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.     

拟穴青蟹GRP78基因的克隆与应激表达

葡萄糖调节蛋白78(glucose regulated protein 78 ku,GRP78)是热休克蛋白70家族成员之一,在调节蛋白质折叠和维持内质网稳态过程中起着分子伴 侣作用。采用RT-PCR、RACE等技术,首次从拟穴青蟹获得了GRP78的cDNA全长序列。该序列全长2 284 bp,开放阅读框(ORF)为1 962 bp,编码653个氨基酸残基。 同源分析显示,该基因编码的蛋白含有HSP70家族的签名序列,C末端为内质网蛋白滞留信号KDEL,与其他物种具有很高相似性。实时荧光定量PCR结果表明,GRP78 基因在拟穴青蟹多个组织中均有表达。第一期仔蟹在不同的温度和盐度下暴露12 h后,GRP78基因表达量随环境温度升高而增加;在高盐(30)条件下GRP78表达量 较高,进而推测拟穴青蟹GRP78参与蛋白质折叠和环境胁迫的应答。     

长牡蛎非整倍体的制备、胚胎发育及存活能力

通过二倍体、三倍体杂交的方法制备长牡蛎(Crassostreagigas)非整倍体。实验组设为2n♀×3n♂组、3n♀×2n♂组和3n×3n组,对照组为2n×2n组。与对照组相比,实验组畸形率高,担轮虫孵化率及D形幼虫生成率等参数均降低,幼虫死亡率高。在担轮幼虫向D形幼虫转化期,实验组发育滞后。培育过程中染色体数为25左右的非整倍体不能存活,整倍体和染色体数与整倍体接近的非整倍体能够存活。实验证实该方法是进行贝类非整倍体制备的有效手段。     

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.     

长牡蛎"海大3号"生长繁殖与营养成分的周年变化

为阐明长牡蛎"海大3号"新品种的生长与繁殖特性,自2016年11月份至2017年10月份对养殖于山东省荣成海区长牡蛎"海大3号"群体的生长、性腺发育及营养成分的周年变化及其与环境因子的关系进行研究。结果显示,在海区水温较低的冬季1—2月份,长牡蛎"海大3号"生长缓慢,性腺处于休止期;随水温升高,在春季3—4月份配子开始发育,雌雄个体发育基本同步,4—5月份壳高生长速率加快,湿重显著增加;在夏季,随配子发育壳高生长速率降低,7月份因配子排放湿重下降;在产卵后的秋季长牡蛎"海大3号"壳高和湿重均显著增长。营养分析结果表明,随性腺发育,外套膜、性腺-内脏团、鳃和闭壳肌中糖原含量呈下降趋势,为配子发育提供能量;脂质和蛋白质作为配子主要结构物质在性腺中不断累积,随配子排放而显著降低;在秋季,4个组织中较高的蛋白质含量为机体的快速生长提供物质基础。同时外套膜作为构成壳物质的分泌器官,在8—9月份其糖原含量和脂质含量显著增加,这暗示机体的快速生长需要一定能量储备。研究表明,长牡蛎"海大3号"的生长和繁殖活动受海区水温季节性变化的影响,并与各组织营养成分密切相关。春季和秋季为机体快速生长时期,夏季为配子集中排放期;糖原是生长繁殖活动的主要能量来源,脂质是配子主要结构物质,蛋白质是机体产后恢复和快速生长的物质基础。     

An insulin-like system involved in the control of Pacific oyster Crassostrea gigas reproduction: hrIGF-1 effect on germinal cell proliferation and maturation associated with expression of an homologous insulin receptor-related receptor

The putative involvement of insulin-like peptides in the control of the reproduction of the Pacific oyster Crassostrea gigas was investigated using different approaches. In conjunction with a monthly histological analysis of the oyster reproductive cycle, in vitro biological effects of the human recombinant IGF-1 (hrIGF-1) on dissociated germinal cells were mesured over 1 year using [³H]-thymidine and [¹⁴C]-amino acid mixture as tracers for DNA and protein synthesis. DNA synthesis was stimulated by hrIGF-1 in November (114 ± 11% for 10^− 7M), December (46 ± 6% for 10^− 7 M) and January, which was identified as the highest gonial mitosis period. A clear dose-effect was observed in January with a maximum activation of 68 ± 7% for 10^− 12 M. Germinal cell protein synthesis was also stimulated in March (20 ± 1% for 10^− 10 M), April (41 ± 5% for 10^− 13 M), May (25 ± 4% for 10^− 13 M), and by almost all of hrIGF-1 doses in June (21.5 ± 2% for 10^− 13 M) and July (34 ± 1% for 10^− 13 M). This suggests the involvement of insulin-like substances in gonadal tubule rebuilding (December), as well as in the development of germinal cells (March, April), and in the summer maturation of gametes (May, June, July). These insulin-like effects conform with the expression pattern of the recently identified C. gigas insulin receptor-related receptor (CIR): It appeared highly expressed in the gonadal area during gonial mitosis phase, but also in maturating oocytes, suggesting the involvement of an insulin-like system in gonial proliferation and maturation. Moreover, CIR showed differential expression during embryogenesis and larval developmental stages. The expression of maternal CIR during the embryonic and early larval development, followed by the increasing zygotic CIR expression from D larvae to 11-day-old veliger larvae, then a decrease until metamorphosis, also suggest that insulin-like peptide is involved in organogenesis.     

