Cloning and expression of a heat shock protein (HSP) 90 gene in the haemocytes of Crassostrea hongkongensis under osmotic stress and bacterial challenge

Heat shock protein 90 (HSP90) is a highly conserved and multi-functional molecular chaperone that plays an essential role in both cellular metabolism and stress response. Here, we report the cloning of the HSP90 homologue in Crassostrea hongkongensis (ChHSP90) through SSH in combination with RACE from cDNA of haemocytes. The full-length cDNA of ChHSP90 is 2459 bp in length, consisting of a 3', 5'-untranslated region (UTR) and an open reading frame of 2169 bp encoding 722 amino acids. The identity analysis of the amino acid sequence of HSP90 revealed that ChHSP90 is highly conserved. Distribution of ChHSP90 mRNA in gonad, heart, adductor muscle, mantle, gill, digestive gland, and haemocytes suggested that ChHSP90 is ubiquitously expressed. The mRNA levels of ChHSP90 under salinity and bacterial challenges were analyzed by real-time PCR. Under hypo-osmotic treatment, ChHSP90 mRNA in gonad, heart and haemocytes were significantly up-regulated on day 2 and onwards; while in gill, digestive gland and adductor muscle it was significantly down-regulated; the expression in mantle was decreased significantly on day 2 and 3 (P < 0.01), and then up-regulated on day 4 (P < 0.05). Under hyper-osmotic treatment, the mRNA level in gonad, heart, adductor muscle was increased on day 2 and onwards; in gill, it was firstly increased, and then gradually decreased, reaching a minimum on day 3. On day 4, the expression level in gill recovered to pre-treatment level; in mantle and digestive gland, the expression levels were decreased, reaching to the minimum on day 3. During Vibrio alginolyticus challenge, the mRNA level of ChHSP90 increased 3-fold at 4 h post-infection, returned to its pre-challenge level at 6 h post-infection, then was further up-regulated from 8 to 36 h post-infection. These experiments demonstrate that ChHSP90 mRNA is constitutively expressed in various tissues and apparently inducible in haemocytes under salinity and bacterial challenges, suggesting its important role in response to both osmotic stress and bacterial invasion.     

Cloning and expression analysis of a HSP70 gene from Pacific abalone (Haliotis discus hannai)

Heat shock protein 70 (HSP70), the primary member of HSPs that are responsive of thermal stress, is found in all multicellular organisms and functions mostly as molecular chaperon. The inducible HSP70 cDNA cloned from Pacific abalone (Haliotis discus hannai) using rapid amplification of cDNA ends (RACE), was highly homologous to other HSP70 genes. The full-length cDNA of the Pacific abalone HSP70 was 2631bp, consisting of a 5'-terminal untranslated region (UTR) of 90bp, a 3'-terminal UTR of 573bp with a canonical polyadenylation signal sequence AATAAA and a poly (A) tail, and an open reading frame of 1968bp. The HSP70 cDNA encoded a polypeptide of 655 amino acids with an ATPase domain of 382 amino acids, the substrate peptide binding domain of 161 amino acids and a C-terminus domain of 112 amino acids. The temporal expression of HSP70 was measured by semi-quantitative RT-PCR after heat shock and bacterial challenge. Challenge of Pacific abalone with heat shock or the pathogenic bacteria Vibrio anguillarum resulted in a dramatic increase in the expression of HSP70 mRNA level in muscle, followed by a recovery to normal level after 96h. Unlike the muscle, the levels of HSP70 expression in gills reached the top at 12h and maintained a relatively high level compared with the control after thermal and bacterial challenge. The upregulated mRNA expression of HSP70 in the abalone following heat shock and infection response indicates that the HSP70 gene is inducible and involved in immune response.     

