Taurine transporter from the giant Pacific oyster <Emphasis Type="Italic">Crassostrea gigas</Emphasis>: function and expression in response to hyper- and hypo-osmotic stress

ABSTRACT:   Taurine is the primary osmolyte in marine molluscs, whose cellular osmo-conforming process is vital for environmental adaptation because of a lack of osmotic homeostasis. Here, cDNA cloning and expression, and functional analyses of taurine transporter (TAUT) from the giant Pacific oyster are reported on. The deduced amino-acid sequence of oyster TAUT (oyTAUT) showed 47–51% identity to those of vertebrate TAUT, whereas identity among the vertebrates is 78–95%. Functional analysis of oyTAUT expressed in Xenopus oocytes revealed that oyTAUT has a lower affinity and specificity for taurine and a requirement for higher NaCl concentration, compared with vertebrate TAUT. Taken together with similar functional properties of TAUT from mussel, indicated by our previous study, it is possible that these functional features reflect the internal environment of the molluscs (i.e. higher taurine and NaCl concentrations). Oyster taurine transporter mRNA expression was induced by not only hyper-osmotic stress, similar to other TAUT, but also hypo-osmotic stress. It is speculated that the expression in response to hypo-osmotic stress was induced by a substantial decrease in tissue taurine content following the decrease in the internal osmolality.     

Expression of HSP70 in response to heat-shock and its cDNA cloning from Mediterranean blue mussel

Expression of HSP70 in response to heat-shock was investigated at the protein and mRNA levels in Mediterranean blue mussel. Western and Northern blot analyses revealed that HSP70 was expressed following heat-shock in the mantle at both protein and mRNA levels, suggesting that gene expression of HSP70 is implicated in the cellular response to heat-shock stress in mussel. It was then attempted to clone HSP70 cDNA in order to determine the primary structure of mussel HSP70. As a result, two full-length cDNA encoding HSP70 were isolated from a cDNA library prepared from the heat-shocked mantle. The isolated cDNA consist of single open reading frames of 2067 bp and 1911 bp which encode proteins of 689 amino acids and 637 amino acids, respectively. Both HSP70 cDNA encode an ATPase do main, and a substrate-binding do main in addition to a Glu-Glu-Val-Asp (EEVD) peptide motif that is specific for cytosolic HSP70. These findings suggest that the cDNA clones obtained in the present study encode cytosolic HSP70.     

Effect of long-term exposure to sulfides on taurine transporter gene expression in the gill of the deep-sea mussel Bathymodiolus platifrons,which harbors a methanotrophic symbiont

Seawater around deep-sea hydrothermal vents and cold seeps contain high levels of hydrogen sulfide, which is toxic to most animals. Invertebrates inhabiting these environments have been reported to accumulate high levels of thiotaurine, a sulfur-containing amino acid. Thiotaurine is likely to play an important role in sulfide detoxification, but its functions in the detoxification process are still unknown. We cloned methane-seep mussel Bathymodiolus platifrons cDNA encoding the taurine transporter (TAUT), which transports thiotaurine and its precursors across the cell membrane. Phylogenetic analyses revealed that the predicted peptide formed a clade with the TAUTs of shallow-water mussel Mytilus galloprovincialis and the hydrothermal vent mussel Bathymodiolus septemdierum that harbors thioautotrophic bacteria. We then reared B. platifrons in the presence or absence of Na₂S and quantified TAUT mRNA using a real-time PCR system. The amount of TAUT mRNA in the gills of B. platifrons increased with rearing in the presence of Na₂S for 69 days, but no change was observed in the absence of sulfide. These results suggest that TAUT plays an important role in sulfide detoxification, even in species that do not harbor thioautotrophic bacteria. The TAUT mRNA level was variable in the mantle and low in the foot throughout the entire rearing period, regardless of the presence/absence of sulfide, suggesting that TAUT gene expression is regulated differentially in each tissue.     

