New type of heat shock protein 70 homologue gene abounds in the genomic sequence of kuruma shrimp Marsupenaeus japonicus

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.     

Characterization of <Emphasis Type="Italic">pax3a</Emphasis> and <Emphasis Type="Italic">pax3b</Emphasis> genes in artificially induced polyploid and gynogenetic olive flounder (<Emphasis Type="Italic">Paralichthys olivaceus</Emphasis>) during embryogenesis

Although chromosome set manipulation techniques including polyploidy induction and gynogentic induction in flatfish are becoming increasingly mature, there exists a poor understanding of their effects on embryonic development. PAX3 plays crucial roles during embryonic myogenesis and neurogenesis. In olive flounder (Paralichthys olivaceus), there are two duplicated pax3 genes (pax3a, pax3b), and both of them are expressed in the brain and muscle regions with some subtle regional differences. We utilized pax3a and pax3b as indicators to preliminarily investigate whether chromosome set manipulation affects embryonic neurogenesis and myogenesis using whole-mount in situ hybridization. In the polyploid induction groups, 94 % of embryos in the triploid induction group had normal pax3a/3b expression patterns; however, 45 % of embryos in the tetraploid induction group showed abnormal pax3a/3b expression patterns from the tailbud formation stage to the hatching stage. Therefore, the artificial induction of triploidy and tetraploidy had a small or a moderate effect on flounder embryonic myogenesis and neurogenesis, respectively. In the gynogenetic induction groups, 87 % of embryos in the meiogynogenetic diploid induction group showed normal pax3a/3b expression patterns. However, almost 100 % of embryos in the gynogenetic haploid induction group and 63 % of embryos in the mitogynogenetic diploid induction group showed abnormal pax3a/3b expression patterns. Therefore, the induction of gynogenetic haploidy and mitogynogenetic diploidy had large effects on flounder embryonic myogenesis and neurogenesis. In conclusion, the differential expression of pax3a and pax3b may provide new insights for consideration of fish chromosome set manipulation.     

Induction of apoptosis in sea bream fibroblasts by Vibrio harveyi haemolysin and evidence for an anti‐apoptotic role of heat shock protein 70

In this study, we exposed black sea bream, Mylio macrocephalus (Basilewsky), fibroblast (BSF) and silver sea bream, Sparus sarba Forsskål, fibroblast (SSF) cell lines to a recombinant Vibrio harveyi haemolysin (VHH) and investigated mechanisms involved in apoptosis. A decrease in mitochondrial membrane potential, followed by an increase in caspase 3 activity, occurred within 2–8 h of VHH exposure, in both cell lines; however, VHH did not alter cellular levels of reactive oxygen species. As heat shock protein 70 (HSP70) is known to prevent the onset of apoptosis in certain mammalian cells, we aimed to test whether such a protective effect is operative in VHH‐exposed fibroblasts. The amounts of HSP70 were elevated in SSF and BSF via an acute heat shock or an acute heat shock followed by a 6 h recovery. It was found that the VHH‐mediated reduction in mitochondrial membrane potential was suppressed in cells that had a 6 h post‐heat shock recovery, and the protective effect of heat shock‐induced HSP70 was attenuated following treatment of cells with the HSP70 inhibitor, quercetin. This study demonstrates how haemolysin causes cell death via induction of apoptosis and provides evidence as to the role of HSP70 as an anti‐apoptotic factor.     

热应激对剑尾鱼HSP70家族两成员基因表达的影响

采用RT-PCR方法扩增实验动物剑尾鱼（Xiphopohorus helleri）纯系RR-B的HSP70家族两个成员的eDNA片段，并将其克隆到PMD18-T载体中测序，将测序结果与GenBank中的核苷酸序列和推导的氨基酸序列进行同源性比较。同时利用RT-PCR半定量方法研究热应激时两个成员在剑尾鱼组织中的表达。通过克隆获得了剑尾鱼的HSP70家族两个新成员的cDNA片段，两序列均包含HSP70家族的特征性签名序列和热休克蛋白的定位序列；通过对克隆片段与已发表的青锵（Oryzias latipes）等鱼类HSP70的核苷酸序列和其编码的氨基酸序列同源性比较，发现核苷酸序列同源性较高，成员一和成员二分别为85％-89％和82％-99％；氨基酸序列同源性更高，成员一和成员二分别为97％-99％和93％-99％。一般条件下，两成员在剑尾鱼肝脏、脾脏、肾脏和心脏中不表达，但热应激能刺激成员一在剑尾鱼脾脏中表达，成员二在肝脏、脾脏、肾脏和心脏中强烈表达，并可能在参与热应激保护方面起更大作用。     