三角帆蚌热休克蛋白70基因克隆及其表达分析

对三角帆蚌HSP70基因序列进行全长克隆及其分子生物学分析,并检测其在不同水温刺激下鳃组织中的表达变化。通过高通量转录组测序获得三角帆蚌HSP70基因（HcHSP70）长片段,采用3'RACE对其进行了3'末端克隆,经拼接得到HcHSP70 cDNA全长序列。采用多种分子生物学软件对HcHSP70 cDNA全长序列进行了特征分析,采用实时荧光定量PCR技术检测了其组织分布,并结合Western-blot技术检测蚌鳃中该基因mRNA与蛋白经不同水温刺激后的表达变化。结果显示,HcHSP70 cDNA全长为2298 bp,其中开放阅读框为1974 bp,编码657个氨基酸。预测分子量大小为71.6 Ku,pH7.0时的理论等电点为5.61。氨基酸序列分析表明,HcHSP70氨基酸序列含HSP70家族的3个标签序列（I₉DLGTTYS₁₆、I₁₉₇FDLGGGTFDVSIL₂₁₀和I₃₃₆ VLVGGSTRIPKVQK₃₅₀）,与长牡蛎及泥蚶的HSP70同源性最高（91%）。实时荧光定量PCR检测结果显示,HcHSP70在鳃、性腺、肝胰腺、外套膜及肌肉等5种被检组织中均有表达,以肝胰腺中的表达水平最高。实时荧光定量PCR与Western-blot技术检测皆表明,蚌鳃组织中HcHSP70基因与蛋白的表达量在37℃时达到最高,而在40℃水温刺激下表达水平下调至正常值,表明其在适应高温刺激时发挥了重要作用。     

受孔雀石绿胁迫的近江牡蛎HSP90基因的表达变化

HSP90是重要抗逆分子,为了探讨近江牡蛎Crassostrea hongkongensis HSP90分子在抗逆中的作用,本研究建立实时荧光定量PCR方法,研究了在孔雀石绿胁迫下近江牡蛎HSP90基因在外套膜、消化腺、鳃、闭壳肌4种器官组织中的表达变化规律.结果显示,在浓度1μg/L的孔雀石绿处理下,4种器官组织中的...     

Cadmium affects the expression of heat shock protein 90 and metallothionein mRNA in the Pacific oyster, Crassostrea gigas

Cadmium (Cd) is a widespread nonessential heavy metal that enters the aquatic environment as a result of natural processes and human activities such as wastewater production, agriculture, and mining. To determine the effects of Cd on organisms, we investigated its time- and dose-related effects on mRNA levels of heat shock protein 90 (HSP90) and metallothionein (MT) in the gill and digestive gland and changes enzyme levels in the hemolymph of the Pacific oyster Crassostrea gigas. Full-length HSP90 cDNA was isolated from C. gigas by rapid amplification of cDNA end (RACE) techniques and found to contain 2154 nucleotides, including an open reading frame, and was predicted to encode a protein of 717 amino acids. BLAST analysis indicated that the HSP90 gene of C. gigas shared high homology with known HSP90 genes of other mollusks. The expression of HSP90 mRNA increased significantly with exposure to 0.01 ppm Cd for 11 days or 0.05 or 0.1 ppm Cd for 7 days. The expression of MT mRNA increased significantly with exposure to 0.01, 0.05, or 0.1 ppm Cd for 11 days. Glutamate oxaloacetate and glutamate pyruvate levels increased significantly with exposure to 0.05 or 0.1 ppm Cd for 7 days. These results indicate that HSP90 and MT play important roles in the physiological changes related to metabolism and cell protection that occur in Pacific oysters exposed to Cd.     

