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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。 相似文献
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合浦珠母贝热休克蛋白hsp70基因的克隆与表达分析 总被引：10，自引：2，他引：8
采用同源克隆和RT-PCR技术对合浦珠母贝(Pinctada fucata)热休克蛋白hsp70基因进行了克隆和表达分析。获得cDNA全长序列2 365 bp,其中3’非编码区域(UTR)为318 bp,5’UTR为88 bp,开放阅读框(ORF)为1 959 bp,编码652个氨基酸,分子量约为71.39 kD,理论等电点为5.22,并含有3个HSP70家族的签名序列IDLGTTYS、DLGGGTFD和EEVD。同源性分析表明,合浦珠母贝HSP70的氨基酸序列与太平洋牡蛎(Crassostrea gigas)等双壳贝类的相似性高达86%以上,基于氨基酸序列的聚类分析表明,合浦珠母贝与牡蛎属种类亲缘关系最近。高温、高盐刺激后,半定量RT-PCR检测发现hsp70基因的表达明显增加,高温刺激的表达量高于高盐刺激,高温刺激组不同组织的表达量由大到小依次为鳃、消化腺、外套膜、肌肉、性腺,高盐刺激组不同组织的表达量由大到小依次为鳃、外套膜、肌肉、消化腺、性腺,表明HSP70参与了机体对刺激的应答过程。该基因的克隆为进一步深入研究合浦珠母贝的抗逆机理及其遗传改良奠定了重要基础。 相似文献
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缢蛏ScHsc70 cDNA的分子特性和表达分析 总被引：2，自引：0，他引：2
从缢蛏(Sinonovacula constricta Lamarck)cDNA文库中筛选出一条热休克蛋白70(Heat shock protein 70,HSP70)同源序列,采用EST扩增和5′RACE(rapid amplification of cDNA ends,RACE)扩增方法获得全长cDNA序列,共2 335 bp,包括76 bp的5′非翻译区和309 bp的3′非翻译区,以及1 950 bp的开放阅读框。阅读框共编码649个氨基酸,推算的分子量约为70.89 kD,理论等电点为5.28。氨基酸序列分析结果表明,该基因的氨基酸序列含有HSP70家族的3个签名序列,2个糖基化位点,1个ATP-GTP结合位点,且C末端具有GGXP四肽重复序列以及细胞质特异性调控基序EEVD。该基因是组成型HSC70(Heat shock cognate 70)亚家族基因成员,被命名为ScHsc70。基于氨基酸序列的双壳贝类聚类分析表明,缢蛏与南极帽贝(Laternula elliptica)、文蛤(Meretrix meretrix)的亲缘关系最近。荧光定量表达分析显示,ScHsc70 mRNA在缢蛏水管、鳃、斧足、外套膜、肝和性腺等组织中以不同水平存在,其中在外套膜中的表达量最低,而在水管和肝中的表达量最高。经副溶血弧菌(Vibrio parahaemolyticus)和副鳗弧菌(V.anguillarum)诱导后,ScHsc70 mRNA在缢蛏肝中的水平呈现先升高后下降的变化趋势,且分别在感染后的第20小时和第30小时达到最高。结果提示,缢蛏ScHsc70基因可能参与了对细菌感染的防御反应。 相似文献
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Optimal dietary taurine level for growth of juvenile Japanese flounder Paralichthys olivaceus 总被引：5，自引：0，他引：5
GWANG-Sic PARK TOSHIO TAKEUCHI MASAHITO YOKOYAMA TADAHISA SEIKAI 《Fisheries Science》2002,68(4):824-829
The requirement for taurine in juvenile Japanese flounder Paralichthys olivaceus was determined by feeding diets containing various levels of taurine and cystine. Test diets supplemented with 0.5, 1.0 and 1.5% of taurine or with 0.5, 1.0 and 1.5% of L -cystine were prepared. The basal diet contained 55% protein from white fish meal. These diets were fed to juvenile Japanese flounder with an initial mean bodyweight of 0.9 g (total length (TL) 48 mm) for 5 weeks. Approximately 1.4% taurine content in the diet was required for optimum growth of juvenile flounder. A positive linear relationship was noted between the content of taurine accumulated in the muscle, liver and brain and the level of taurine in the diet. However, there was no increased taurine content in tissues of fish fed the cystine-supplemented diet. In contrast, the fish fed control and cystine-supplemented diets showed higher contents of cystathionine in the tissues. The concentration of cystathionine in tissues rapidly decreased with an increase of taurine in the diet. It was also observed that for each of the dietary groups, a trace amount of taurine was excreted. These results suggest that the taurine content in the diet affects the sulfur amino acid metabolism of juvenile Japanese flounder, and indicate that juvenile flounder are unable to biosynthesize taurine from cystine. 相似文献
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MD Shah ALAM Shin-Ichi TESHIMA Manabu ISHIKAWA Shunsuke KOSHIO 《Fisheries Science》2002,68(3):509-516
ABSTRACT: To investigate the effects of disproportionate levels of dietary arginine and lysine on juvenile Japanese flounder Paralichthys olivaceus , growth performance and biochemical parameters were evaluated by feeding five test diets, comprising different levels of arginine and lysine, to triplicate groups of juveniles (initial bodyweight 1.85 g) for 40 days. Crystalline amino acids were supplemented to test diets to correspond to the amino acid pattern found in the whole body protein of the Japanese flounder, except for arginine and lysine. After the feeding trials, plasma arginine, lysine and urea levels, excreted ammonia-N, and liver arginase activity were