Molecular cloning and characterisation of prophenoloxidase (ProPO) cDNA from Fenneropenaeus chinensis and its transcription injected by Vibrio anguillarum

The prophenoloxidase(ProPO) gene was cloned from haemocytes of Chinese shrimp Fenneropenaeus chinensis by Rapid Amplification Complementary DNA Ends (RACE) method. The full-length cDNA of prophenoloxidase gene consists of 3040 bp with a 2061 bp Open Reading Frame (ORF), encoding 686 amino acids. Phylogenetic analysis revealed that it belongs to insect-type invertebrate prophenoloxidase gene family. To understand ProPO reaction for pathogeny's challenge in shrimp, the expressions of ProPO in different tissues were studied by real-time PCR after challenged by Vibrio anguillarum. The results showed that the expression level of ProPO gene in haemocytes was highest among three studied tissues including haemocytes, lymphoid organ and hepatopancreas. The time-course change of ProPO mRNA levels in challenge experiment showed that ProPO mRNA transcripts had the biggest change extent in lymphoid organ.

Molecular cloning and characterisation of a pattern recognition protein, lipopolysaccharide and beta-1,3-glucan binding protein (LGBP) from Chinese shrimp Fenneropenaeus chinensis

A pattern recognition protein (PRP), lipopolysaccharide and beta-1,3-glucan binding protein (LGBP) cDNA was cloned from the haemocyte of Chinese shrimp Fenneropenaeus chinensis by the techniques of homology cloning and RACE. Analysis of nucleotide sequence revealed that the full-length cDNA of 1,275 bp has an open reading frame of 1,098 bp encoding a protein of 366 amino acids including a 17 amino acid signal peptide. Sequence comparison of the deduced amino acid sequence of F. chinensis LGBP showed a high identity of 94%, 90%, 87%, 72% and 63% with Penaeus monodon BGBP, Litopenaeus stylirostris LGBP, Marsupenaeu japonicus BGBP, Homarus gammarus BGBP and Pacifastacus leniusculus LGBP, respectively. The calculated molecular mass of the mature protein is 39,857 Da with a deduced pI of 4.39. Two putative integrin binding motifs, RGD (Arg-Gly-Asp) and a potential recognition motif for beta-1,3-linkage of polysaccharides were observed in LGBP sequence. RT-PCR analysis showed that LGBP gene expresses in haemocyte and hepatopancreas only, but not in other tissues. Capillary electrophoresis RT-PCR method was used to quantify the variation of mRNA transcription level during artificial infection with heat-killed Vibrio anguillarum and Staphylococcus aureusin. A significant enhancement of LGBP transcription was appeared at 6 h post-injection in response to bacterial infection. These results have provided useful information to understand the function of LGBP in shrimp.

Molecular cloning and stress-dependent expression of a gene encoding Delta(12)-fatty acid desaturase in the Antarctic microalga Chlorella vulgaris NJ-7

The psychrotrophic Antarctic alga, Chlorella vulgaris NJ-7, grows under an extreme environment of low temperature and high salinity. In an effort to better understand the correlation between fatty acid metabolism and acclimation to Antarctic environment, we analyzed its fatty acid compositions. An extremely high amount of Delta(12) unsaturated fatty acids was identified which prompted us to speculate about the involvement of Delta(12) fatty acid desaturase in the process of acclimation. A full-length cDNA sequence, designated CvFAD2, was isolated from C. vulgaris NJ-7 via reverse transcription polymerase chain reaction (RT-PCR) and RACE methods. Sequence alignment and phylogenetic analysis showed that the gene was homologous to known microsomal Delta(12)-FADs with the conserved histidine motifs. Heterologous expression in yeast was used to confirm the regioselectivity and the function of CvFAD2. Linoleic acid (18:2), normally not present in wild-type yeast cells, was detected in transformants of CvFAD2. The induction of CvFAD2 at an mRNA level under cold stress and high salinity is detected by real-time PCR. The results showed that both temperature and salinity motivated the upregulation of CvFAD2 expression. The accumulation of CvFAD2 increased 2.2-fold at 15A degrees C and 3.9-fold at 4A degrees C compared to the alga at 25A degrees C. Meanwhile a 1.7- and 8.5-fold increase at 3 and 6% NaCl was detected. These data suggest that CvFAD2 is the enzyme responsible for the Delta(12) fatty acids desaturation involved in the adaption to cold and high salinity for Antarctic C. vugaris NJ-7.