黄颡鱼HSC70基因及其组织表达分析

热休克蛋白70（HSP70）与生物体的抗胁迫能力密切相关。本文采用RACE (Rapid amplification of cDNA ends) 技术,从黄颡鱼Pelteobagrus fulvidraco克隆到一种组成型热休克蛋白（HSC70）基因及其cDNA。该cDNA全长2245bp,包括5′非编码区82bp,3′非编码区225bp,开放阅读框(ORF) 1938bp,编码645个氨基酸组成的蛋白质。黄颡鱼HSC70基因含有8个内含子,与人、鼠、虹鳟和花斑溪鳉的HSC70基因内含子数目相同,位置相似。其中,最长内含子（873bp）位于5′端非编码区,其余内含子（长度在80－251bp之间不等）均在编码区以内。黄颡鱼HSC70基因编码的氨基酸序列与南方鲶的相似度最高,达96.13%,与欧洲银鲫和团头鲂的相似度分别为94.45%和94.14%。RT-PCR检测显示,正常情况下黄颡鱼HSC70在血细胞、心脏、肝、头肾、脾、鳃、肌肉和脑中均有表达,但表达量在鳃中最高,肌肉中最低;统计结果显示,热激后HSC70在血细胞、肝、头肾和脑中的表达量显著上升(p<0.05),而在其余组织中热激前后的表达差异不显著(p>0.05)。     

The cDNA cloning and mRNA expression of heat shock protein 70 gene in the haemocytes of bay scallop (Argopecten irradians, Lamarck 1819) responding to bacteria challenge and naphthalin stress

Heat shock protein 70 (HSP70) is an important member of the heat shock protein superfamily, and it plays a key role in the process of protecting cells, facilitating the folding of nascent peptides and responding to stress. The cDNA of bay scallop Argopecten irradians HSP70 (designated AIHSP70) was cloned by the techniques of homological cloning and rapid amplification of cDNA end (RACE). The full length of AIHSP70 cDNA was 2651bp in length, having a 5' untranslated region (UTR) of 96bp, a 3' UTR of 575bp, and an open reading frame (ORF) of 1980bp encoding a polypeptide of 659 amino acids with an estimated molecular mass of 71.80kDa and an estimated isoelectric point of 5.26. BLAST analysis revealed that the AIHSP70 gene shared high identity with other known HSP70 genes. Three classical HSP signature motifs were detected in AIHSP70 by InterPro analysis. 3-D structural prediction of AIHSP70 showed that its N terminal ATPase activity domain and C terminal substrate-binding domain shared high similarity with that in human heat shock protein 70. The results indicated that the AIHSP70 was a member of the heat shock protein 70 family. A semi-quantitive RT-PCR method was used to analyse the expression of AIHSP70 gene after the treatment of naphthalin which is one kind of polycyclic aromatic hydrocarbon (PAH) and the challenge of bacteria. mRNA expression of AIHSP70 in scallop was up-regulated significantly after the stimulation of naphthalin and increased with increasing naphthalin concentration. A clearly time-dependent expression pattern of AIHSP70 was observed after the scallops were infected by Vibrio anguillarum, and the mRNA expression reached a maximum level at 8h and lasted to 16h, and then dropped progressively. The results indicated that AIHSP70 could play an important role in mediating the environmental stress and immune response in scallop.     

克氏原螯虾一种诱导型HSP70基因克隆及分析

克氏原螯虾是我国淡水虾类养殖的重要品种,具有很强的抵御各种环境胁迫和各种刺激的能力。本文以该虾为对象,通过基因克隆以及从基因水平探讨HSP70s与环境应激之间的关系,为深入研究水生无脊椎动物HSP70s功能提供基础。采用RT-PCR和RACE方法从克氏原螯虾（Procambarus clarkii）心脏组织中克隆得到一种HSP70 cDNA（scHSP70）,其全长为2271bp,包括1902bp的完整编码序列、142bp的5′及221bp的3′端非翻译区, GenBank登陆号DQ301506。基因组DNA扩增表明该基因仅由一个外显子组成。根据cDNA序列推导出scHSP70由635个氨基酸组成,分子量为69.6kD,理论等电点为5.34。该序列存在真核细胞HSP70家族的三个特征标签。SWISS-MODEL蛋白三维结构预测显示scHSP70在N端形成ATP酶结构域,在近C端形成底物肽结合结构域。克氏原螯虾在系统发生树上的进化地位与传统分类学相一致。半定量RT-PCR实验表明,scHSP70有广泛的组织分布,在心脏中表达量最高,在血液中最少。热激后该基因大量表达,说明该基因是一种诱导型HSP70。这为从蛋白水平研究克氏原螯虾HSP70与环境应激之间的关系提供基础。     

