hybridization (SSH). Subsequently, the full length cDNA of carp SLP-76 was obtained by means of 3' RACE and 5' RACE, respectively. The full length cDNA of carp SLP-76 was 2007 bp, consisting of a 5'-terminal untranslated region (UTR) of 285 bp, a 3'-terminal UTR of 240 bp, and an open reading frame of 1482 bp. Sequence comparison showed that the deduced amino acid sequence of carp SLP-76 had an overall similarity of 34-73% to that of other species homologues, and it was composed of an NH2-terminal domain, a central proline-rich domain, and a C-terminal SH2 domain. Amino acid sequence analysis indicated the existence of a Gads binding site R-X-X-K, a 10-aa-long sequence which binds to the SH3 domain of LCK in vitro, and three conserved tyrosine-containing sequence in the NH2-terminal domain. Then we used PCR to obtain a genomic DNA which covers the entire coding region of carp SLP-76. In the 9.2k-long genomic sequence, twenty one exons and twenty introns were identified. RT-PCR results showed that carp SLP-76 was expressed predominantly in hematopoietic tissues, and was upregulated in thymus tissue of four-month carp compared to one-year old carp. RT-PCR and virtual northern hybridization results showed that carp SLP-76 was also upregulated in thymus tissue of GH transgenic carp at the age of four-months. These results suggest that the expression level of SLP-76 gene may be related to thymocyte development in teleosts.     

赤眼鳟Toll样受体3基因cDNA全长克隆及表达分析

为探究赤眼鳟（Squaliobarbus curriculus）Toll样受体3（Toll-like receptor 3,ScTLR3）基因是否参与抗病毒免疫反应,实验运用RACE技术,克隆得到ScTLR3基因cDNA全长序列,并进行了生物信息学分析;通过Real-Time qPCR技术,检测了ScTLR3 mRNA在健康赤眼鳟10个组织中的分布以及感染草鱼呼肠孤病毒（GCRV）后肝脏、脾脏、体肾和头肾中的表达特征。结果表明:ScTLR3基因cDNA序列全长4043 bp,包括5-非编码区（UTR）216 bp,开放阅读框（ORF）2715 bp和3-UTR 1112 bp;ScTLR3共编码904个氨基酸残基,推导的蛋白分子量102.67 kD,理论等电点8.76;SMART结构域预测显示,ScTLR3由N端的信号肽（SP）、富亮氨酸结构域（LRRs）、跨膜结构域（TM）和C端的Toll/白介素-1受体结构域（TIR）组成。实时荧光定量结果显示,ScTLR3mRNA在检测的各组织中均有表达,肝脏中的相对表达量极显著高于其他组织（P0.01）;感染GCRV后,肝脏、脾脏、体肾和头肾组织中ScTLR3 mRNA均上调表达,肝脏、脾脏和体肾组织中的相对表达量在24h达到峰值,分别为对照组的5倍、7倍和6倍。研究表明,ScTLR3具有TLRs家族基因的典型结构特征,并能被GCRV诱导表达,推测其在赤眼鳟抗GCRV入侵免疫反应中发挥了重要作用。     

Molecular cloning and responsive expression to injury stimulus of a defender against cell death 1 (DAD1) gene from bay scallops <Emphasis Type="Italic">Argopecten irradians</Emphasis>

Apoptosis is an active process of cell death, which is an integral part of growth and development in multicellular organisms. The defender against cell death 1 (DAD1), the regulatory protein to inhibit the apoptosis process, was first cloned from the bay scallop Argopecten irradians by randomly sequencing a whole tissue cDNA library and rapid amplification of cDNA end (RACE). The full-length cDNA of the A. irradians DAD1 was 607 bp, consist of a 5'-terminal untranslated region (UTR) of 63 bp, a 3'-terminal UTR of 205 bp with a canonical polyadenylation signal sequence AATAAA and a poly (A) tail, and an open reading frame of 339 bp. The deduced amino acid sequence of the A. irradians DAD1 showed 75.5% identity to Araneus ventricosus, 74.5% to Drosophila melanogaster, and 73.6% to Homo sapiens, Sus scrofa, Mesocricetus auratus, Rattus norvegicus and Mus musculus. Excluding the Saccharomyces cerevisiae DAD1 homologue, all animal DAD1 including A. irradians DAD1 homologue formed a subgroup and all plant DAD1 proteins formed another subgroup in the phylogenetic analysis. The A. irradians DAD1 was expressed in all examined tissues including adductor muscle, mantle, gills, digestive gland, gonad and hemolymph, suggesting that A. irradians DAD1 is expressed in most body tissues. Furthermore, the mRNA expression levels of A. irradians DAD1 gene of hemolymph were particularly high after injury, suggesting that the gene is responsive to injury stimuli.     