Molecular characterization and effect of RNA interference of retinoid X receptor (RXR) on E75 and chitinase gene expression in Chinese shrimp Fenneropenaeus chinensis

Retinoid X receptor (RXR)/ultraspiracle (USP) is the heterodimeric partner of ecdysteroid receptor and is required for the molting process of arthropods. To better understand the molecular aspects governing the process of molting in shrimp, the full-length cDNA of two RXRs, named as FcRXR-1 and FcRXR-2 were obtained from Chinese shrimp Fenneropenaeus chinensis which were of 1715 and 1700 bp long, revealed a 1315 and 1300 bp open reading frame (ORF) respectively. Quantitative Real time PCR analysis showed a marked tissue-specific difference in the expression of FcRXR transcript, which revealed that the expression of FcRXR Could be regulated in a tissue-specific manner. Moreover, high expression of FcRXR mRNAs was observed in late pre-molt period (D3) and post molt stages (A-B) of shrimp. Among the two isoforms, FcRXR-2 appeared in a considerably high level in all the stages compared to the FcRXR-1. In addition, we examined the temporal expression of two chitinase genes: FcChitinase (FcChi) and FcChitinase-1 (FcChi-1) during the molt cycle of F chinensis. Both the FcChi and FcChi-1 transcripts were detected in all stages of molting, although considerable fluctuations observed through the molt cycle. Injection of double stranded RXR (dsRXR) into juvenile shrimp resulted in a maximum silencing effect at 48 h post injection. We analyzed the expression levels of FcChi, FcChi-1 and the ecdysone inducible gene E75 (FcE75) in samples of dsRXR injected shrimp. Significant reduction in levels of both FcE75, FcChi and FcChi-1 transcripts Occurred in the silenced shrimp. This correlation suggested that RXR might involve in the downstream regulation of E75 and chitinase gene transcription in the ecdysone signaling pathway of decapod crustaceans. (C) 2009 Published by Elsevier Inc.

Molecular characterization and mRNA transcript profile of vitellogenin in Chinese shrimp, Fenneropenaeus chinensis

A full-length cDNA encoding vitellogenin (Vg) was cloned from Chinese shrimp, Fenneropenaeus chinensis using RACE method. The full-length cDNA consist of 7,942 nucleotides including a 7,761 bp open reading frame, which encodes 2,587 amino acid residues. The deduced amino acid sequence showed high (from 94% to 37%) identity with other known crustacean Vgs. In addition, a consensus cleavage site (R-X-K/R-R) recognized by an endopeptidase and a member of subtilisin family of serine protease were identified in the deduced Vg precursor. RT-PCR analysis shown that Vg mRNA can be detected in both ovary and hepatopancreas of vitellogenic females but not in other experimental tissues including muscle, heart, lymph organ, gill, haemocytes and intestine. These results suggest that the Vg gene may be expressed exclusively in mature females, and both ovary and hepatopancreas are the possible tissues for Vg synthesis in F. chinensis. In addition, Vg gene is detected in genomic DNA of both females and males.

