三疣梭子蟹Profilin基因全长cDNA的克隆与表达分析

三疣梭子蟹（Portunus trituberculatus）是我国沿海重要养殖品种之一,近年来养殖病害呈逐年上升趋势,制约了三疣梭子蟹养殖产业的健康可持续发展。克隆三疣梭子蟹免疫相关基因,研究免疫基因的功能和作用机制,可为三疣梭子蟹养殖病害的防治奠定基础。本研究从三疣梭子蟹血细胞全长cDNA文库中克隆了742 bp 的profilin基因全长cDNA。Profilin全长cDNA中开放阅读框长375 bp,编码125 aa。推导的三疣梭子蟹profilin理论等电点pI 5.87,氨基酸序列与冈比亚按蚊（Anopheles gambiae）profilin同源性最高,序列一致性为42.9%。荧光定量RT-PCR分析结果显示在正常的三疣梭子蟹机体中,血细胞profilin表达水平最高,其次为肝胰脏;在致病菌副溶血弧菌（Vibrio parahaemolyticus）诱导后,血细胞中profilin表达量显著上升（P<0.01）,表明profilin可能参与了三疣梭子蟹的免疫防御反应,是一个免疫相关因子。     

A C-type lectin like-domain (CTLD)-containing protein (PtLP) from the swimming crab Portunus trituberculatus

C-type lectins play important roles in the non-self innate immune system of invertebrates. In this study, we isolated the full-length cDNA of the C-type lectin like-domain (CTLD)-containing protein, designated PtLP, from the hepatopancreas of the swimming crab Portunus trituberculatus, one of the most common edible crabs of East Asia. The PtLP cDNA consists of 923bp and encodes a polypeptide of 164 amino acids containing a well-conserved C-type lectin like-domain (CTLD). The deduced amino acid sequence of PtLP shows 29-36% amino acid sequence identity to other crustacean C-type lectin sequences. A phylogenetic analysis revealed that PtLP is in a large cluster together with black tiger shrimp PmAV, a gene involved in virus resistance of shrimp, and all of the C-type lectins from the various shrimps. Quantitative RT-PCR analysis showed that the PtLP mRNA was expressed highly in hepatopancreas and moderately in gills, hemocytes, and ovary of normal swimming crabs.     

A single WAP domain-containing protein from Litopenaeus vannamei hemocytes

A cDNA clone coding for a single WAP domain (SWD) protein was isolated from a hemocyte cDNA library of Litopenaeus vannamei. The full-length cDNA sequence is 0.4kb long and encodes a 93-amino acid protein. Using this sequence as a probe a similar clone coding for a 92-amino acids protein was found in a cDNA library from Penaeus monodon hemocytes. The mRNA size was confirmed by Northern blot as well as that gene is expressed in hemocytes, but not in hepatopancreas. mRNA levels of the shrimp SWD protein were modified after injection of Vibrio alginolyticus, indicating the probable role of this protein in the immune response. Although amino acid sequence seems to be similar to those of other WAP domain-containing proteins, shrimp SWD protein does not have any other functional domain, similar to a mouse single WAP motif (SWAM) protein reported in mouse; however, the phylogenetic analysis shows that shrimp SWD is more related to other WAP proteins than to mouse SWAM.     

Cloning and sequencing analysis of three amylase cDNAs in the shrimpPenaeus vannamei (Crustacea decapoda): evolutionary aspects

