Isolation of a cDNA encoding a putative cellulase in the red claw crayfish Cherax quadricarinatus

Amino acid sequences of cellulases have been determined in insects, nematodes, plants, slime moulds and bacteria but not in crustaceans. However, cellulase activity has been demonstrated in the hepatopancreas of the red claw crayfish, Cherax quadricarinatus. In order to obtain information on the nature of this cellulase, a C. quadricarinatus hepatopancreas cDNA library was screened with a PCR product generated using degenerate oligonucleotide primers derived from conserved regions of known cellulases. Two identical 1.56 kb cDNAs with sequence similarities to known cellulases, particularly the termite endoglucanases, were identified and sequenced. The clones contain the complete cDNA open reading frame for an endo-1,4-beta-glucanase of 469 amino acids termed Cherax quadricarinatus endoglucanase (CqEG). The endogenous origin of the gene was confirmed by PCR amplification and sequencing of a 1012 bp PCR product from genomic DNA. This fragment contains four exon sequences identical to the cDNA and is interrupted by three introns of 371, 102, 194 bp respectively, with one intron exhibiting typical eukaryotic splice sites. The isolation of an endo-1,4-beta-glucanase encoding cDNA from the crayfish C. quadricarinatus provides the first endogenous cellulase sequence in a crustacean species.     

Molecular cloning,expression pattern and phylogenetic analysis of myosin light chain 2 gene from Antheraea pernyi: A potential marker for phylogenetic inference

Myosin light chain 2 (MLC-2) gene was isolated and characterized from Antheraea pernyi, a well-known wild silkmoth. The isolated cDNA sequence is 905 bp in length with an open reading frame of 612 bp encoding a polypeptide of 203 amino acids. Semi-quantitative RT-PCR analysis showed that the MLC-2 gene was transcribed during four developmental stages (egg, larva, pupa, and moth), and present in all tissues tested. Alignment analysis revealed that the deduced protein sequence has over 95% identity to myosin light chain 2 of lepidopteran species, and 57–88% identity to other insect species, suggesting that insect MLC-2 proteins are highly conserved throughout evolution. The protein sequence was used to construct phylogenetic trees with other known vertebrate and invertebrate MLC-2 sequences, and the obtained trees demonstrated similar topology with the classical systematics, indicating the potential value of MLC-2 gene in phylogenetic study.     

Characterization of emY162 encoding an immunogenic protein cloned from an adult worm-specific cDNA library of Echinococcus multilocularis

A cDNA library based on mRNA from adult worms of Echinococcus multilocularis was constructed. One cDNA clone, emY162, was isolated from this cDNA library. The putative protein from emY162 cDNA consists of 153 amino acids and has a predicted molecular weight of 17.0 kDa. The amino acid sequences of EMY162 are predicted to be a hydrophobic N-terminus conserving a secretory signal, and a hydrophobic C-terminus encoding a transmembrane domain or glycosyl-phosphatylinositol membrane anchor, and to have single fibronectin type III-like domain. In addition, it was shown that the emY162 gene (1738 bp) in the E. multilocularis genome DNA consists of three exons and two introns, and that emY162 is expressed in all four stages (protoscoleces, cultured metacestodes, immature adult worms and mature adult worms). Moreover, immunity to recombinant EMY162, which comprises the fibronectin type III-like domain on the EMY162 protein, was examined. Immune responses to the recombinant EMY162 were studied by using serum from dogs infected with E. multilocularis. Strong IgG immune responses were detected in Western blots.     

Molecular characterization of a 14-3-3 zeta gene from Plodia interpunctella: A potential marker for phylogenetic inference

The 14-3-3 proteins are a large family of approximately 30 kDa acidic proteins and acting in the regulation of many biological processes. In this study, a 14-3-3 zeta (Pi14-3-3z) gene from the Indian meal moth, Plodia interpunctella (Lepidoptera, Pyralidae) was isolated and characterized. The full-length cDNA of Pi14-3-3z is 1382 bp, including a 5'-untranslated region (UTR) of 141 bp, 3′-UTR of 497 bp and an open reading frame (ORF) of 744 bp encoding a polypeptide of 247 amino acids which contains a 14-3-3 homologues domain (PF00244). The deduced Pi14-3-3z protein sequence has 81%–100% identity with the homologues in comparison to with other individuals. qPCR analysis revealed that Pi14-3-3z was expressed at the four developmental stages and in all tissues tested. Based on the amino acid of 14-3-3z, phylogenetic analysis demonstrated a similar topology with the traditional classification, suggesting 14-3-3z protein has the potential value in phylogenetic inference.     

