Acclimation-dependent expression of heat shock protein 70 in Pacific abalone (Haliotis discus hannai Ino) and its acute response to thermal exposure

Heat shock protein 70 (Hsp70) is one important member of heat shock protein (Hsp) family that is responsible for various stresses, especially thermal stress. Here we examined the response of Hsp70 gene to both chronic and acute thermal exposure in Pacific abalone (Haliotis discus hannai Ino). For the chronic exposure, abalones were maintained at 8, 12, 20, and 30°C for four months and their mRNA levels were measured. The highest mRNA level of Hsp70 gene relative to actin gene was detected in the 30°C-acclimated group, followed by the 8°C-acclimated group and then the 12°C- and 20°C-acclimated groups. After the long-term acclimation, gills from each of the above acclimation groups were dissected and exposed to different temperatures between 8°C and 38°C for 30 min. Hsp70 expression in gills acclimated to different temperatures responded differentially to the same temperature exposure. The incubation temperature that induced maximum Hsp70 mRNA expression was higher in the higher temperature acclimation groups than lower temperature groups. Pacific abalones could alter the expression pattern of Hsp70 gene according to environmental thermal conditions, through which they deal with the stress of thermal variations.     

Histological change and heat shock protein 70 expression in different tissues of Japanese flounder Paralichthys olivaceus in response to elevated temperature

High temperature influences the homeostasis of fish.We investigated the effects of elevated temperature on tissues of Japanese flounder {Paralichthys olivaceus) by analyzing the histology and heat shock protein 70(hsp70) expression of fish reared in warm conditions.In this study,temperature was increased at l±0.5℃/day starting at 24±0.5℃,and was kept at that temperature for 5 days before the next rise.After raising temperature at the rate up to 32±0.5℃,tissue samples from midgut,spleen,stomach,liver,muscle,gill,heart,trunk kidney and brain were collected for histological analysis and mRNA assay.Almost all the tissues showed changes in morphological structure and hsp70 level at 32±0.5℃.Histological assessment of the tissues indicated that the gill had the most serious damage,including highly severe epithelial lifting and edema,curved tips and hyperemia at the ending of the lamellars,desquamation and necrosis.The next most severe damage was found in liver and kidney.The hsp70 levels in all the tissues first increased and then decreased.The gut,stomach,muscle,heart,and brain had the highest expressions in 6 h,whereas the spleen,liver,gill and kidney had the highest expressions in 2 h.Therefore,tissues with the most significant lesions(especially gill and liver) responded much earlier(2 h) in hsp70 expression than other tissues,and these tissues demonstrated the most marked histological disruption and elevated mRNA levels,making them ideal candidates for further studies on the thermal physiology of this species.     

Gene cloning and prokaryotic expression of recombinant outer membrane protein from Vibrio parahaemolyticus

Gram-negative Vibrio parahaemolyticus is a common pathogen in humans and marine animals. The outer membrane protein of bacteria plays an important role in the infection and pathogenicity to the host. Thus, the outer membrane proteins are an ideal target for vaccines. We amplified a complete outer membrane protein gene (ompW) from V. parahaemolyticus ATCC 17802. We then cloned and expressed the gene into Escherichia coli BL21 (DE3) cells. The gene coded for a protein that was 42.78 kDa. We purified the prote...     

Effects of temperature on the respiratory metabolism,feeding and expression of three heat shock protein genes in Anadara broughtonii

Anadara broughtonii is one of the main commercially important species of shellfish in northern China.A.broughtonii lives in relatively stable subtidal zone where the clam could re spond to environmental changes with minimum energy.Therefore,slight fluctuations in water environment may have a great impact on physiological processes such as feeding and metabolism.Large-scale mortality often occurs during the intermediate rearing processes,and high temperatures in summer are considered the leading cause of mortality.To understand the physiological and molecular response patterns of A.broughtonii at high temperatures and to estimate the appropriate metabolism temperature for A.broughtonii,the effects of temperature on the respiratory metabolism and food intake at different growth stages were studied.The response patterns of heat shock protein genes to heat stress and post-stress recovery were also explored.Results show that the temperature and growth stage of A.broughtonii were two important factors affecting metabolism and feeding.The optimum temperature for feeding and physiological activities in each shelllength group was 24℃.The temperature at which the peak metabolic rate occurred in each shell-length group increased with the increase in shell length.With the increase in heat stress,the expression of three heat shock protein genes(Abhsp60,Abhsp70,and Abhsp90) in the tissues of two size groups of A.broughtonii increased significantly when temperature was above 24℃ and reached a peak at 30℃.After heat shock at30℃ for 2 h,the expression of the three heat shock protein genes rapidly increased,peaked at 2 h after the heat shock,and then gradually decreased to the level of the control group at 48 h after the heat shock.The three heat shock protein genes respond rapidly to heat stress and can be used as indicators to the cellular stre ss response in A.broughtonii under an environmental stre ss.     