The effects of dietary lipid levels on performance and heat‐shock protein response of juvenile white seabass,Atractoscion nobilis

A feeding trial was conducted to study the effect of dietary lipid on growth performance and heat‐shock protein (HSP70 and HSP60) response of white seabass (WSB), Atractoscion nobilis. Five diets were formulated to contain 440 g kg^?1 protein from 300 g kg^?1 fish meal, 240 g kg^?1 soybean meal and 100 g kg^?1 soy protein concentrate with different levels of lipid: 100, 120, 140, 160 or 180 g kg^?1. At the end of the trial, heat shock response based on HSP70 and HSP60 was measured in liver and white muscle from fish at ambient temperature and temperature shock conditions. Final weight and percent gain were significantly higher for fish fed the 100 g kg^?1 lipid diet than for fish fed the rest of the diets (P ≤ 0.05). Feed conversion ratio was lowest for fish fed the 100 g kg^?1 lipid diet. The HSP70 and HSP60 responses were positively correlated to dietary lipid levels following temperature shock. At ambient temperature, HSP60 and HSP70 responses in muscle and HSP60 response in liver increased with dietary lipid level. Temperature shock significantly increased the HSP response of fish in all treatments. Results of this study demonstrated that a moderate (110–120 g kg^?1) level of dietary lipids would be recommended for production diets but a higher dietary lipid level may be required for optimal stress tolerance.     

Molecular characterization and expression analysis of heat shock proteins 40, 70 and 90 from kuruma shrimp <Emphasis Type="Italic">Marsupenaeus japonicus</Emphasis>

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.     

Molecular cloning and expression of the heat shock protein 70 gene in the Kumamoto oyster <Emphasis Type="Italic">Crassostrea sikamea</Emphasis>

Episodes of summer mortality of the Kumamoto oyster Crassostrea sikamea are a major problem for its cultivation. Expression of the heat shock protein 70 (HSP70) is induced by various environmental stresses, including heat. We cloned and sequenced hsp70 complementary DNA from C. sikamea to investigate the relationship between hsp70 expression and heat tolerance in this oyster. Quantitative real-time polymerase chain reaction was performed using gill tissue dissected from oysters before and after heat shock for 1 h. The results showed hsp70 expression was faster and greater in oysters cultured at 20–22 °C than at 10–12 °C, and survival was lower among oysters cultured at 20–22 °C than at 10–12 °C. Moreover, heat tolerance was investigated by a 1-h pre-heat treatment, followed by exposure to heat shock conditions 5 days later. Survival was higher and hsp70 expression was notably lower in oysters that received the pre-heat treatment compared with those that did not. We conclude that a pre-heat treatment of only 1 h may be useful for inducing heat tolerance in C. sikamea, and that a low level of hsp70 expression after heat shock is an important index in selecting for high heat tolerance in these oysters.     

Total substitution of fish oil by vegetable oils in Senegalese sole (Solea senegalensis) diets: effects on fish performance, biochemical composition, and expression of some glucocorticoid receptor-related genes