近江牡蛎HSP70基因对溶藻弧菌感染的反应

采用实时荧光定量RT-PCR方法,检测了注射溶藻弧菌(Vibiro alginolyticus)后近江牡蛎鳃,闭壳肌,消化腺,外套膜,心脏以及血细胞中HSP70基因的表达变化。结果显示近江牡蛎这五种器官组织中的HSP70基因表达量均出现显著性高表达,且在鳃、外套膜和血细胞中的HSP70基因表达变化规律表现为典型的时间依赖性。血细胞中,显著高表达的峰值出现在24h,至72h恢复到对照水平,高表达持续时间最长:鳃中表达峰值出现时间较早,在第3h,随后在第12h便恢复到对照水平;外套膜,消化腺以及心脏中的峰值分别出现在6h,6h和3h,而在闭壳肌组织中,没出现显著性高表达。由此可见,近江牡蛎HSP70s可能在机体抗菌免疫过程中起了重要作用。     

Reduction of HSP70 and HSC70 Heat Shock mRNA Induction by Pentobarbital After Transient Global Ischemia in Gerbil Brain

Abstract: The effect of pentobarbital on the induction of heat shock protein (HSP) 70 and heat shock cognate protein (HSC) 70 mRNAs after transient global ischemia in gerbil brains was investigated by in situ hybridization using cloned cDNA probes selective for each mRNA species. In sham control brains, HSP70 mRNA was scarcely present, whereas HSC70 mRNA was present in most cell populations. After a 5-min occlusion of bilateral common carotid arteries, HSP70 and HSC70 mRNAs were induced together in several cells and were especially dense in hippocampal dentate granule cells at 3 h, but the strong hybridization of the mRNAs continued only in hippocampal CA1 cells by 2 days. At 7 days after the ischemia, CA1 neuronal cell death was apparent, and the HSP70 mRNA disappeared and HSC70 mRNA content returned to the sham level, except for in the CA1 cells. Pretreatment with pentobarbital (40 mg/kg, i.p.) greatly reduced or inhibited the induction of HSP70 and HSC70 mRNAs at both early (3-h) and late (2-day) phases after ischemia. The drug also prevented CA1 cell death at 7 days along with the maintenance of expression of HSC70 mRNA at the sham control level. Hypothermic effects of pentobarbital were noted at 30 and 60 min after the reperfusion, whereas at 2 h there was no statistical significance between the control and drug-treated groups. The great reduction of HSP70 and HSC70 mRNA induction at both early and late phases after ischemia suggests that pentobarbital reduces intra- and/or postischemic stress and may protect CA1 cells from ischemic damage. These effects of the drug may be mainly due to its specific action rather than its hypothermic effects.     