Molecular cloning,tissue expression of gene Muc2 in blunt snout bream Megalobrama amblycephala and regulation after re-feeding

Mucins are important components of mucus,which form a natural,physical,biochemical and semipermeable mucosal layer on the epidermis of fish gills,skin,and the gastrointestinal tract. As the first step towards characterizing the function of Muc2,we cloned a partial Megalobrama amblycephala Muc2 cDNA of 2 175 bp,and analyzed its tissue-specific expression pattern by quantitative real-time PCR(qPCR). The obtained sequence comprised 41 bp 5′-untranslated region(5′-UTR),2 134 bp open reading frame encoding a protein of 711 amino acids. BLAST searching and phylogenetic analysis showed that the predicted protein contained several common secreted mucin-module domains(VWD-C8-TIL-VWD-C8) and had high homology with mucins from other vertebrates. Among four candidate reference genes(β- Actin,RPI13α,RPII,18S) for the qPCR,R PII was chosen as an appropriate reference gene because of its lowest variation in different tissues. M. amblycephala Muc2 was mainly expressed in the intestine,in the order(highest to lowest) middle-intestine fore-intestine hind-intestine. Muc2 was expressed relatively poorly in other organs(brain,liver,kidney,spleen,skin and gill). Furthermore,after 20-days of starvation,M. amblycephala Muc2 expressions after refeeding for 0h,3 h,16 h,3 d,and 10 d were significantly decreased in the three intestinal segments(P 0.05) at 16 h,and were then upregulated to near the initial level at 10 d.     

Molecular characterization and expression analysis of Lily-type lectin (SmLTL) in turbot Scophthalmus maximus,and its response to Vibrio anguillarum

A full-length lily-type lectin(Sm LTL) was identified from turbot(Scophthalmus maximus) in this study. By searching database for protein identification and function prediction, Sm LTL were confirmed. The full-length c DNA of Sm LTL is composed of 569 bp and contains a 339 bp ORF that encodes 112 amino acid residues. The Sm LTL peptide is characterized by a specific β-prism architecture and contains three mannose binding sites in a three-fold internal repeat between amino acids 30–99; two of the repeats share the classical mannose binding domain(Qx Dx Nx Vx Y) while the third binding site was similar to other fish-specific binding motifs(Tx Tx Gx Rx V). The primary, secondary, and tertiary structures of Sm LTL were predicted and analyzed, indicating that the S m LTL protein was hydrophilic, contained 5.36% α-helices, 39.29% extended strands, 16.07% β-folds, and 39.29% random coils, and three β-folds. Quantitative realtime polymerase chain reaction(qPCR) analysis revealed that the Sm LTL mRNA was abundantly expressed in skin, gill, and intestine. Low levels of Sm LTL expression were observed in other tissues. The expression of Sm LTL in gill, skin and intestine increased at m RNA level after stimulation of V ibrio anguillarum, our results suggest that Sm LTL serve as the first line of defence against microbial infections and play a pivotal role in the innate mucosal immune system. The current study indicates that Sm LTL is a member of the lilytype lectin family and the information reported here will provide an important foundation for future research on the role of this protein.     

