Effect of lead on cytoskeletal protein stability in crucian carp <Emphasis Type="Italic">Carassius auratus</Emphasis>

Inorganic lead （Pb） is one of the most common environmental pollutants. Much evidence indicates that Pb exposure could directly affect fish growth and development. In this study, we investigated the cytotoxic effects of Pb on cytoskeletal protein stability at both protein and mRNA level in crucian carp Carassius auratus. Pb（NO3）2 treatment in concentration of 100 μmol/L resulted in decreased expression of both α- and β-tubulin but γ-tubulin as assayed with SDS-PAGE, Western Blot, and ELISA. In vivo and in vitro analyses on protein expression of tubulins are consistent. The effect of Pb on mRNA expression varied among different tissues. Our results suggest that cytotoxicity of Pb at protein translation level is stronger than at mRNA expression level.     

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.     

RNA/DNA ratio and LPL and MyoD mRNA expressions in muscle ofOreochromis niloticus fed with elevated levels of palm oil

Palm oil is of great potential as one of the sustainable alternatives to fish oil (FO) in aquafeeds. In this present study, five isonitrogenous diets (32% crude protein) with elevated palm oil levels of 0%, 2%, 4%, 6% and 8% were used during an 8-week feeding trial to evaluate its effects on RNA/DNA ratio and lipoprotein lipase (LPL) and MyoD mRNA expressions in muscle of Oreochromis niloticus. The results showed that RNA, DNA content as well as ratio of RNA to DNA were significantly affected (P < 0.05), in each case the highest was recorded in fish group subjected to 6% palm oil level. There was a strong positive correlation between nucleic acid concentration (RNA concentration and RNA: DNA ratio) and specific growth rate (SGR), protein efficiency ratio (PER), while a negative correlation existed between nucleic acid concentration (RNA concentration and RNA: DNA ratio) and feed conversion ratio (FCR). The mRNA expressions of LPL and MyoD in muscle were not significantly affected by the different palm oil levels, although the highest expression was observed in fish fed with 6% palm oil level. There also existed a strong positive correlation between the mRNA expression of LPL, MyoD and SGR, PER, while their correlation with FCR was negative. In conclusion, elevated palm oil affected the RNA, DNA concentration as well as RNA/DNA ratio significantly, although the mRNA expression of LPL and MyoD were not affected significantly by elevated palm oil levels.     

Identification and characterization of a DnaJ gene from red alga <Emphasis Type="Italic">Pyropia yezoensis</Emphasis> (Bangiales,Rhodophyta)

Members of the DnaJ family are proteins that play a pivotal role in various cellular processes, such as protein folding, protein transport and cellular responses to stress. In the present study, we identified and characterized the full-length DnaJ cDNA sequence from expressed sequence tags of Pyropia yezoensis (PyDnaJ) via rapid identification of cDNA ends. This cDNA encoded a protein of 429 amino acids, which shared high sequence similarity with other identified DnaJ proteins, such as a heat shock protein 40/DnaJ from Pyropia haitanensis. The relative mRNA expression level of PyDnaJ was investigated using real-time PCR to determine its specific expression during the algal life cycle and during desiccation. The relative mRNA expression level in sporophytes was higher than that in gametophytes and significantly increased during the whole desiccation process. These results indicate that PyDnaJ is an authentic member of the DnaJ family in plants and red algae and might play a pivotal role in mitigating damage to P. yezoensis during desiccation.     

DNA Methylation of pacap Gene Is Involved in Growth Superiority of Female Half Smooth Tongue Sole(Cynoglossus semilaevis) in Different Salinities

Pituitary adenylate cyclase activating polypeptide(PACAP)and growth hormone-releasing hormone(GHRH)play important roles in the GH/IGF growth axis in fishes.To determine whether epigenetic change is involved in the regulation of pacap and ghrh responses to low salinity stress in Cynoglossus semilaevis,the correlation between growth traits,DNA methylation status and gene expression level in low salinity(15,S15)and optimal salinity(30,S30)at day 7(D7)and day 60(D60)were analyzed.Results showed that exposure to low salinity for 60 days attenuated C.semilaevis growth rate.Under low salinity,DNA methylation level of pacap promoter increased in females and decreased in males at day 7,but inverted at day 60.Additionally,pacap expression was up-regulated in both males and females.The pacap promoter methylation level was higher and expression level was lower in female than in male.The results suggest that pacap promoter methylation level is negatively correlated to mRNA level and positively correlated to body weight,while gene expression level is negatively related with body weight.With the decrease of salinity,DNA methylation level of ghrh promoter and exon1,as well as its gene expression displayed minor changes.Overall,pacap gene seems to play an important role in fish growth,contributing to female growth superiority,while ghrh gene seems not pertinent under salinity stress.The results indicate that low salinity potentially affects fish growth through regulating DNA methylation in pacap promoter.This study expands the understanding of the molecular mechanism of how salinity modulates fish growth from the epigenetic perspective.     