Cloning and expression pattern analysis of a lipopolysaccharide -and beta-1,3-glucan-binding protein in Portunus trituberculatus

The lipopolysaccharide -and beta-1,3-glucan-binding protein (LGBP) is a pattern recognition receptor, which is fundamental for the innate immune response of crustaceans. A LGBP gene was cloned from the haemocytes of Portunus trituberculatus using SMART RACE methods. The full-length LGBP cDNA (1 378 bp) had a 1 095 bp open reading frame encoding a protein of 365 amino acid residues including a 16 amino acid residues signal peptide, a 138 bp 5' untranslated region (UTR) and a 144 bp untranslated region in the 3' UTR with a 29 bp polyA tail. The calculated molecular mass of the mature protein (349 amino acid residues) is 39,825.24 with an estimated pI of 4.49. The gene sequence and secondary structure of LGBP were analyzed by bio-informatics. Additionally, a Glyco hydro 16 domain was identified. The expression of P. trituberculatus in various tissues were detected through RT-PCR methods. The results showed that the LGBP gene expressed in all the tissues detected, including haemocytes, hepatopancreas, heart, gills and muscle. In response to the challenge of Staphyloccocus aureus and Vibrio alginolyticus, the LGBP gene expression in haemocytes of the group challenged with mixed bacteria were higher than the control group within 48 h. It suggested that the LGBP gene plays an active role in immunologic process against bacterial infection.     

Erk2 in ovarian development of green mud crab Scylla paramamosain

We identified extracellular signal-regulated kinase 2 (erk2) from green mud crab, Scylla paramamosain, in this article. It was originally identified from an expressed sequence tag fragment from a normalized gonadal cDNA library. 5' Rapid amplification of cDNA end (RACE) technique was used to obtain the 5' untranslated region (UTR). The full-length cDNA of Sp-erk2 is 1516 bp, including a 5'-terminal UTR of 19 bp, an open-reading frame of 1098 bp, and a 3'-terminal UTR of 399 bp. The translated protein is 365 amino acids in length with a predicted molecular weight of 42 kDa, which is the same as other species. It is the first time that the expression of Sp-erk2 in different stages of ovary development of crustacean was analyzed, and the result showed that the expression of Sp-erk2 increased gradually with ovarian development, with a peak in the mature phase. In situ hybridization histochemistry was used to clarify the detail of expression. Positive signals illustrated that Sp-erk2 mRNA is present in follicular cells when the ovary is in early stages, and in both follicular cells and oocytes when it is in mature phases. All above suggest that Sp-erk2 is important for ovarian development.     

凡纳滨对虾MT基因序列及其在卵巢发育和低温胁迫中的表达分析

在低温处理仔虾全长cDNA文库的筛选测序中, 获得凡纳滨对虾(Litopenaeus vannmei)金属硫蛋白基因全长cDNA序列, 该序列含有425个碱基, 包含177 bp开放阅读框, 上游98 bp的非编码区及下游150 bp 的非编码区, 编码58个氨基酸, 其中半胱氨酸含量丰富, 富含金属硫蛋白典型的Cys-X(1-3)-Cys 结构。多序列比对表明, 凡纳滨对虾MT蛋白序列与美洲螯龙虾(Homarus americanus)MT蛋白序列具最高同源性72.4%。Real-time PCR结果表明, 凡纳滨对虾MT基因在卵巢组织中呈优势表达, 在不同发育期的卵巢中的表达量都很高, 在低温处理凡纳滨对虾肝胰腺组织中上调表达。实验所得结果为研究凡纳滨对虾金属硫蛋白基因在生殖发育和低温应激中的功能提供了参考。       