Characterization of defensin gene from abalone Haliotis discus hannai and its deduced protein

Defensin is one of preserved ancient host defensive materials formed in biological evolution. As a regulator and effector molecule, it is very important in animals' acquired immune system. This paper reports the defensin gene from the mixed liver and kidney cDNA library of abalone Haliotis discus hannai Ino. Sequence analysis shows that the gene sequence of full-length cDNA encodes 42 mature peptides (including six Cys), molecular weight of 4 323 Da, and pI of 8.02. Amino acid sequence homology analysis shows that the peptides are highly similar (70% in common) to other insects defensin. Because of a typical insect-defensin structural character of mature peptide in the secondary structure, the polypeptide named Haliotis discus defensin (hd-def), a novel of antimicrobial peptides, belongs to insects defensin subfamily. The RT-PCR result of Haliotis discus defensin shows that the gene can be expressed only in the hepatopancreas by Gram-negative and positive bacteria stimulation, which is ascribed to inducible expression. Therefore, it is revealed that the Haliotis discus defensin gene expression was related to the antibacterial infection of Haliotis discus hannai Ino.     

Molecular characterization and expression of the SiUCP2 gene in sea urchin Strongylocentrotus intermedius

Uncoupling protein 2(UCP2) is a proton transporter located in the inner mitochondrial membrane,and inhibits the formation of adenosine triphosphate and reactive oxygen species by uncoupling oxidative phosphorylation.To provide a theoretical basis for the role of SiUCP2 in lipid metabolism,a2 341-bp full-length cDNA of Si UCP2 from sea urchin Strongylocentrotus intermedius,which encodes 323 amino acids(predicted MW 3 6.11 kDa) was obtained,and the structure and function of the Si UCP2 gene and its expre ssion at the mRNA and protein level were studied.Si UCP2 had high homology with UCP2 of other species.Expression of SiUCP2 was detected in the order of tube feet gonads coelomocytes intestines.The expression level was the highest in prismatic larvae and lowest in the two-cell stage.Moreover,using in-situ hybridization,we found that SiUCP2 protein was expressed in the gonads and intestine.This study provided a theoretical basis for subsequent studies on the role of SiUCP2 and its regulatory mechanism in lipid metabolism,and for the improvement of gonad quality to obtain a higher economic value from sea urchins.     

Gene cloning and prokaryotic expression of recombinant flagellin A from Vibrio parahaemolyticus

The Gram-negative Vibrio parahaemolyticus is a common pathogen in humans and marine animals. Bacteria flagellins play an important role during infection and induction of the host immune response. Thus, flagellin proteins are an ideal target for vaccines. We amplified the complete flagellin subunit gene (flaA) from V. parahaemolyticus ATCC 17802. We then cloned and expressed the gene into Escherichia coli BL21 (DE3) cells. The gene coded for a protein that was 62.78 kDa. We purified and characterized the protein using Ni-NTA affinity chromatography and Anti-His antibody Western blotting, respectively. Our results provide a basis for further studies into the utility of the FlaA protein as a vaccine candidate against infection by Vibrio parahaemolyticus. In addition, the purified FlaA protein can be used for further functional and structural studies.     

Molecular cloning and mRNA expression analysis of myosin heavy chain (MyHC) from fast skeletal muscle of grass carp, Ctenopharyngodon idella

The myosin heavy chain (MyHC) is one of the major structural and contracting proteins of muscle. We have isolated the cDNA clone encoding MyHC of the grass carp, Ctenopharyngodon idella. The sequence comprises 5 934 bp, including a 5 814 bp open reading frame encoding an amino acid sequence of 1 937 residues. The deduced amino acid sequence showed 69% homology to rabbit fast skeletal MyHC and 73%–76% homology to the MyHCs from the mandarin fish, walleye pollack, white croaker, chum salmon, and carp. The putative sequences of subfragment-1 and the light meromyosin region showed 61.4%–80% homology to the corresponding regions of other fish MyHCs. The tissue-specific and developmental stage-specific expressions of the MyHC gene were analyzed by quantitative real-time PCR. The MyHC gene showed the highest expression in the muscles compared with the kidney, spleen and intestine. Developmentally, there was a gradual increase in MyHC mRNA expression from the neural formation stage to the tail bud stage. The highest expression was detected in hatching larva. Our work on the MyHC gene from the grass carp has provided useful information for fish molecular biology and fish genomics.     