To study the substitution of fish oil by vegetable oils in fish diets, juveniles Senegalese sole (Solea senegalensis) were fed diets (56 % crude protein, 12 % crude lipid) containing either linseed (100LO) or soybean (100SO) oils in comparison with a 100 % fish oil-based diet (100FO) for 90 days. Samples of muscle, liver, and intestine were collected for biochemical analysis and for glucocorticoid receptor-related genes, including GR1 and GR2, and the associated heat shock proteins HSP70, HSP90AA, and HSP90AB. Besides, basal levels of plasma cortisol were also determined. After the feeding period, a stress test, consisting on 5 min of net chasing, was applied to a selected population of each dietary group. Total replacement of fish oil by vegetable oils did not induced changes in fish growth and performance, but affected fatty acid profile of muscle, liver, and intestine, reflecting those tissues the characteristic fatty acids of each type of dietary oil. A tendency to conserve the ARA/EPA ratio could be observed in the different tissues, despite of the level of these fatty acids in diet. Chasing stress induced an increase of muscle GR1 and a reduction in intestinal GR2 relative expressions at any of the experimental diets assayed. In liver, chasing stress induced an increase in both GR1 and GR2 gene expression in fish fed fish oil diets. Similarly, chasing stress induced an increase of muscle HSP70 and decrease of HSP90AB in liver at any of the experimental diet assayed. Besides, vegetable oils decreased the expression of HSP70 in intestine, being the relative expression of liver HSP90AA increased by the inclusion of linseed oil in the diet, at any of the experimental conditions assayed.     

Heat shock protein 70 expression in different tissues of Cirrhinus mrigala (Ham.) following heat stress

Members of heat shock protein 70 (HSP70) are highly conserved proteins of about 70 kDa and play important roles in protein folding. Levels of these proteins increase when cells are under stress. Environmental temperature influences both the basal and induced levels of HSPs. However, studies on HSPs in fishes from a tropical country such as India are lacking. In the present study, Indian major carp (IMC) Cirrhinus mrigala (Ham.) acclimatized at 25±2°C had high levels of HSP70, viz., 1.2–1.3 ng μg^?1 total protein in kidney and gill and 4.2–5.3 ng μg^?1 total protein in liver and brain tissues, indicating the presence of biochemically significant levels of stress. However, maintenance of acclimatized fish at 17°C for up to 48 h did not lead to a significant decrease in stress protein levels. A heat shock at 37°C for up to 48 h resulted in only two to threefold increase in HSP70 levels in these organs. Although the increase in HSP70 levels was apparent from the first hour of heat stress in all these tissues, the increase was significant from the second hour in the brain, the sixth hour in liver and kidney and the 20th hour in the gills.     

罗非鱼HSP70(tHSP70)蛋白的生物信息学分析、真核表达及质谱鉴定

运用生物信息学技术分析罗非鱼热休克蛋白70(tilapia heat shock protein,tHSP70)的理化特性、糖基化位点、跨膜区域、蛋白的细胞定位及信号肽与虹鳟等其他动物HSP70的相似性,以人工合成cDNA为模板,通过聚合酶链式反应扩增其完整CDS,并将此DNA片段与真核表达载体pGAPZa-A连接,构建pGAPZa-HSP70表达质粒,在毕赤酵母GS115中表达tHSP70蛋白,对表达上清液进行SDS-PAGE及Western-blotting分析后,采用Ni²⁺IDA层析脱盐纯化目的蛋白,并对所表达的蛋白进行糖基化PAS染色鉴定。对表达的蛋白进行SDS-PAGE后,将所获得的非预定大小(约100 ku)条带进行电离飞行时间质谱鉴定,并确定其蛋白种类。结果表明:tHSP70所含氨基酸数量为640,分子量为70 274.5 u,等电点为5.49。在哺乳动物网织红细胞(体外)的半衰期为30 h,在酵母内半衰期大于20 h,而在大肠杆菌内的半衰期也大于10 h,不稳定指数为36.64,脂肪族指数为85.58。可能分别含有6个N-和O-糖基化位点,所克隆tHSP70基因与目的基因完整编码区序列完全一致,所构建的pGAPZa-HSP70质粒能在GS115中成功表达,诱导表达上清液经SDS-PAGE及Western-blotting分析后发现,除70 ku目的蛋白外,还出现一条100 ku条带。经糖基化PAS染色证明,此100 ku蛋白可能是HSP70糖基化的结果,且能被人的兔抗HSP70抗体识别,质谱鉴定证明该条带即是tHSP70蛋白。本研究所表达tHSP70蛋白为后续罗非鱼细胞学及抗原提呈等免疫学研究奠定了基础。     