Molecular identification,expression pattern,and in-vitro bioactivity analysis of insulin-like growth factor 2 in olive flounder Paralichthys olivaceus

Insulin-like growth factors (IGFs) are key regulators of development and growth. Here, we characterized the igf2 gene from olive flounder (Paralichthys olivaceus) and determined its temporal and spatial expression. We set up an in-vitro protein expression system in eukaryotic human embryonic kidney (HEK293T) cells and explored its effects on cell proliferation. The flounder igf2 cDNA contained a 648-bp open reading frame (ORF) encoding a protein of 215 amino acids (aa), which spanned the complete signal peptide (47 aa), mature peptide (70 aa), and E domain (98 aa). In adult flounder, igf2 mRNA was detected in all selected tissues. In early development, igf2 mRNA was detected throughout development from unfertilized eggs to hatching-stage embryos. In-situ hybridization analysis indicated that igf2 mRNA was specially expressed in the brain region, floor plate, hypochord, otic vesicle, and pectoral fin during embryogenesis. Western blotting analysis indicated that the soluble recombinant flounder IGF2 protein was successfully produced through eukaryotic expression in HEK293T cells. In addition, the recombinant IGF2 protein significantly promoted the proliferation of human cervical carcinoma (HeLa) and HEK293T cells. These results provide new information about the structural and functional conservation, expression patterns, and biological activity of the igf2 in teleosts.

     

Glutamate dehydrogenase and Na+-K+ ATPase expression and growth response of Litopenaeus vannamei to different salinities and dietary protein levels

Improvement in the osmoregulation capacity via nutritional supplies is vitally important in shrimp aquaculture.The effects of dietary protein levels on the osmoregulation capacity of the Pacific white shrimp(L.vannamei) were investigated.This involved an examination of growth performance,glutamate dehydrogenase(GDH) and Na+-K+ ATPase mRNA expression,,and GDH activity in muscles and gills.Three experimental diets were formulated,containing 25%,40%,and 50% dietary protein,and fed to the shrimp at a salinity of 25.After 20 days,no significant difference was observed in weight gain,though GDH and Na+-K+ ATPase gene expression and GDH activity increased with higher dietary protein levels.Subsequently,shrimp fed diets with 25% and 50% dietary protein were transferred into tanks with salinities of 38 and 5,respectively,and sampled at weeks 1 and 2.Shrimp fed with 40% protein at 25 in salinity(optimal conditions) were used as a control.Regardless of the salinities,shrimp fed with 50% dietary protein had significantly higher growth performance than other diets;no significant differences were found in comparison with the control.Shrimp fed with 25% dietary protein and maintained at salinities of 38 and 5 had significantly lower weight gain values after 2 weeks.Ambient salinity change also stimulated the hepatosomatic index,which increased in the first week and then recovered to a relatively normal level,as in the control,after 2 weeks.These findings indicate that in white shrimp,the specific protein nutrient and energy demands related to ambient salinity change are associated with protein metabolism.Increased dietary protein level could improve the osmoregulation capacity of L.vannamei with more energy resources allocated to GDH activity and expression.     

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.     

Characterization,expression and function analysis of DAX1 gene of scallop (Chlamys farreri jones and preston 1904) during its gametogenesis

DAX1, a member of nuclear receptor superfamily, has a function in the sex determination and gonadal differentiation of several vertebrate species. However, little information about DAX1 of invertebrates is available. Here we cloned a homolog of scallop (Chlamys farreri Jones and Preston 1904) dax1, Cf-dax1, and determined its expression characteristics at mRNA and protein levels. The cDNA sequence of Cf-dax1 was 2093 bp in length, including 1404 bp open reading frame (ORF) encoding 467 amino acids. Unlike those of vertebrates, no conserved LXXLL-related motif was found in the putative DNA binding region of Cf-DAX1. Fluorescence in situ hybridization showed that Cf-dax1 located on the short arm of a pair of subtelocentric chromosomes. Tissue distribution analysis using semi-quantitative RT-PCR revealed that Cf-dax1 expressed widely in adult scallop tissues, with the highest expression level found in adductor muscle, moderate level in mantle, gill and testis, and low level in kidney, ovary and hepatopancreas. The result of quantitative real-time PCR indicated that the expression of Cf-dax1 was significantly higher (P<0.05) in testis than in ovary at the same stage, showing a sex-dimorphic expression pattern. Furthermore, immunohistochemical detection found that Cf-DAX1 mainly located in spermatogonia and spermatocytes of testis and in oogonia and oocytes of ovary, implying that DAX1 may involve in gametogenesis of bivalves.     