Molecular cloning and mRNA expression analysis of myosin heavy chain (MyHC) from fast skeletal muscle of grass carp, Ctenopharyngodon idella

The myosin heavy chain(MyHC)is one of the major structural and contracting proteins of muscle.We have isolated the cDNA clone encoding MyHC of the grass carp,Ctenopharyngodon idella. The sequence comprises 5 934 bp,including a 5 814 bp open reading frame encoding an amino acid sequence of 1 937 residues.The deduced amino acid sequence showed 69%homology to rabbit fast skeletal MyHC and 73%–76%homology to the MyHCs from the mandarin fish,walleye pollack,white croaker,chum salmon,and carp.The putative sequences of subfragment-1 and the light meromyosin region showed 61.4%–80%homology to the corresponding regions of other fish MyHCs.The tissue-specific and developmental stage-specific expressions of the MyHC gene were analyzed by quantitative real-time PCR.The MyHC gene showed the highest expression in the muscles compared with the kidney,spleen and intestine.Developmentally,there was a gradual increase in MyHC mRNA expression from the neural formation stage to the tail bud stage.The highest expression was detected in hatching larva.Our work on the MyHC gene from the grass carp has provided useful information for fish molecular biology and fish genomics.     

Construction of a genomic DNA library with a TA vector and its application in cloning of the phytoene synthase gene from the cyanobacteriumSpirulina platensis M-135

INTRODUCTIONThesignalproaningschemeofmostpnsentsonarsyStasuChaseChosounder,fishfinder,etc.,deteCtsthesignaIsaanrdingt0theamPlitudethasholdafterthefilter.However,inacomplicatalandfrequenhychangingunderotCfacousticalchanne,thesta-bilityandreiabilityofthiskindofsonarsySteIndroeshamlyasanysySthenoisewhleadt0anindedion.AmplitudefaderesultingfromstrongsignalfluCtuationcauseslossofdata.InsomesyStetnS,suchasndnelocatingsonar,highrangingamCyisneded,soasinglededionschernecann0tadapttoit.Resul…     

Cold-adaptive alkaline protease from the psychrophilic Planomicrobium sp.547: enzyme characterization and gene cloning

A psychrophilic bacterium strain 547 producing cold-adaptive alkaline protease was isolated from the deep sea sediment of Prydz Bay, Antarctica. The organism was identified as a Planomicrobium species by 16S rRNA analysis. The optimal and highest growth temperatures for strain 547 were 15℃ and 30℃, respectively. The extracellular protease was purified by ammonium sulfate precipitation and DEAE cellulose-52 chromatography. The optimal temperature and pH for the activity of the purified enzyme were 35 ℃ and pH 9.0, respectively. The enzyme retained approximately 40% of its activity after 2 h of incubation at 50℃. The enzymatic activity was inhibited by 1 mmol/L phenylmethyl sulfonylfluoride (PMSF) and hydrochloride 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF), indicating that it was a serine protease. The presence of Ca2+ and Mn2+ increased the activity of the enzyme. The protease gene with a size of 1 269 bp was cloned from Planomicrobium sp. 547 using primers designed based on the conserved sequences of proteases in GenBank. The Planomicrobium sp. 547 protease contained a domain belonging to the peptidase S8 family, which has a length of 309 amino acid (AA) residues. The alignment and phylogenetic analysis of the AA sequence indicated that the protease belonged to the subtilisin family.     

Transient expression of the enhanced green fluorescent protein(egfp) gene in Sargassum horneri

Sargassum horneri is a macroalga widespread in North Asia-Pacific region, and these years its bloom has caused huge damage to the environment and the economic in China. To make up the blank on genetic engineering research, a transient transformation system for the multicellular marine brown alga S. horneri was established in this research. The algae used in this research were collected from the Yellow Sea of China and verified as a same species S. horneri with analysis of molecular markers. The S. horneri parietal leaves were transformed with the enhanced green fluorescent gene as the reporter by micro-particle bombardment. The results show that green fluorescent protein(GFP) is an ef fective transgene reporter for S.horneri and that particle bombardment is a suitable method for transformation of S. horneri. Through selection of four dif ferent promoters for EGFP and six groups' bombardment characters, the highest transformation e ciency approximately 1.31% was got with the vector pEGFP-N1 at bombardment characters 900 spi and6 cm distance. This research paves a way for the further research and application of S. horneri.