analysed. Survival, specific growth rate, feed conversion efficiency, and apparent protein retention were adversely affected ( P < 0.05) by the deficiency in dietary arginine or lysine concentrations. An excess of either lysine or arginine in the diet did not depress growth when the diets contained adequate levels of either arginine or lysine, proving that there is no evidence for an arginine–lysine antagonism. Results for plasma arginine, lysine and urea levels, excreted ammonia, and liver arginase activity also demonstrated that Japanese flounder juveniles are not sensitive to excess dietary levels of lysine and arginine. 相似文献
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Masaya TAKAGI Takeshi KIKKO Masatomi HOSOI Isao HAYASHI Haruhiko TOYOHARA 《Fisheries Science》2004,70(4):682-687
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. 相似文献
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Shin-Kwon Kim Hiroyuki Matsunari Kazuharu Nomura Hideki Tanaka Masahito Yokoyama Yuko Murata Kenji Ishihara Toshio Takeuchi 《Fisheries Science》2008,74(4):875-881
ABSTRACT: The effects of dietary taurine levels and lipid contents on the conjugated bile acid composition and growth performance of juvenile Japanese flounder Paralichthys olivaceus were investigated. Six types of diet (three different levels of taurine at two different levels of lipid) were fed to juveniles (average body weight, 0.04 g). Fishmeal that was washed with 70% ethanol to remove taurine was used as the sole protein source. Feeding experiments were carried out at 20°C for 6 weeks. At the end of the experiments, fish were weighed and analyzed for free amino acids in the body and the composition of the conjugated bile acids. The body weight and percent weight gain of the juveniles were improved by the dietary taurine supplementation. The taurine contents of the whole body and tissues increased with the increase of the dietary taurine contents. The conjugated bile acids in the gall bladder consisted of taurocholic acid and taurochenodeoxycholic acid, which increased with the increase of the dietary taurine level. Taurocholic acid accounted for more than 95% of the total conjugated bile acids. This indicates that taurine is the sole amino acid to conjugate bile acid in Japanese flounder. 相似文献
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Seven isonitrogenous and isoenergetic experimental diets were formulated to investigate the effect of low molecular weight fish protein hydrolysate (FPH) in diets on growth performance, feed utilization and liver IGF‐I mRNA levels in Japanese flounder (38.80 ± 1.11 g) fed with high plant protein diets. Fish meal protein was, respectively, replaced by 6% (FPH6), 11% (FPH11), 16% (FPH16), 21% (FPH21), 26% (FPH26) FPH of total dietary protein. FPH diets contained a constant high level of plant protein (690 g kg?1) from soybean meal. As a positive control diet, FM2 contained about 590 g kg?1 plant protein and 410 g kg?1 fish meal protein, while negative control diet FM1 contained about 690 g kg?1 plant protein and 310 g kg?1 fish meal protein. The expression levels of liver IGF‐I mRNA were evaluated using real‐time PCR normalized against the 18S rRNA gene. The results showed that moderate low molecular weight FPH (FPH11) improved growth performance and protein retention. Fish fed with FPH11 and control diet FM2 had similar growth and feed utilization, while high‐level low molecular weight FPH did not improve growth performance and protein retention, and depressed liver IGF‐I mRNA expression in Japanese flounder. 相似文献