InPenaeus vannamei, α-amylase is the most important glucosidase and is present as at least two major isoenzymes which have been purified. In order to obtain information on their structure, a hepatopancreas cDNA library constructed in phage lambda-Zap II (Strategene) was screened using a synthetic oligonucleotide based on the amino acid sequence of a V8 staphylococcal protease peptide ofP. vannamei α-amylase. Three clones were selected: AMY SK 37 (EMBL sequence accession number: X 77318) is the most complete of the analyzed clones and was completely sequenced. It contains the complete cDNA sequence coding for one of the major isoenzymes of shrimp amylase. The deduced amino acid sequence shows the existence of a 511-residue-long pre-enzyme containing a highly hydrophobic signal peptide of 16 amino acids. Northern hybridization of total RNA with the amylase cDNA confirms the size of the messenger at around 1,600 bases. AMY SK 28, which contains the complete mature sequence of amylase, belonged to the same family characterized by a common 3′ terminus and presented four amino acid changes. Some other variants of this family were also partially sequenced. AMY SK 20 was found to encode a minor variant of the protein with a different 3′ terminus and 57 amino acid changes. Phylogenetic analysis established with the conserved amino acid regions of the (β/α) eight-barrel domain and with the total sequence ofP. vannamei showed close evolutionary relationships with mammals (59–63% identity) and with insect α-amylase (52–62% identity). The use of conserved sequences increased the level of similarity but it did not alter the ordering of the groupings. Location of the secondary structure elements confirmed the high level of sequence similarity of shrimp α-amylase with pig α-amylase. Correspondence to: A. Van Wormhoudt     

Structural and functional differences of Litopenaeus vannamei crustins

Penaeid crustins were described in Litopenaeus vannamei and L. setiferus as proteins belonging to an antibacterial peptide family with similar sequences but different sizes. Six crustin-coding clones were isolated from a cDNA library from L. vannamei hemocytes, sequenced and compared. Two different isoforms (named I and P) were found, based on two nucleotide differences that produce one change in amino acid sequence (Ile/Pro). Other single differences in nucleotide sequences were also noted, but they did not change the translated product. The mRNA steady state levels of crustin I, but not of crustin P, were down regulated by Vibrio alginolyticus inoculation. Thus, the differences among penaeid crustins seem to be associated with one amino acid substitution, which affects their expression after bacterial inoculation. By structural similarity, shrimp crustins seem to belong to an antibacterial WAP-domain containing protein family.     

Thermolabile alkaline phosphatase from Northern shrimp (Pandalus borealis): protein and cDNA sequence analyses

Sequence analysis of short fragments resulting from trypsin digestion of the thermolabile shrimp alkaline phosphatase (SAP) from Northern shrimp Pandalus borealis formed the basis for amplification of its encoding cDNA. The predicted protein sequence was recognized as containing the consensus alkaline phosphatase motif comprising the active site of this protein family. Protein sequence homology searches identified several eukaryote alkaline phosphatases with which the 475-amino acid SAP polypeptide revealed shares 45% amino acid sequence identity. Residues for potential metal binding seem to be conserved in these proteins. The predicted 54-kDa molecular mass of SAP is smaller than previously reported, but is consistent with our recent SDS-PAGE analysis of the native protein. Compared to its homologs, the shrimp enzyme has a surplus of negatively charged amino acids, while the relative number of prolines is lower and the frequency of aromatic residues is higher than in mesophilic counterparts.     

Transcriptional regulation of extracellular copper zinc superoxide dismutase from white shrimp Litopenaeus vannamei following Vibrio alginolyticus and WSSV infection

The cDNA encoding an extracellular copper zinc superoxide dismutase (LvECSOD) was cloned from the hepatopancreas of white shrimp Litopenaeus vannamei. It consisted of 915 bp nucleotides with an open reading frame corresponding to a deduced protein of 178 amino acids. The LvECSOD contains a putative signal peptide of 16 amino acids, two potential N-linked glycosylation sites (N(115)GTA and N(135)ITG) and a copper zinc superoxide dismutase family signature sequence (G(162)NAGaRvACctI(173)). It was found that four copper binding sites, four zinc binding sites and two cysteines involving in the formation of the disulfide bridge were conserved in the protein. LvECSOD shared 33-58% identity to ECSODs from other organisms. Expression analysis revealed that LvECSOD mRNA was widely distributed in all the tissues examined. When the shrimp challenged with Vibrio alginolyticus or white spot syndrome virus (WSSV), expression of LvECSOD mRNA in the hepatopancreas and hemocytes was mediated responsively. Our results suggested that LvECSOD was implicated in the immune response induced by V. alginolyticus and WSSV.     