Isolation and Characterization of the PKAr Gene From a Plant Pathogen,Curvularia lunata

By using EST database from a full-length cDNA library of Curvularia lunata, we have isolated a 2.9 kb cDNA, termed PKAr. An ORF of 1,383 bp encoding a polypeptide of 460 amino acids with molecular weight 50.1 kDa, (GeneBank Acc. No. KF675744) was cloned. The deduced amino acid sequence of the PKAr shows 90 and 88 % identity with cAMP-dependent protein kinase A regulatory subunit from Alternaria alternate and Pyrenophora tritici-repentis Pt-1C-BFP, respectively. Database analysis revealed that the deduced amino acid sequence of PKAr shares considerable similarity with that of PKA regulatory subunits in other organisms, particularly in the conserved regions. No introns were identified within the 1,383 bp of ORF compared with PKAr genomic DNA sequence. Southern blot indicated that PKAr existed as a single copy per genome. The mRNA expression level of PKAr in different development stages were demonstrated using real-time quantitative PCR. The results showed that the level of PKAr expression was highest in vegetative growth mycelium, which indicated it might play an important role in the vegetative growth of C. lunata. These results provided a fundamental supporting research on the function of PKAr in plant pathogen, C. lunata.     

Cloning and characterization of the 14-3-3 protein gene from the halotolerant alga Dunaliella salina

Previous studies have demonstrated that 14-3-3 proteins exist in all the eukaryotic organisms studied; however, studies on the 14-3-3 proteins have not been involved in the halotolerant, unicellular green alga Dunaliella salina so far. In the present study, a cDNA encoding 14-3-3 protein of D. salina was cloned and sequenced by PCR and rapid amplification of cDNA end (RACE) technique based on homologous sequences of the 14-3-3 proteins found in other organisms. The cloned cDNA of 1485 bp in length had a 29.2 kDa of molecular weight and contained a 774 bp of open reading frame encoding a polypeptide of 258 amino acids. Like the other 14-3-3 proteins, the deduced amino acid sequences of the D. salina 14-3-3 protein also contained two putative phosphorylation sites within the N-terminal region (positions 62 and 67). Furthermore, an EF hand motif characteristic for Ca²⁺-binding sites was located within the C-terminal part of this polypeptide (positions 208–219). Analysis of bioinformatics revealed that the 14-3-3 protein of D. salina shared homology with that of other organisms. Real-time quantitative PCR demonstrated that expression of the 14-3-3 protein gene is cell cycle-dependent.     

Molecular cloning and expression profiles of nitric oxide synthase (NOS) in mud crab Scylla paramamosain

The importance of the nitric oxide synthase (NOS) gene family is demonstrated by many studies in vertebrates and invertebrates in recent years. However, it keeps unknown of nitric oxide (NO) system and NOS gene family in mud crab Scylla paramamosain, an important cultured commercial crustacean in China and Pacific area. In this report, the cDNA of NOS containing full-length ORF was cloned from mud crab, S. paramamosain. It was of 4424 bp, including a 5′-terminal untranslated region (UTR) of 239 bp, a 3′-terminal UTR of 540 bp, which contained two ATTTA motifs, and an open reading frame (ORF) of 3645 bp encoding a polypeptide of 1214 amino acids. Structural analysis indicated that NOS contained a typical NO synthase domain at the N-terminal, next to a flavodoxin 1 domain, a flavin adenine dinucleotide (FAD) binding domain, respectively, and a conservative nicotinamide adenine dinucleotide (NAD) binding domain structure at the C-terminal. Quantitative real-time PCR analysis revealed S. paramamosain NOS (SpNOS) to be expressed in all tissues examined, with the highest expression in midintestine and the weakest level in heart and eyestalk. The expression profiles of SpNOS indicated that the NOS expression levels were significantly induced in midintestine, hepatopancrease and hemocytes after challenged with Vibrio Parahaemolyticus, the synthetic double-stranded RNA polyinosinic polycytidylic acid (poly I:C) and lipopolysaccharides (LPS). The NOS activity in hemocytes showed significant increase during at 24 h-48 h time period after immune challenges with V. Parahaemolyticus, poly I:C and LPS. Results here may suggest that the inducible NOS play an important role in mud crab’s defense against pathogenic infection.     