Temperature Influences on the Expression of GFP Promoted by the Upstream Sequence of cpcB from Arthrospiraplatensis

In order to investigate the regulation mechanism of the phycocyanin gene,a series of functional analyses of the upstream sequence of cpcB gene from Arthrospira platensis were conducted in E. coli with green fluorescent protein encoding gene(gfp) as the reporter. Results showed that the gfp gene could express at a high level under the promotion of the upstream sequence,suggesting the existence of some strong promoter elements in it. The expression of GFP was influenced by temperature. Higher temperature led to higher expression level. The bioinformatics analyses followed by mutation analyses on the secondary structure of translation ini-tiation region(TIR) revealed that RNA thermosensor might account for the temperature regulation.     

Seasonal and spatial distribution of trace metals in alpine soils of Eastern Tibetan Plateau,China

The distribution of trace metals in remote alpine region is an effective way to understand the impacts of regional human activity and vegetation on the alpine ecosystem. In this study, the concentrations of cadmium (Cd), copper (Cu), lead (Pb), and zinc (Zn) in the soils of Gongga Mountain, Eastern Tibetan Plateau, were investigated to reveal their seasonal and spatial distribution and enrichment state, and to decipher the effects of atmospheric deposition and vegetation on their distributions among five vegetation zones. The results showed that the concentrations of Cd, Pb, and Zn were higher in the O horizon than in other horizons despite the seasonal changes, whereas Cu was enriched in the C horizon. The enrichment states of the metals studied in the soils decreased in the order of Cd > Pb > Zn > Cu. Cd and Pb were mainly sourced from atmospheric deposition; Zn was from both atmospheric deposition and parent materials, whereas Cu was mainly from parent materials. Seasonally, the trace metals were generally higher in May and December but lower in September, implying the impact of vegetation on the distribution of trace metals under the plant uptake and the litter decomposition. Spatially, the higher enrichment of Cu, Pb, and Zn in the soils existed in the mixed broadleaf-coniferous forests and coniferous forests (approximately 3000 m above sea level). The results suggested that atmospheric deposition and biological processes are main factors controlling the seasonal and spatial distribution of trace metals in the soils of the remote alpine ecosystem.     

Cloning, Expression and Characterization of Translationally Controlled Tumor Protein （TCTP） Gene from Flatfish Turbot （Scophthalmus maximus）

A full-length cDNA encoding translationally controlled tumor protein of marine flatfish turbot (Scophthalmus maximus), SmTCTP, was isolated with rapid amplification of cDNA Ends (RACE). SmTCTP consisted of a 5' untranslated region (UTR) of 84 bp, a 3' UTR of 451 bp and an open reading flame (ORF) of 513 bp, encoding a protein of 170 amino acid residues, which contained two signature sequences of TCTP family. The 5'UTR of SmTCTP started with a 5'-terminal oligopyrimidine tract (5'-TOP), a typical feature for translationaily controlled mRNAs. The deduced amino acid sequence of SmTCTP was similar to the other known verte-brate TCTPs in a range of 58.8% to 64.1%. The length offish TCTPs was diverse among species, e.g., TCTP of turbot and sea perch (Lateolabrax japonicus) is 170 aa in length, while that of zebrafish (Danio rer/o) and rohu (Labeo rohita) is 171 aa in length. North-ern blot analysis revealed that SmTCTP has only one type of mRNA. Its expression level in albino skin was slightly higher than that in normal skin. We constructed the pET3Oa-SmTCTP expression plasmid. The recombinant protein of His-tag SmTCTP was over-expressed in E. coli, purified and identified with peptide mass fingerprinting. These results may pave the way of further inves-tigation of the biological function of TCTP in fish.     

Expression and localization of a novel phosducin-like protein from amphioxus Branchiostoma belcheri

A full length amphioxus cDNA, encoding a novel phosducin-like protein (Amphi-PhLP),was identified for the first time from the gut cDNA library of Branchiostoma belched. It is comprised of 1 550 bp and an open reading frame (ORF) of 241 amino acids, with a predicted molecular mass of approximately 28 kDa. In situ hybridization histocbemistry revealed a tissue-specific expression pattern of Amphi-PhLP with the high levels in the ovary, and at a lower level in the hind gut and testis, hepatic caecum, gill, endostyle, and epipharyngeal groove, while it was absent in the muscle, neural tube and notochord. In the Chinese Hamster Ovary (CHO) cells transfected with the expression plasmid pEGFP-NIIAmphi-PhLP, the fusion protein was targeted in the cytoplasm of CHO cells, suggesting that Amphi-PhLP is a cytosolic protein. This work may provide a framework for further understanding of the physiological function of Amphi-PhLP in B. belcheri.     