不同盐度低渗胁迫处理对龙须菜切段再生能力及光合生理的影响

为探究龙须菜对低渗胁迫的生理性适应，分析了龙须菜主枝切段在不同盐度低渗培养液(盐度19.500、13.000、6.500、0.000)下耐受不同胁迫时间(1、3、6、12和20 h)后，继续恢复正常盐度(盐度26.000)培养过程中的形态发生、光合生理指标和细胞器亚显微结构变化。结果显示，短于12 h的低渗(盐度0～19.500)胁迫可以促进龙须菜切段的出芽，其总出芽数均高于对照组(盐度26.000)。3 h的淡水胁迫后恢复至正常盐度培养，藻段再生过程的出芽数目较多，鲜重增加最多，表现出了明显的生长优势。龙须菜藻段经淡水胁迫3 h后恢复至正常盐度培养28 d，藻段的相对生长率(RGR)为0.91%/d，较对照组RGR提高了61.27%。1和3 h淡水胁迫对龙须菜藻段的光合生理指标无明显的负面效应，且恢复至正常盐度培养后藻段的光合生理活性增强，提示低渗胁迫可能增强了藻段的细胞代谢活力。透射电镜观察表明，经淡水胁迫3 h后，表皮细胞内的红藻淀粉颗粒、质体小球及脂质体等为藻体生长发育提供能量的物质和质体再生所需中性脂原料明显增加，适应于藻段再生过程的新生芽形成。相反，长时间的淡水胁迫对色素体...     

饲料牛磺酸含量对斜带石斑鱼幼鱼生长性能、体成分、TauT mRNA表达量及牛磺酸合成关键酶活性的影响

为探讨饲料牛磺酸含量对斜带石斑鱼幼鱼生长性能、体成分、TauT mRNA表达量及牛磺酸合成关键酶(CSD和CDO)活性的影响,实验在以酪蛋白和明胶为蛋白源的基础饲料(0DT)中分别添加0.5%(0.5DT)、1.0%(1.0DT)、1.5%(1.5DT)的牛磺酸,配制成4种不同牛磺酸含量的饲料。将平均体质量为(13.85±0.25) g的320尾斜带石斑鱼幼鱼随机分为4组,每组4个循环水族箱,每箱放养20尾鱼,每组分别投喂一种相同实验饲料,每天定时投喂实验饲料至表观饱食状态,实验为期84 d。结果显示,在0DT饲料中补充外源牛磺酸能显著提高饲料效率、摄食率、增重率和全鱼粗蛋白含量,而显著降低肝体比和全鱼粗脂肪含量。组织中肝脏、肌肉、肠道TauT mRNA表达量在1.0DT组时达到最大值,且显著高于其他各组,当饲料牛磺酸含量继续增加至1.5DT组时明显降低,但仍显著高于0DT和1.0DT组。斜带石斑鱼幼鱼血浆、肝脏、肠道和肌肉中牛磺酸含量与饲料牛磺酸含量之间呈正相关。饲料中补充外源牛磺酸能够显著降低肝脏、肌肉中CSD活性,同时降低血浆、肝脏、肠道和肌肉中CDO活性,但对血浆CSD活性无显著影响。研究表明,饲料中补充外源牛磺酸能够明显促进斜带石斑鱼幼鱼生长,增加鱼体蛋白沉积,同时降低鱼体脂肪沉积,上调组织中TauT mRNA表达水平及提高牛磺酸蓄积,降低牛磺酸合成关键酶活性。研究表明,以增重率为目标,通过二次多项式回归分析,饲料中牛磺酸的适宜含量为0.92%。     