Effects of mass and position of artificial fouling added to the upper valve of the mangrove oyster Crassostrea rhizophorae on its growth and survival

We ran an experiment on mangrove oysters Crassostrea rhizophorae to evaluate the effects of adding different masses of artificial fouling to the upper valve, either to the umbo region or the ventral edge of the shell. Growth and survival were quantified after a 30 d period in suspended culture in the La Restinga Lagoon, Venezuela. The artificial fouling was cement weighing 0.5, 1, 2 or 3 fold the mass of the upper valve. No fouling was added to a control group. Fouling mass, but not the position of the artificial fouling, affected growth in shell length. However, only the heaviest fouling (3 times the mass of the upper valve) had a significant effect. In contrast, there was no affect of either fouling mass or position on tissue growth. Finally, our data indicated that mortality could be affected by the position where we added artificial fouling (greater mortality when fouling was added to the ventral edge of the shell), but not by fouling mass. Our study indicates it is unlikely that the levels of natural fouling that develop on oysters in suspended culture would be sufficient to affect either growth or survival.     

Settlement of Ostrea edulis is determined by the availability of hard substrata rather than by its nature: Implications for stock recovery and restoration of the European oyster

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Molecular cloning of heat shock protein 60 from Marsupenaeus japonicus and its expression profiles at early developmental stages and response to heat stress

Water temperature is an essential environmental factor in aquaculture that affects many aspects of organism, and a rise in water temperature stresses aquatic animals. HSP60 is a major component of the chaperone system and plays an important role in stress response. In this study, the full‐length cDNA sequence of HSP60 from Marsupenaeus japonicus was cloned for the first time, and the tissue distributions and expression profiles of MjHSP60 at the early developmental stages of M. japonicus and under heat stress were verified by qRT‐PCR. The full‐length cDNA sequence of MjHSP60 was 2,303 bp with the deduced amino acid (AA) sequence of 579 AA. MjHSP60 was expressed in all eight tested tissues of M. japonicus and was mainly expressed in the hepatopancreas under normal condition. MjHSP60 was expressed in all of the early developmental stages examined from the fertilized egg to the post‐larval stage, and the expression level of MjHSP60 peaked at zoea II and reached a trough at the transition periods between each two stages (N and M1) and showed a relatively stable expression level at the post‐larval stage. The expression level of MjHSP60 was significantly up‐regulated in the gills, muscle, heart, hepatopancreas and stomach at 3 hr post‐heat‐stress and showed varied sensitivity to heat stress. The expression profile of MjHSP60 in the hepatopancreas and gill showed a time‐dependent manner post‐heat‐stress and peaked at 3 hr post‐heat‐stress. The present study provided a basis for further studies for elucidating the function of MjHSP60 in the heat stress response and the early developmental stages of M. japonicus.     

The induction of Hsp70 synthesis by non‐lethal heat shock confers thermotolerance and resistance to lethal ammonia stress in the common carp,Cyprinus carpio (Linn)

Exposure to heat‐shock protein (Hsp) stimulating factors induces Hsp accumulation and confers tolerance to lethal ammonia stress on the common carp Cyprinus carpio. This study investigated whether a non‐lethal heat shock bestowed similar protective effects against ammonia and induced thermotolerance, both thought to be rendered by increased amounts of Hsps. The 30‐min lethal temperature (30 min LHT) and 1‐h lethal ammonia concentration (1 h LCT) for this species occurred at 41°C and 14.2 mg/L NH₃ respectively. Heating juvenile carp (5 cm) from 28°C to 32, 34 and 38°C, with a subsequent 8‐h recovery period augmented tolerance to lethal heat and ammonia perturbation by two to threefold as compared with animals held at 28°C. Protection occurred in conjunction with Hsp70 accumulation in gills, substantiating the role of this Hsp in enhancing the stress tolerance of common carp.