Cloning and expression of glucose regulated protein 78 (GRP78) in<Emphasis Type="Italic"> Fenneropenaeus chinensis</Emphasis>

GRP78 (78 kDa glucose-regulated protein), also known as BiP (immunoglobulin heavy-chain-binding protein), is an essential regulator of endoplasmic reticulum (ER) homeostasis because of its multiple functions in protein folding, ER calcium binding, and controlling of the activation of transmembrane ER stress sensors. In this report, we cloned the full length cDNA of GRP78 (FcGRP78) from Chinese shrimp Fenneropenaeus chinensis. This cDNA revealed a 2,325 bp with 1,968 bp open reading frame encoding 655 amino acids. This is the first reported GRP78 gene in Crustacea. The deduced amino acid sequence of FcGRP78 shared high identity with previously reported insect GRP78s: 86, 87 and 85% identity with GRP78s of Drosophila melanogaster, Aedes aegypti and Bombyx mori, respectively. Northern blot analysis shows that FcGRP78 is ubiquitously expressed in tissues of shrimp. Heat shock at 35°C significantly enhanced the expression of FcGRP78 at the first hour, reached the maximum at 4 h post heat shock, dropped after that and resumed to the normal level until 48 h of post recovery at 25°C. Additionally, differential expression of FcGRP78 was detected in haemocytes, hepatopancreas and lymphoid organ when shrimp were challenged by white spot syndrome virus (WSSV). We inferred that FcGRP78 may play important roles in chaperoning, protein folding and immune function of shrimp.     

A novel C-type lectin (Cflec-3) from Chlamys farreri with three carbohydrate-recognition domains

C-type lectins are a superfamily of carbohydrate-recognition proteins which play crucial roles in the innate immunity. In this study, the gene of a C-type lectin with multiple carbohydrate-recognition domains (CRDs) from scallop Chlamys farreri (designated as Cflec-3) was cloned by rapid amplification of cDNA ends (RACE) approach based on expression sequence tag (EST) analysis. The full-length cDNA of Cflec-3 was of 2256 bp. The open reading frame encoded a polypeptide of 516 amino acids, including a signal sequence and three CRDs. The deduced amino acid sequence of Cflec-3 showed high similarity to members of C-type lectin superfamily. By fluorescent quantitative real-time PCR, the Cflec-3 mRNA was mainly detected in hepatopancreas, adductor, mantle, and marginally in gill, gonad and hemocytes of healthy scallops. After scallops were challenged by Listonella anguillarum, the mRNA level of Cflec-3 in hemocytes was up-regulated and was significantly higher than that of blank at 8 h and 12 h post-challenge. The function of Cflec-3 was investigated by recombination and expression of the cDNA fragment encoding its mature peptide in Escherichia coli BL21 (DE3)-pLysS. The recombined Cflec-3 (rCflec-3) agglutinated Gram-negative bacteria Pseudomonas stutzeri. The agglutinating activity was calcium-dependent and could be inhibited by d-mannose. These results collectively suggested that Cflec-3 was involved in the immune response against microbe infection and contributed to nonself-recognition and clearance of bacterial pathogens in scallop.     

Molecular cloning and expression analysis of a heat shock protein (Hsp90) gene from black tiger shrimp (<Emphasis Type="Italic">Penaeus monodon</Emphasis>)

The techniques of homology cloning and anchored PCR were used to clone the Hsp90 gene from black tiger shrimp. The full length cDNA of black tiger shrimp Hsp90 (btsHsp90) contained a 5′ untranslated region (UTR) of 72 bp, an ORF (open reading frame) of 2160 bp encoding a polypeptide of 720 amino acids with an estimated molecular mass of 83-kDa and a 3′ UTR of 288 bp. The sequence of the coding region showed 90 and 84% homology with that of the Chiromantes haematocheir and Homo sapiens, respectively. Conserved signature sequences of Hsp90 gene family were found in the btsHsp90 deduced amino acid sequence. The temporal expressions of Hsp90 gene were constitutively in the black tiger shrimp tissues including liver, ovary, muscle, brain stomach, and heart, and their levels were markedly enhanced after 30-min heat treatment at 37°C. In ovarian maturation stages, the expression of btsHsp90 was strongest in the second stage, weaker in the fourth and first stage.