三唑磷短期暴露对翡翠贻贝的毒性效应

采用半静态毒性试验方法研究了三唑磷（triazophos,OP）短期暴露下对翡翠贻贝（Perna viridis）的毒性效应。结果显示,在OP胁迫下,翡翠贻贝内脏团和外套膜超氧化物歧化酶（SOD）活性在胁迫前期均被抑制,随时间延长活性被诱导。低质量浓度0P（0．4mg·L^-1和2mg·L^-1）使翡翠贻贝内脏团和外套膜谷胱甘肽-S转移酶（GST）活性先降低后升高;高质量浓度（10mg·L^-1）GST活性降低。OP能诱导还原型谷胱甘肽（GSH）的产生,而高质量浓度（10mg·L^-1）也能抑制GSH产生。受0P胁迫影响,翡翠贻贝内脏团和外套膜丙二醛（MDA）均升高。内脏团总抗氧化能力（T—AOC）显著降低;而外套膜T—AOC会被OP所诱导。转入清水恢复72h后仅外套膜MDA有所恢复。结果表明OP短期暴露会对翡翠贻贝抗氧化系统造成损伤。     

Comparative analysis of total carotenoid content in tissues of purple and white inner-shell color pearl mussel,Hyriopsis cumingii

The freshwater pearl mussel Hyriopsis cumingii is the most important mussel species for freshwater pearl production in China. Mussel shell color is an important indicator of pearl quality. The objective of this study was to assess whether total carotenoid content (TCC) in H. cumingii is related to shell color. TCC of different tissues (gonad, gill, hepatopancreas, kidney, axe foot, mantle, and adductor muscle) of purple and white inner-shell H. cumingii was determined by UV–Vis spectrophotometry. The results revealed that TCC ranged from 12.91 ± 0.78 to 56.30 ± 0.74 μg/g. In general, TCC was higher in the hepatopancreas, followed by the mantle, gonad, gill, kidney, axe foot, and adductor muscle. TCC in gonad, gill, hepatopancreas, kidney, and mantle of purple mussels was significantly higher than that of white mussels. TCC in mussel tissues of H. cumingii was significantly different (P < 0.001) with respect to shell color. There were significant positive correlations between TCC in mussel mantle and shell color intensity. Future studies will assess the biological roles of carotenoids in shell color formation.     

Isolation and characterization of a novel 45 kDa calponin-like protein from anterior byssus retractor muscle of the mussel Mytilus galloprovincialis

SUMMARY: A calponin-like protein of 45 kDa was isolated from mussel anterior byssus retractor muscle (ABRM) and its inhibitory effects on actomyosin Mg²⁺-ATPase was demonstrated. The 2-D electrophoresis for ABRM myofibrils gave a spot of 45 kDa protein in addition to myofibrillar proteins such as myosin and actin. The 45 kDa protein, which was more basic and showed a slightly higher molecular weight than actin, was isolated by ion-exchange chromatography and subjected to chymotryptic digestion. N-terminal amino acid sequencing of polypeptide fragments produced gave two sequences, ASQKGMTSFGAVRHH and GMDRALISKMGSKYDSGL, both of which showed a high homology to those of vertebrate calponins and invertebrate calponin-related proteins. Furthermore, the 45 kDa protein strongly reacted with commercially available antibody raised against chicken smooth muscle calponin, demonstrating that the mussel ABRM 45 kDa protein is a new member of the calponin family. Then, actomyosin Mg²⁺-ATPase activity of ABRM was measured in the presence and absence of the 45 kDa protein. The 45 kDa protein clearly inhibited actomyosin Mg²⁺-ATPase activity in a dose-dependent manner as in the case of other vertebrate calponins. These results indicate that the 45 kDa calponin-like protein is involved in the thin filament-associated regulation of molluscan smooth muscle contraction, possibly of a unique contraction called catch.     