Identification of Porphyra lines (Rhodophyta) by AFLP DNA fingerprinting and molecular markers

Twenty-seven Porphyra lines from 5 classes, including lines widely used in China, wild lines, and lines introduced to China from abroad in recent years, were screened by means of amplified fragment length polymorphism (AFLP) with 24 primer pairs. From the generated AFLP products, 13 bands that showed stable and repeatable AFLP patterns amplified by primer pairs M-CGA/E-AA and M-CGA/E-TA were scored and used to develop the DNA fingerprints of the 27 Porphyra lines. Moreover, the DNA fingerprinting patterns were converted into computer language expressed with digitals 1 and 0, which represented the presence (numbered as 1) or absence (numbered as 0) of the corresponding band. On the basis of these results, computerized AFLP DNA fingerprints were constructed in which each of the 27 Porphyra lines has its unique AFLP,fingerprinting pattern and can be easily distinguished from others. Software called PGI-AFLP (Porphyra germplasm identification-AFLP) was designed for identification of the 27 Porphyra lines. In addition, 21 specific AFLP markers from 15 Porphyra lines were identified; 6 AFLP markers from 4 Porphyra lines were sequenced, and 2 of them were successfully converted into SCAR (sequence characterized amplification region) markers. The developed AFLP DNA fingerprinting and specific molecular markers provide useful ways for the identification, classification, and resource protection of the Porphyra lines.

Molecular cloning, characterization and expression analysis of a putative C-type lectin (Fclectin) gene in Chinese shrimp Fenneropenaeus chinensis

Lectin is regarded as a potential molecule involved in immune recognition and phagocytosis through opsonization in crustacean. Knowledge on lectin at molecular level would help us to understand its regulation mechanism in crustacean immune system. A novel C-type lectin gene (Fclectin) 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 1482 bp with an 861 bp open reading frame, encoding 287 amino acids. The deduced amino acid sequence contains a putative signal peptide of 19 amino acids. It also contains two carbohydrate recognition domains/C-type lectin-like domains (CRD1 and CRD2), which share 78% identity with each other. CRD1 and CRD2 showed 34% and 30% identity with that of mannose-binding lectin from Japanese lamprey (Lethenteron japonicum), respectively. Both CRD1 and CRD2 of Fclectin have I I amino acids residues, which are relatively invariant in animals' C-type lectin CRDs. Five residues at Ca2+ binding site I are conserved in Fclectin. The potential Ca2+/carbohydrate-binding (site 2) motif QPD, E, NP (Gln-Pro-Asp, Glu, Asn-Pro) presented in the two CRDs of Fclectin may support its ability to bind galactose-type sugars. It could be deduced that Fclectin is a member of C-type lectin superfamily. Transcripts of Fclectin were found only in hemocytes by Northern blotting and RNA in situ hybridization. The variation of mRNA transcription level in hemocytes during artificial infection with bacteria and white spot syndrome virus (WSSV) was quantitated by capillary electrophoresis after RT-PCR. An exploration of mRNA expression variation after LPS stimulation was carried out in primarily cultured hemocytes in vitro. Expression profiles of Fclectin gene were greatly modified after bacteria, LPS or WSSV challenge. The above-stated data can provide us clues to understand the probable role of C-type lectin in innate immunity of shrimp and would be helpful to shrimp disease control. (c) 2006 Elsevier Ltd. All rights reserved.

Molecular characteristics and expression analysis of calreticulin in Chinese shrimp Fenneropenaeus chinensis

Calreticulin (CRT), as an endoplasmic reticulum luminal resident protein, plays important roles in Ca2+ homeostasis and molecular chaperoning. CRT on the surface of the cell can modulate cell adhesion, phagocytosis and integrin-dependent Ca2+ signaling. The full length cDNA of calreticulin (FcCRT) was cloned from Chinese shrimp Fenneropenaeus chinensis. It consists of 1672 by with an open reading frame of 1221 bp, encoding 406 amino acids. This is the first reported cDNA sequence of calreticulin in Crustacea. The deduced amino acid sequence of FcCRT showed high identity with those of Bombyx mori (88%), Drosophila melanogaster (83%), Mus musculus (82%) and Homo sapiens (82%). Highest expression of FcCRT was detected in ovary by Northern blot and in situ hybridization. Different mRNA levels of FcCRT were detected at various molting stages. Expression of FcCRT was induced significantly after 3 h of heat shock treatment, reached the maximum at 4 h and dropped after that. Differential expression profiles of FcCRT were observed in hepatopancreas and haemocytes when shrimp were challenged by white spot syndrome virus (WSSV). From the above results, we inferred that FcCRT might play important roles in Ca2+ homeostasis, chaperoning and immune function in shrimp. (c) 2007 Elsevier Inc. All rights reserved.