A 150-year isotopic record of lead deposition in Yancheng coastal wetland,China

Radioactive markers are useful in dating lead(Pb) deposition patterns from industrialization in sedimentary archives. As a well-known natural reserve in the world, Yancheng coastal wetland in Jiangsu Province is one of areas most sensitive to global sea level change and is located in the most developed and polluted region of China. Two cores were collected in Yancheng wetland in October 2013 and dated using ~(210)Pb and ~(137)Cs radiometric techniques. Sediments in both cores were sectioned into depth bands and examined systematically for dry bulk density, water content, magnetic susceptibility and grain-size. Multiple elements including Pb were also measured using inductively coupled plasma systems. Unsupported ~(210)Pb activities decreased with depth in both of the two cores, and ~(210)Pb chronologies were established(covering 150 years) using the constant rate of supply(CRS) model. The measured Pb contents ranged from 14.97 mg/kg to 29.40 mg/kg with average values of 17.17–22.79 mg/kg, and the Pb fluxes ranged from 41.70 mg/(m~2·yr) to 172.70 mg/(m~2·yr) with averages of 95.59–123.41 mg/(m~2·yr). Temporal variations of Pb flux, enrichment factors and Pb isotopes show a gradual and continuous increase over time and clearly reflect increased emissions from anthropogenic activities in the region. The Pb isotopic compositions show that most of Pb deposition in Yancheng wetland is input from natural sources by water flows and has the same levels of Pb as in the surface sediment of the Yangtze River and the Pacific mineral aerosol. We also stress the anthropogenic Pb contribution in Yangcheng wetland sediment and the reason of our Pb isotopes not showing anthropogenic signature is likely the instability of anthropogenic Pb in high Fe/Mn oxide conditions. Therefore, more attention should be paid to current local pollution problems, and society should take action to seek a balance between economic development and environmental protection.     

Characterization of ribulose-1, 5-bisphosphate carboxylase/oxygenase and transcriptional analysis of its related genes in Saccharina japonica (Laminariales,Phaeophyta)

Saccharina japonica is a common macroalga in sublittoral communities of cold seawater environments,and consequently may have highly effi cient ribulose-1,5-bisphosphate carboxylase/oxygenase(Rubisco)activity for carbon assimilation.In our study,we cloned the full-length Rubisco gene from S.japonica(SJ-rbc).It contained an open reading frame for a large subunit gene(SJ-rbcL)of 1 467 bp,a small subunit gene(SJ-rbcS)of 420 bp,and a SJ-rbcL /S intergenic spacer of 269 bp.The deduced peptides of SJ-rbcL and SJ-rbcS were 488 and 139 amino acids with theoretical molecular weights and isoelectric points of 53.97 kDa,5.81 and 15.84 kDa,4.71,respectively.After induction with 1 mmol/L isopropyl-β-Dthiogalactopyranoside for 5 h and purifi cation by Ni 2+ affi nity chromatography,electrophoresis and western blot detection demonstrated successful expression of the 55 kDa SJ-rbcL protein.Real-time quantitative PCR showed that the mRNA levels of SJ-rbcL in gametophytes increased when transferred into normal growth conditions and exhibited diurnal variations: increased expression during the day but suppressed expression at night.This observation implied that Rubisco played a role in normal gametophytic growth and development.In juvenile sporophytes,mRNA levels of SJ-rbcL,carbonic anhydrase,Calvin-BensonBassham cycle-related enzyme,and chloroplast light-harvesting protein were remarkably increased under continuous light irradiance.Similarly,expression of these genes was up-regulated under blue light irradiance at 350 μmol/(m 2·s).Our results indicate that long-term white light and short-term blue light irradiance enhances juvenile sporophytic growth by synergistic effects of various photosynthetic elements.     

Characterization of SNAP-25 gene from marine teleostean, Lateolabrax japonicus

The t-SNARE protein SNAP-25 (synaptosome-associated protein of 25 kDa) plays an essential role in regulating fusion between the vesicle and plasma membranes during exocytosis. To clone and characterize SNAP-25 gene, the first step in the functional study of SNARE proteins in marine teleostean, was to obtain the cDNA of sea perch SNAP-25 (SPsn25) by RT-PCR and RACE-PCR amplification of a Japanese sea perch. The full-length cDNA of 831bp contains a CDS of 615 bp, coding 204 amino acid residues, and a 5′UTR of 219bp. Bioinformatic analysis revealed that SPsn25 corresponds with SNAP-25a isoform and shares 91.1% identity with SNAP-25a of a goldfish and a zebrafish. The SPsn25 expression in both mRNA and protein levels in the Japanese sea perch had been identified through semi-quantitative RT-PCR and Western Blot assay. Together, these data again confirmed the nerve tissue specificity of the fish SNAP-25 gene expression.