淇河鲫cyp19a1b基因的克隆表达及芳香化酶抑制剂对其表达的影响

为探索脑型芳香化酶基因(cyp19a1b)在雌核发育三倍体鱼淇河鲫性别决定与分化过程中的作用,利用RACE方法克隆淇河鲫cyp19a1b基因cDNA全长序列,采用Real-time PCR分析其在不同组织、胚胎及胚后不同发育时期的表达情况,同时检测芳香化酶抑制剂Letrozole诱导性逆转及腹腔注射人绒毛膜促性腺激素(hCG)后cyp19a1b在脑中的表达情况。结果显示,淇河鲫cyp19a1b cDNA全长2 984 bp(GenBank ID:MF926270),包含132 bp5′非编码区,1 319 bp 3′非编码区,1 533 bp开放读码框,编码510个氨基酸残基;氨基酸序列比对及系统进化分析结果显示,淇河鲫Cyp19a1b与其他鲤科鱼类同源性较高,与哺乳类、爬行类等脊椎动物同源性较低,这与其分类地位一致;组织分布检测结果显示,cyp19a1b基因在淇河鲫脑中表达量最高,在卵巢等其他组织中表达量较低;Realtime PCR结果表明,在胚胎发育过程中cyp19a1b在外源精子刺激后从囊胚期开始上调,神经胚期达到最高,随后降低;出膜后随着发育的进行,该基因在脑中表达量逐步上调,在躯体中一直维持在较低水平;除此之外,伴随着Letrozole诱导的性逆转,cyp19a1b在脑中表达量降低;腹腔注射hCG可以促进cyp19a1b在脑中表达。研究表明,淇河鲫cyp19a1b基因可能通过参与神经系统的形成及神经内分泌活动,在性别决定与分化过程中起到一定的作用。     

Characterization of the isoforms of type IIb sodium-dependent phosphate cotransporter (Slc34a2) in yellow catfish, <Emphasis Type="Italic">Pelteobagrus fulvidraco</Emphasis>, and their vitamin D<Subscript>3</Subscript>-regulated expression under low-phosphate conditions

In this study, two isoforms slc34a2 genes (type IIb sodium-dependent phosphate cotransporter), slc34a2a2 and slc34a2b, were cloned from intestine and kidney of yellow catfish (Pelteobagrus fulvidraco), with rapid amplification of cDNA ends. The structure differences and the regulation effects of dietary VD₃ under low phosphorus were compared among three isoforms of slc34a2 in yellow catfish. The predicted Slc34a2a2 and Slc34a2b proteins match 65 % and 53.8 % sequence identity, with Slc34a2a1, respectively. The membrane-spanning domains were different among these three isoforms. Intestinal Slc34a2a1 and Slc34a2a2 proteins had eight and eleven transmembrane domains, while renal Slc34a2b protein had nine. The tissue distribution study showed that same as slc34a2a1, slc34a2a2 mRNA was mainly distributed in intestine and slc34a2b mRNA in kidney. The effect of vitamin D₃ (VD₃) level on slc34a2 subfamily expression under low-phosphate conditions, induced by the addition of 0 (VD0), 324 (VD1), 1243 (VD2), 3621 (VD3), 8040 (VD4), or 22700 (VD5) IU VD₃/kg feed, was assessed by qPCR. The dose-responsive expression of intestinal slc34a2a2 and high expression of intestinal slc34a2a2 in VD5 together with peak expression of kidney slc34a2b in VD3 coincided with the accumulation of body phosphate content. These data suggested that appropriate level of dietary VD₃ up-regulated slc34a2a1, slc34a2a2, and slc34a2b mRNA levels, which increased phosphate retention. In conclusion, the current study provided another possible approach to improve dietary phosphate utilization by adding appropriate level of VD₃ to a low-phosphate diet to regulate intestinal and renal slc34a2 gene expression and thus minimize the excretion of phosphorus in yellow catfish.