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.

Molecular cloning and characterization of a thioester-containing protein from Zhikong scallop Chlamys farreri

Thioester-containing proteins are a family of proteins characterized by the unique intrachain beta-cysteinyl-gamma-glutamyl thioester, which play important roles in innate immune responses. The cDNA of Zhikong scallop Chlamys farreri thioester-containing protein (designated as CfTEP) was cloned by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of CfTEP was of 4616 bp, consisting of a 5 '-terminal untranslated region (UTR) of 30 bp and a 3 ' UTR of 140 bp with a polyadenylation signal sequence AATAAA and a poly(A) tail. The CfTEP cDNA encoded a polypeptide of 1481 amino acids with the theoretical isoelectric point of 5.98 and the predicted molecular weight of 161.4 kDa. The deduced amino acid sequence of CfTEP contained the canonical thioester motif GCGEQ, nine potential N-glycosylation sites and a C-terminal distinctive cysteine signature. It also contained a presumed catalytic histidine and proteolytic cleavage sites that were similar to C3 molecules. The high similarity of CfTEP with the thioester-containing proteins in other organisms, such as the TEPs from insects, the complement component C3, C4, C5 and the protease inhibitor alpha(2)-macroglobulin indicated that CfTEP should be a member of TEP family. The phylogenetic analysis revealed that CfTEP was closely related to TEPs from mollusc, nematodes and insects, and they formed a separate branch apart from the branches of complements factors and alpha(2)-macroglobulins. The spatial expression of CfTEP transcripts in healthy and bacterial challenged scallops was examined by semi-quantitative RT-PCR. The CfTEP transcripts were mainly detected in the tissues of hepatopancreas and gonad, and remarkably up-regulated by Microbial challenge, which suggested that CfTEP was a constitutive and inducible acute-phase protein involved in immune defense. These results provided new insights into the role of CfTEP in scallop immune responses, as well as the evolutionary origin of this important, widespread and functionally diversified family of proteins. (c) 2007 Published by Elsevier Ltd.

Molecular cloning, expression of a peroxiredoxin gene in Chinese shrimp Fenneropenaeus chinensis and the antioxidant activity of its recombinant protein

Peroxiredoxin (Prx) is known to be an antioxidant protein that protects the organisms against various oxidative stresses and functions in intracellular signal transduction. A Prx gene was firstly isolated in the crustacean, Chinese shrimp Fenneropenaeus chinensis. The full-length cDNA consists of 942 bp with a 594 bp open reading frame, encoding 198 amino acids. The molecular mass of the deduced amino acid is 22041.17 Da with an estimated pI of 5.17. Sequence comparison showed that Prx of F. chinensis shares 76%, 73% and 72% identity with that of Aedes aegypti, Branchiostoma belcheri tsingtaunese and Drosophila melanogaster, respectively. Northern blot analysis revealed the presence of Prx transcripts of F chinensis in all tissues examined. Real-time PCR analysis indicated that the Prx showed different expression profiles in shrimp hemocytes and hepatopancreas after artificial infection with Vibrio anguillarum. In addition, a fusion protein containing Prx was produced in vitro. LC-ESI-MS analysis showed that four peptide fragments of the recombinant protein were identical to the corresponding sequence of F. chinensis Prx. And the purified recombinant proteins were shown to reduce H2O2 in the presence of dithiothreitol. (c) 2007 Elsevier Ltd. All rights reserved.