Ubiquitous increase in taurine transporter mRNA in tissues of tilapia (Oreochromis mossambicus) during high-salinity adaptation

We have isolated a cDNA encoding the taurine transporter from a tilapia (Oreochromis mossambicus) gill cDNA library. Transient expression of the cDNA in COS-7 cell indicates that the clone encodes a Na⁺- and Cl^–-dependent and -amino acid-specific taurine transporter. By the transfer of tilapia cultured in freshwater to 70% artificial seawater, plasma osmolality increased by up to 100–135 mOsm/kgH₂O along with the marked increase in the taurine transporter mRNA level in all the tissues examined i.e., kidney, stomach, intestine, gill, eye, liver, fin, muscle and brain. In most tissues, time-dependent change in the taurine transporter mRNA level corresponds to that in plasma osmolality. However, fin showed an acute and muscle showed a delayed increase in taurine transporter mRNA compared to changes in plasma osmolality. The taurine transporter mRNA level in tilapia embryos also increased after transfer from freshwater to 100% artificial seawater. Increase in taurine transporter expression leads to the activation of cellular uptake of taurine from plasma and the accumulation of taurine in the cell. Thus the results in the present study suggest that taurine plays an important role as an osmolyte in the ubiquitous tissues of tilapia during high-salinity adaptation.     

低盐胁迫对刺参 4 个盐度调节相关基因表达丰度的影响

从刺参(Apostichopus japonicus)低盐转录组数据库中选取与应激(热休克蛋白70基因)和离子传递(甘氨酸转运蛋白基因、锌转运蛋白基因、神经乙酰胆碱受体基因)相关的4个差异表达基因,利用qRT-PCR技术分析这4个基因在不同组织中的表达水平及低盐对其表达丰度的影响.结果表明甘氨酸转运蛋白基因在刺参呼吸树中表达水平最高,肠次之,体腔液表达较低;锌指蛋白基因和神经乙酰胆碱受体基因均在体腔液中表达最高,肠次之,在呼吸树中不表达;热休克蛋白70基因在体腔液中表达量最高,其次是呼吸树和肠组织.低盐胁迫下这4种基因的表达量均随着胁迫时间的延长呈波动性增减,其中甘氨酸转运蛋白在体腔液中的表达低于正常表达水平,在胁迫后3h时达到最低表达量.神经乙酰胆碱受体基因除在体腔液中48 h出现明显的上调外,在体腔液其他时间点和肠组织中处于下调表达状态.低盐胁迫下这4个基因表达丰度的变化,说明这些基因或作为功能蛋白直接参与机体的代谢调节,或作为调控蛋白调节胁迫功能蛋白的表达和活性来提高刺参对低盐胁迫的耐受能力.研究结果可为刺参盐度调节适应机制的研究奠定基础.     

三角帆蚌外套膜表达的免疫相关基因筛选

利用三角帆蚌(Hyriopsis cumingii)外套膜cDNA文库EST序列,通过BLAST分析注释基因功能,发现35个与免疫防御功能相关的基因。根据其功能,这些免疫相关基因可划分为7类,即细胞免疫过程(2个)、蛋白酶和蛋白酶调节子(9个)、压力蛋白(7个)、抗菌肽(5个)、溶酶体酶(2个)、粘着蛋白(3个)及细胞凋亡和细胞周期调控相关基因(7个)。免疫相关基因在三角帆蚌外套膜中的广泛表达表明外套膜是重要的免疫组织。该研究为三角帆蚌外套膜分子免疫机理研究和抗病育种工作提供了基础性资料。     

<Emphasis Type="Italic"> Xenostrobus securis </Emphasis>(Lamarck, 1819) (Mollusca: Bivalvia): first report of an introduced species in Galician waters

The presence of the non-indigenous species, the black-pygmy mussel Xenostrobus securis, is reported here for the first time in an intense shellfish farming area off Galicia (NW Spain). Very high concentrations of this mytilid bivalve have colonized estuarine waters located at the inner part of the Ria de Vigo. The invasive role of X. securis is discussed in the context of the wide ecological tolerance of the species and the recent finding of settlements of this species on numerous colonies of the economically-important blue mussel Mytilus galloprovincialis. The mode of introduction of the black-pygmy mussel is also discussed in relation to human management activities.     

cDNA cloning of oyster matrix metalloproteinase and its possible involvement in hypoxic adaptation