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ABSTRACT: Three diets supplemented with taurine, β-alanine andGABA and a control diet were fed to juvenile and fingerling Japaneseflounder to investigate the effects of the diets on growth and metabolicchanges of free amino acids in whole body and tissues. In experimentI, three diets supplemented with 1% each of taurine, β-alanineand GABA and a control diet were fed to juvenile Japanese flounderwith an initial mean body weight of 0.4 g for 4 weeks at20°C. In experiment II, the taurine-supplemented diet anda control diet were fed to fingerling Japanese flounder with an initialmean body weight of 15 g for 4 weeks at 20°C.Only supplementation of taurine in the diet of juvenile flounderimproved their growth performance in experiment I, but fingerlinggrowth performance of experiment II was not significantly relatedto taurine supplementation in the experimental diet. These resultssuggest that there is a greater requirement for taurine for thegrowth of juvenile Japanese flounder than fingerling Japanese flounder. 相似文献
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Heat shock proteins (HSPs) are a group of highly conserved molecular chaperones. Shrimp HSPs have recently been a topic of increasing interest because of their roles in shrimp immunity and homeostasis. In penaeid shrimp, HSP70s and the cognate forms, heat shock cognate (HSC) 70s, have been reported, but their responses towards various stimulations are different. We found a novel type HSP70 (MjHSP70-2) from the hyperexpansion of the large segmental duplication that is present in kuruma shrimp Marsupenaeus japonicus, which shows about 60 % identity with reported shrimp HSP70s. In a phylogenetic tree, MjHSP70-2 formed a sister clade with eukaryote HSP70 family while MjHSP70 was located close to the shrimp HSP70 and HSC70 group. MjHSP70-2 gene expression was not significantly increased by heat shock or pathogen challenge by Vibrio penaeicida, but it was significantly increased by infection with white spot syndrome virus. In contrast, MjHSP70 gene expression was increased by heat shock but decreased by infection with V. penaeicida. The kuruma shrimp genome was found to have 400-fold more copies of the MjHSP70-2 gene than the putative single-copy gene transglutaminase. In conclusion, our results reveal the presence of a novel type HSP70 gene, HSP70-2, from kuruma shrimp. There are multiple forms of HSP70 in crustaceans, and these HSP70s behave differently under various stressors. 相似文献
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ABSTRACT: The effect of dietary taurine on juvenile Japanese flounder was determined by feeding three taurine-supplemented experimental diets (TAU) and a commercial diet (CD) to evaluate a practical diet for juvenile Japanese flounder. Juvenile Japanese flounder were reared on the three experimental diets supplemented with taurine at 0, 0.5, 1.0% and CD. These diets were fed to juvenile Japanese flounder of an initial mean body weight of 0.2 g for 6 weeks at 20°C and the taurine contents of the whole body and tissues were analyzed. The final average body weight of juvenile Japanese flounder fed the 1.0% TAU was significantly higher than that of the other groups. Taurine contents in the whole body and tissues increased with the increase in dietary taurine level. These results indicate that juvenile Japanese flounder require at least 15 mg/g taurine in the diet, even though a combined mix of fish, krill and squid meal was the main protein source in the experimental diets. 相似文献
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. Shah Alam Shin-ichi Teshima Dedy Yaniharto Shunsuke Koshio Manabu Ishikawa 《Aquaculture (Amsterdam, Netherlands)》2002,210(1-4):359-369
A feeding trial using five semi-purified diets (50% crude protein) was conducted to investigate the effects of different dietary amino acid patterns on growth and body composition of juvenile Japanese flounder. The control diet contained casein and gelatin as intact protein sources and four other diets contained 30% casein–gelatin (2:1, w/w) and 20% crystalline amino acids (CAA). CAA were added to the diets to simulate the amino acid pattern found in red sea bream egg protein (REP), Japanese flounder larvae whole body protein (FLP), Japanese flounder juvenile whole body protein (FJP), and brown fish meal protein (BFP), respectively. The test diets were fed to triplicate groups of juveniles (2.75±0.05 g) twice a day for 40 days to evaluate weight gain, survival, feed conversion efficiency (FCE), protein efficiency ratio (PER), and apparent protein utilization (APU). The apparent retention of total dietary amino acids in the whole body and A/E ratios of the whole body were also evaluated. The highest weight gain was observed in fish fed the diet containing the dietary amino acid pattern of BFP followed by fish fed the control, FJP, FLP and the REP diets. Percent survival, FCE, PER and APU were also significantly (P<0.05) affected by the amino acid pattern in the diets, indicating the highest value in fish fed the BFP diet. Except for a few amino acids, the amino acid composition of the whole body did not show marked differences with different dietary amino acid pattern. Results suggest that BFP could be more suitable as a reference amino acid pattern in the diet of juvenile Japanese flounder compared to the amino acid pattern of FLP, FJP or REP. 相似文献
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Distribution of three GnRHs in the brain and pituitary of the wild Japanese flounder Paralichthys olivaceus 总被引：1，自引：1，他引：1
Ky Xuan Pham Masafumi Amano Noriko Amiya Yutaka Kurita Kunio Yamamori 《Fisheries Science》2006,72(1):89-94
ABSTRACT: Wild adult maturing and immature female Japanese flounder Paralichthys olivaceus were collected in June 2004 and January 2005, respectively, to clarify a possible role of gonadotropin-releasing hormones (GnRHs) in reproduction. Levels of salmon GnRH (sGnRH), chicken GnRH-II (cGnRH-II) and sea bream GnRH (sbGnRH) in the brain and pituitary were examined by time-resolved fluoroimmunoassay. Three forms of GnRHs were detected in the discrete brain at various levels. In the pituitary of both maturing and immature fish, sbGnRH was abundant together with a pronounced amount of sGnRH, whereas cGnRH-II was almost below the detectable limit. In maturing fish, levels of sbGnRH were high in the telencephalon, hypothalamus and pituitary, while levels of sbGnRH of immature fish were very low in these regions. These results indicate that sbGnRH is mainly responsible for gonadotropin secretion, and that sbGnRH in the anterior part of the brain is associated with gonadal maturation in the Japanese flounder. 相似文献
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Threechada Danwattananusorn Fernand F. Fagutao Aiko Shitara Hidehiro Kondo Takashi Aoki Reiko Nozaki Ikuo Hirono 《Fisheries Science》2011,77(6):929-937
Heat shock proteins (HSPs) are proteins that are expressed more strongly when the cells are exposed to physiological and stressful
conditions. In this study, the full-length cDNAs of heat shock proteins 40 (MjHSP40), 70 (MjHSP70) and 90 (MjHSP90) were cloned from kuruma shrimp Marsupenaeus japonicus. The open reading frames (ORFs) of the cDNA clones have lengths of 1,191, 1,959 and 2,172 bp and encode 396, 652 and 723
amino acid residues, respectively. The predicted MjHSP40 amino acid sequence contains a J domain, a glycine/phenylalanine-rich region, and a central domain containing four repeats
of a CxxCxGxG motif, indicating that it is a type I HSP40 homolog. The signature sequences of the HSP70 and HSP90 gene families
are conserved in the MjHSP70 and MjHSP90 amino acid sequences. The deduced amino acid sequences of MjHSP70 and MjHSP90 share high identity with previously reported shrimp HSP70s and HSP90s, respectively. The expression of MjHSP90 mRNA increased at 32°C. Additionally, the expressions of MjHSP40, MjHSP70 and MjHSP90 mRNAs increased in defense-related tissues (i.e., hemocytes and lymphoid organ) when the shrimp were challenged with
white spot syndrome virus. 相似文献
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Kazuharu TAKEUCHI Satoshi KUBOTA Masato KINOSHITA Haruhiko TOYOHARA Morihiko SAKAGUCHI 《Fisheries Science》2002,68(3):610-617
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. 相似文献