Molecular cloning of the black tiger shrimp (Penaeus monodon) elongation factor 2 (EF-2): sequence analysis and its expression on the ovarian maturation stage

The techniques of homology cloning and anchored PCR were used to clone the elongation factor 2 (EF-2) gene from black tiger shrimp (Penaeus monodon). The full length cDNA of black tiger shrimp EF-2 (btsEF-2) contained a 5' untranslated region (UTR) of 73 bp, an ORF of 2541 bp encoding a polypeptide of 846 amino acids with an estimated molecular mass of 95 kDa, and a 3( UTR of 112 bp. The searches for protein sequence similarities with BLAST analysis indicated that the deduced amino acid sequence of btsEF-2 was homological to the EF-2 of other species and even the mammalians. The conserved signature sequence of EF-2 gene family, GTPase effector domain and ADP-ribosylation domain were found in the btsEF-2 deduced amino acid sequence. The temporal expressions of gene in the different ovarian stages were measured by real time PCR. The mRNA expressions of the gene were constitutively expressed in ovary and different during the maturation stages. The result indicated that EF-2 gene was constitutively expressed and could play a critical role in the ovarian maturation stage.     

First report of invertebrate Mx: cloning, characterization and expression analysis of Mx cDNA in disk abalone (Haliotis discus discus)

Myxovirus resistance (Mx) protein is one of the most studied antiviral proteins. It is induced by the type I interferon system (IFN alpha/beta) in various vertebrates, but its expression has not been identified or characterized in mollusks or other multi-cellular invertebrates to date. In this study, we isolated the Mx gene from a disk abalone (Haliotis discus discus) normalized cDNA library. Mx cDNA was sequenced, cloned and compared to other known Mx proteins. The full-length 1664 bp of abalone Mx cDNA contained a 1533-bp open reading frame that codes for 511 amino acids. Within the coding sequence of abalone Mx, characteristic features were found, such as a tripartite guanosine-5'-triphosphate (GTP)-binding motif and a dynamin family signature. In addition, leucine residues in the C-terminal region displayed a special leucine domain at L(468), L(475), L(489) and L(510), suggesting that abalone Mx may have a similar oligomerization function as other leucine zipper motifs. Abalone Mx protein exhibited 44% amino acid similarity with channel catfish Mx1, rainbow trout Mx2 and Atlantic halibut Mx. Abalones were injected intramuscularly with the known IFN inducer poly I:C and RT-PCR was performed for Mx mRNA analysis. The results showed enhanced Mx expression in abalone gill and digestive tissues 24h as well as 48 h after injection of poly I:C. Mx mRNA was expressed in gill, digestive gland, mantle and foot tissues in healthy abalone, suggesting that the basal level of Mx expressed is tissue-specific. There is no known Mx protein closely related to abalone Mx according to phylogenetic analysis. Abalone Mx may have diverged from a common gene ancestor of fish and mammalian Mx proteins, since abalone Mx showed high similarity in terms of conserved tripartite GTP-binding, dynamin family signature motifs and poly I:C enhancement of Mx mRNA expression.     

Characterization of interleukin-1 receptor-associated kinase 1 binding protein 1 gene in small abalone Haliotis diversicolor

Interleukin receptor-associated kinase (IRAK)-1 binding protein 1 (IRAK1BP1) is a critical factor in preventing dangerous overproduction of proinflammatory cytokines by the innate immune system and in influencing the specificity of TLR responses. In this study, a first molluscan IRAK1BP1 gene, saIRAK1BP1, was cloned from the small abalone (Haliotis diversicolor). Its full-length cDNA sequence is 1047bp, with a 747bp open reading frame encoding a protein of 249 aa. The molecular mass of the deduced protein is approximately 28.1kDa with an estimated pI of 8.87, and shows highest identity (52%) to acorn worm Saccoglossus kowalevskii. Amino acid sequence analysis revealed that saIRAK1BP1 shares a conserved SIMPL domain. Quantitative real-time PCR was employed to investigate the tissue distribution of saIRAK1BP1 mRNA, and its expression in abalone under bacteria challenge and larvae at different developmental stages. The saIRAK1BP1 mRNA could be detected in all examined tissues, with the highest expression level in hemocytes, and was up-regulated in gills, kidneys and hemocytes after bacteria injection. Additionally, saIRAK1BP1 was constitutively expressed at all examined developmental stages. These results indicate that saIRAK1BP1 play an important role in the adult abalone immune system and might be essential in embryo and larval development in abalone.     