ABSTRACT:   We have cloned a cDNA encoding the matrix metalloproteinase (MMP) from oyster Crassostrea gigas . The clone contains a 1797 bp open reading frame encoding a protein of 599 amino acids. Oyster MMP (Cg-MMP) has a transmembrane domain at its C-terminal and a furin/prohormone convertase cleavage site at the end of a propeptide domain, which are commonly observed in membrane-type MMPs (MT-MMPs). This suggests that Cg-MMP is an MT-MMP. The deduced amino acid sequence of oyster MMP shares approximately 30% identity with human MT4-MMP and MMPs from fruit fly and hydra. Cg-MMP mRNA was detected in the gill, mantle and adductor muscle, and more intense signals in the northern blot analysis were recognized in the gill and adductor muscle. Similar tissue distribution was observed for tissue inhibitor of metalloproteinase (Cg-TIMP) in oyster. In response to hypoxic stress, the abundance of Cg-MMP mRNA was elevated in the gill, while that of Cg-TIMP mRNA remained almost constant. These findings suggest that promotion of collagen metabolism may be implicated in the hypoxic adaptation in oyster.     

三角帆蚌内脏团培育圆形有核珍珠的试验

报导了三角帆蚌(Hyriopsis cumingii)内脏团培育圆形有核珍珠的试验结果。结果显示:插植珠核的7362只手术蚌植核后一个月内成活率99.2%。经23个月养殖后,随机抽取50只。结果留核率达94%,成珠率为86%,其中中高档珠占37.2%。有核珍珠直径在9.4~11.6 mm之间,其中10 mm以上占88.4%。11.6%的珍珠表面有小于3 mm的凸起,未发现带扁而长尾巴的珍珠。试验表明:三角帆蚌内脏团内能培育出高品质的有核珍珠。此外还发现,脱核或核片分离的细胞小片仍可在内脏团内形成珍珠。     

池蝶蚌组织蛋白酶L1-like基因的克隆及表达特征分析

为了解淡水贝类是否存在组织蛋白酶L的亚型及其亚型的免疫相关作用,本实验利用已构建的池蝶蚌血细胞全长c DNA文库,筛选获得与之同源的EST序列,结合RACE技术进一步克隆了池蝶蚌一个新的组织蛋白酶L基因的c DNA全长,命名为Hs Cts L1-like基因(Gen Bank登录号为KF015273)。该序列全长为1280 bp,5′-非翻译区(5′UTR)为31 bp,3′-非翻译区(3′UTR)为256 bp,开放阅读框区(ORF)为993 bp,编码330个氨基酸,预测蛋白相对分子量为36.86 ku,理论等电点为6.23。序列分析结果显示,Hs Cts L1-like与其他软体动物相对应序列具有共同结构特征,包含信号肽、前肽抑制域和成熟肽三部分,在其他物种中已鉴定的Cts L签名序列标签(ERF/WNIN、GNFD、GCXGG和QCHN等)在Hs Cts L1-like中均可找到。其氨基酸序列同缢蛏Cts L1(AGL33704.1)同源性最高,达67%;与报道的三角帆蚌Cts L(ADV03094)和池蝶蚌中另一个Cts L(AEX88474)仅均为52.91%;系统进化分析表明,Hs Cts L1-like与缢蛏、长牡蛎和合浦珠母贝的Cts L1聚为一分支,推测Hs Cts L1-like属于Cts L家族中的亚型1。实时荧光定量PCR(q RT-PCR)检测显示,Hs Cts L1-like m RNA在肝脏中表达量最高,其次是卵巢和精巢。注射鳗弧菌后,血细胞和肝脏Hs Cts L1-like m RNA转录水平显著升高,暗示其是一个免疫有关的基因,参与了池蝶蚌的先天免疫应答反应。     

Effect of Washing on the Quality of Surimi-Like Preparation Obtained from Soft Tissue of Freshwater Mussel Sinanodonta woodiana (Lea, 1834)