Molecular cloning of an invertebrate goose-type lysozyme gene from Chlamys farreri, and lytic activity of the recombinant protein

Lysozyme is a widely distributed hydrolase possessing lytic activity against bacterial peptidoglycan, which enables it to protect the host against pathogenic infection. In the present study, the cDNA of an invertebrate goose-type lysozyme (designated CFLysG) was cloned from Zhikong scallop Chlamys farreri by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) techniques. The full-length cDNA of CFLysG consisted of 829 nucleotides with a canonical polyadenylation signal sequence AATAAA and a poly(A) tail, and an open reading frame (ORF) of 603 bp encoding a polypeptide of 200 amino acid residues with a predicted molecular weight of 21.92 kDa and theoretical isoelectric point of 7.76. The high similarity of CFLysG with goose-type (g-type) lysozymes in vertebrate indicated that CFLysG should be an invertebrate counterpart of g-type lysozyme family, which suggested that the origin of g-type lysozyme preceded the emergence of urochordates and even preceded the emergence of deuterostomes. Similar to most g-type lysozymes, CFLysG possessed all conserved features critical for the fundamental structure and function of g-type lysozymes, such as three catalytic residues (Glu 82, Asp 97, Asp 108). By Northern blot analysis, mRNA transcript of CFLysG was found to be most abundantly expressed in the tissues of gills, hepatopancreas and gonad, weakly expressed in the tissues of haemocytes and mantle, while undetectable in the adductor muscle. These results suggested that CFLysG could possess combined features of both the immune and digestive adaptive lysozymes. To gain insight into the in vitro lytic activities of CFLysG, the mature peptide coding region was cloned into Pichia pastoris for heterogeneous expression. Recombinant CFLysG showed inhibitive effect on the growth of both Gram-positive and Gram-negative bacteria with more potent activities against Gram-positive bacteria, which indicated the involvement of CFLysG in the innate immunity of C. farreri. (c) 2006 Elsevier Ltd. All rights reserved.

Molecular cloning, expression of a big defensin gene from bay scallop Argopecten irradians and the antimicrobial activity of its recombinant protein

Antimicrobial peptides are important components of the host innate immune responses by exerting broad-spectrum microbicidal activity against pathogenic microbes. The first mollusk big defensin (designated AiBD) cDNA was cloned from bay scallop Argopecten irradians by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) techniques. The scallop AiBD consisted of 531 nucleotides with a canonical polyadenylation signal sequence AATAAA and a poly(A) tail, encoding a polypeptide of 122 amino acids. The high similarity of AiBD deduced amino acid sequence with big defensin from Tachypleus tridentatus and Branchiostoma belcheri tsingtaunese indicated that AiBD should be a member of big defensin family. The expression of AiBD in various tissues was measured by using Northern blotting analysis. mRNA transcripts of AiBD could be detected in haemocytes of unchallenged scallops. The temporal expression of AiBD in haemolymph after Vibrio anguilarum challenge was recorded by quantitative real time PCR. The relative expression level of AiBD in haemolymph was up-regulated evenly in the first 8 h, followed by a drastic increase, and increased 131.1-fold at 32 h post-injection. These results indicated that AiBD could be induced by bacterial challenge, and it should participate in the immune responses of A. irradians. Biological activity assay revealed that recombinant AiBD could inhibit the growth of both Gram-positive and Gram-negative bacteria, and also showed strong fungicidal activity towards the expression host. Recombinant expression of AiBD made it possible to further characterize its functions involved in immune responses, and also provided a potential therapeutic agent for disease control in aquaculture. (c) 2006 Elsevier Ltd. All rights reserved.

Molecular cloning and responsive expression to injury stimulus of a defender against cell death 1 (DAD1) gene from bay scallops Argopecten irradians

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.

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.