Purification and cDNA cloning of a cellulase from abalone Haliotis discus hannai.

A cellulase [endo-beta-1,4-D-glucanase (EC 3.2.1.4)] was isolated from the hepatopancreas of abalone Haliotis discus hannai by successive chromatographies on TOYOPEARL CM-650M, hydroxyapatite and Sephacryl S-200 HR. The molecular mass of the cellulase was estimated to be 66 000 Da by SDS/PAGE, thus the enzyme was named HdEG66. The hydrolytic activity of HdEG66 toward carboxymethylcellulose showed optimal temperature and pH at 38 degrees C and 6.3, respectively. cDNAs encoding HdEG66 were amplified by the polymerase chain reaction from an abalone hepatopancreas cDNA library with primers synthesized on the basis of partial amino-acid sequences of HdEG66. By overlapping the nucleotide sequences of the cDNAs, a sequence of 1898 bp in total was determined. The coding region of 1785 bp located at nucleotide position 56-1840 gave an amino-acid sequence of 594 residues including the initiation methionine. The N-terminal region of 14 residues in the deduced sequence was regarded as the signal peptide as it was absent in HdEG66 protein and showed high similarity to the consensus sequence for signal peptides of eukaryote secretory proteins. Thus, matured HdEG66 was thought to consist of 579 residues. The C-terminal region of 453 residues in HdEG66, i.e. approximately the C-terminal three quarters of the protein, showed 42-44% identity to the catalytic domains of glycoside hydrolase family 9 (GHF9)-cellulases from arthropods and Thermomonospora fusca. While the N-terminal first quarter of HdEG66 showed 27% identity to the carbohydrate-binding module (CBM) of a Cellulomonas fimi cellulase, CenA. Thus, the HdEG66 was regarded as the GHF9-cellulase possessing a family II CBM in the N-terminal region. By genomic PCR using specific primers to the 3'-terminal coding sequences of HdEG66-cDNA, a DNA of 2186 bp including three introns was amplified. This strongly suggests that the origin of HdEG66 is not from symbiotic bacteria but abalone itself.     

罗氏沼虾Rab11基因cDNA克隆及其组织表达分析

为了发掘罗氏沼虾(Macrobrachium rosenbergii)免疫相关基因, 研究Rab蛋白(Ras-related proteins inbrain)在罗氏沼虾免疫应答中发挥的作用, 研究采用RACE-PCR技术克隆了罗氏沼虾Rab11基因全长cDNA序列, 记为MrRab11。全长1381 bp, 包括226 bp的5'UTR, 511 bp的3'UTR和645 bp的开放阅读框, 编码214个氨基酸, 含有一个Rab结构域。氨基酸序列比对显示, 罗氏沼虾与冈比亚按蚊(Anopheles gambiae)、蚤状溞(Daphniapulex)、叶蝉(Homalodisca vitripennis) Rab11一致性分别为82%、83%和82%。软件预测, MrRab11编码的蛋白分子量约为23.75 kD, 等电点约为5.34。实时荧光定量表达分析表明, MrRab11基因在罗氏沼虾各组织中都有表达, 肝胰腺中的表达量最高, 其次是肌肉和肠道。在阴沟肠杆菌(Enterobacter cloacae)感染12h后, 罗氏沼虾肝胰腺中MrRab11的表达量上升, 显著高于对照组(P0.05), 推测这是MrRab11对阴沟肠杆菌的应激表达,MrRab11在肝胰腺中参与了罗氏沼虾免疫应答过程。