ABSTRACT

The research analyses the influence of the number of washings and different washing solutions, on the recovery of dry matter and protein in a preparation made from freshwater mussel Sinanodonta woodiana, (Lea, 1834). The study involved qualitative and quantitative characterisation of mussel protein preparation (MPP) obtained by electrophoretic separation and differential scanning calorimetry (DSC). The type of washing agent had the greatest influence on dry matter and protein content. Washing the homogenate with NaCl solutions and water was more effective on protein extraction than using only water. Two bands of proteins with molecular weights of 270–273 kDa and 42–43 kDa were characterized by the highest band intensity in electropherograms. The DSC analysis showed that on all denaturation curves of the samples there was one main peak of transformation ranging 56.69–62.94°C. The research revealed that washing homogenates with NaCl solutions caused the appearance of additional peaks at lower transformation temperatures, i.e. from 19.68°C to 36.45°C. The highest enthalpy value (4.53 Jg^?1) was observed for the sample washed once NaCl solution and then water. The number of washings (1 or 2) and the use of a 0.5% NaCl solution or water were found to be the most favorable parameters for MPP production.     

Effects of integrated combination and quicklime supplementation on growth and pearl yield of freshwater pearl mussel, Hyriopsis cumingii (Lea, 1852)

The effects of integrated combination and quicklime supplementation on growth and pearl yield of a freshwater mussel, Hyriopsis cumingii (Lea, 1852), were examined through a 137-day growout in land-based enclosures. The integrated combinations examined were either mussel, bighead carp and gibel carp or mussel and bighead carp. Each combination was treated either with or without quicklime supplementation. One half of the mussels in each enclosure were grafted with pieces of the mantle epithelium while the other half were not. During the experiment, gibel carp were fed formulated feed while the mussel and bighead carp were fed natural live food. Quicklime was regularly provided in the enclosures as calcium replenishment. The species composition in the integrated system significantly affected growth in shell size and wet weight of the mussels regardless of the graft and pearl yield, while no significant effects of quicklime supplementation were detected. Growth rates in shell size and wet weight of both grafted and non-grafted mussels and pearl yield were slightly higher in the enclosures with mussel, bighead carp and gibel carp than those with mussel and bighead carp, although these differences were not statistically significant. The non-grafted mussel exhibited faster growth in shell size and wet weight than the grafted mussel within the same treatment. Results of the present study indicate that species combination in an integrated system can affect growth and pearl yield of H. cumingii . The species combination of mussel, bighead carp and gibel carp is recommended for commercial H. cumingii farming.     

Cloning and characterization of cDNA for carp matrix metalloproteinase 9

ABSTRACT: We have cloned a cDNA encoding the MMP-9 from a carp epidermal cell (EPC) cDNA library. The clone contains a 2025-base pair (bp) open reading frame encoding a protein of 674 amino acids. The deduced amino acid sequence shares 68% and 69% identity with medaka and Japanese flounder MMP-9. The hinge domain of the carp MMP-9, like those of the other non-mammalian species, lacks a type V collagen-like region that is typical of mammalian MMP-9. Gelatin zymography and immunoblot analysis of conditioned media of EPC cells and cDNA-transfected COS-7 cells detected a 76-kDa gelatinase. The apparent molecular mass of the carp zymogen is much smaller than those of its mammalian counterparts while almost identical with that of chicken 75-kDa gelatinase B-like enzyme. Although hypo-osmotic stress induced the elevation of MMP-9 mRNA level in EPC cells, no significant change in the protein in conditioned medium was detected during hypo-osmotic stress. Northern blot analysis detected a large amount of MMP-9 mRNA in carp kidney and spleen, suggesting the high expression of MMP-9 in blood cells, neutrophils, and macrophages. The smaller amount of MMP-9 mRNA was detected in gill, heart, fin, and eye, whereas none of the mRNA was detected in the hepatopancreas, intestine, brain, muscle, and skin.