Cloning and expression of HSP70 gene of sipuncula Phascolosoma esculenta

In this study the gene encoding HSP70 was isolated from Phascoloma esculenta by homologous cloning and rapid amplification of cDNA ends (RACE). The full-length of cDNA (2520 bp) consists of a 5′-terminal untranslated region (UTR) (125 bp), a 3′-terminal UTR (421 bp) with a canonical polyadenylation signal sequence (AATAAA), a poly (A) tail, and an open reading frame (ORF) (1974 bp). The predicted molecular mass and isoelectric point for HSP70 is 71.6 kDa and 5.15, respectively. BLAST analysis showed that P. esculenta HSP70 gene shared high similarity. Classical HSP signature motifs, ATP/GTP-Binding Site Motif A, Bipartite Nuclear Targeting Sequence, the cytosolic HSP70 could be expressed in Escherichia coli BL21. After purification, the recombinant pET-HSP70 protein was used to produce the polyclonal antibody in mice and the specificity of the antibody was confirmed by Western blot analysis. Fluorescent real-time quantitative PCR analysis showed that expression of Hsp70 in sipuncula was increased significantly after exposure to 10 mM Zn for12 h, Cd for 24 h, Cu for 48 h, and was exposure to 37 °C for 24 h sea water.     

Molecular cloning and expression analysis of a squalene synthase gene from a medicinal plant, Euphorbia pekinensis Rupr.

Euphorbia pekinensis Rupr., which is also known as a medicinal plant, produces a large amount of alkaloids, phytosterols and triterpenes. In this study, we reported on the cDNA cloning and characterization of a novel squalene synthase (SQS) from E. pekinensis. Squalene synthase catalyzes the condensation of two molecules of farnesyl diphosphate (FPP) to produce squalene (SQ), the first committed precursor for sterol and triterpene biosynthesis. The full length cDNA named EpSQS (Genbank Accession Number JX509735) contained 1,614 bp with an open reading frame of 1,236 bp encoding a polypeptide of 411 amino acids. The deduced amino acid sequence of the EpSQS named EpSQS exhibited a high homology with other plant SQSs, and contained a single domain surrounded by helices. Phylogenetic analysis showed that EpSQS belonged to the plant SQS kingdom. Tissue expression analysis revealed that EpSQS expressed strongly in roots, weakly in stems and leaves, implying that EpSQS was a constitutive expression gene. The recombinant protein was expressed in Escherichia coli and detected by SDS-PAGE and western blot. The high performance liquid chromatography (HPLC) analysis showed that EpSQS could catalyze the reaction from farnesyl diphosphate (FPP) to squalene.     

Differentiation of Species and Populations of Aphelenchoides and of Ditylenchus angustus Using a Fragment of Ribosomal DNA

The polymerase chain reaction (PCR) was used to amplify a fragment of the ribosomal DNA (rDNA) from species and undescribed populations of Aphelenchoides and Ditylenchus angustus. The PCR primers used were based on conserved sequences in the 18S and 26S ribosomal RNA genes of Caenorhabditis elegans. In C. elegans, these primers amplify a 1,292 base pair (bp) fragment, which consists of the two internal transcribed spacers and the entire 5.8S gene. Amplification products from crude DNA preparations of 12 species and populations of Aphelenchoides and from D. angustus ranged in size from approximately 860-1,100bp. Southern blots probed with a cloned ribosomal repeat from C. elegans confirmed the identity of these amplified bands as ribosomal fragments. In addition to the differing sizes of the amplified rDNA fragments, the relative intensity of hybridization with the C. elegans probe indicated varying degrees of sequence divergence between species and populations. In some cases, amplified rDNA from the fungal host was evident. Storage of A. composticola at - 45 C for 2 years did not affect the ability to obtain appropriate amplified products from crude DNA preparations. Amplified rDNA fragments were cut with six restriction enzymes, and the restriction fragments produced revealed useful diagnostic differences between species and some undescribed populations. These results were consistent with previous studies based on morphology and isoenzymes. Three undescribed populations of Aphelenchoides were found to be different from all the species examined and from each other.