大菱鲆组成型热休克蛋白70的全长cDNA克隆及表达分析

根据感染哈维氏弧菌(Vibrio harveyi)的大菱鲆(Scophthalmus maximus)的差减cDNA文库中hsp70EST序列设计引物,用RACE方法首次克隆到长2188bp的大菱鲆HSC70(Heat-shock cognate protein 70)全长cDNA序列,包括1956bp的开放阅读框及5′和3′非翻译区。在预测的651个氨基酸序列中发现了Dnak特征性基序,胞质HSP70特征基序以及四肽简并重复序列。与其他真核生物HSP70家族成员进行同源性比较,发现大菱鲆HSC70与牙鲆(Paralichthys olivaceus)HSC71、虹鳟(Oncorhynchus mykiss)HSC71、人(Homo sapiens)HSC70、家鼠(Mus musculus)HSC70、烟草天蛾(Manduca sexta)HSC70的氨基酸相似性分别是97%,95%,94%,93%,86%,表现出较高的保守性。表达分析显示,hsc70mRNA在大菱鲆正常肝脏、鳃、肠、脾脏、头肾、肾等组织中以不同的水平存在,呈组成型表达;被哈维氏弧菌感染后,大菱鲆肝脏和脾脏组织hsc70mRNA表达水平分别在24h和12h出现上调(2.5-fold和1.6-fold);注射生理盐水组与未注射组之间差异不显著。       

Molecular characterization and expression analysis of lipopolysaccharide and β-1,3-glucan-binding protein (LGBP) from pearl oyster <Emphasis Type="Italic">Pinctada fucata</Emphasis>

The lipopolysaccharide and β-1,3-glucan-binding protein (LGBP) plays an important function in the innate immune response of invertebrates as a pattern recognition receptor (PRR). Herein, we described the isolation and characterization of pearl oyster Pinctada fucata LGBP (designated as poLGBP). The poLGBP cDNA was 2,075 bp long and consisted of a 5′-untranslated region (UTR) of 18 bp, a 3′-UTR of 299 bp with one cytokine RNA instability motifs (ATTTA), and an open reading frame (ORF) of 1,758 bp encoding a polypeptide of 585 amino acids with an estimated molecular mass of 65.1 kDa and a theoretical isoelectric point of 5.80. Homology analysis of the deduced amino acid sequence of the poLGBP with other known LGBP sequences by MatGAT software revealed that the poLGBP shared 26.3–56.7% identity and 40.5–70.9% similarity to the other known LGBP sequences. SMART and alignment analysis revealed that the poLGBP possessed a potential polysaccharide-binding motif, a glucanase motif, a LPS-binding site, a β-1,3-linkage of polysaccharide, a glycine-rich region, a threonine-rich region and two N-glycosylation sites. In healthy pearl oyster, the poLGBP mRNA was specifically expressed in digestive gland, and not detected in gill, adductor muscle, gonad, intestine, mantle and hemocytes. However, after bacteria stimulation, the expression of the poLGBP mRNA was significantly up-regulated in digestive gland and also weakly detected in haemocytes, gonad and intestine. After LPS stimulation, the poLGBP mRNA expression was significantly up-regulated at 8 and 12 h in digestive gland, and the expression level was 10.7-fold higher than the PBS group at 12 h. After bacteria stimulation, the expression level of the poLGBP mRNA was also significantly up-regulated in digestive gland and was 12.9-fold higher than the PBS group at 8 h. However, during the experiment, the poLGBP mRNA expression was not detected in gill after LPS or bacteria stimulation. The tissue-specific expression and the expression up-regulation after LPS or bacteria stimulation in digestive gland suggested that the poLGBP was an inducible acute-phase protein and might play an important function in digestion as digestive enzyme and pattern recognition receptor.     

双齿围沙蚕诱导型热休克蛋白70基因的克隆及Cu胁迫下的表达分析

本研究主要评估了双齿围沙蚕热休克蛋白70(HSP70)基因的分子特征,记录了其对于液态Cu²⁺胁迫的基因表达情况,并通过测序获得的HSP70 cDNA序列与其他沙蚕及无脊椎动物HSP70同源性比对来判定蛋白特性.结果表明: 该HSP70基因全长cDNA序列共2161 bp,包括5′非翻译区48 bp,3′非翻译区142 bp,一个多聚腺苷酸信号序列(AATAAA)和Poly A尾巴以及开放阅读框1971 bp.阅读框共编码656个氨基酸,总分子量为71.43 kD,理论等电点为5.15.该氨基酸序列中含有HSP70家族的3个签名序列——IDLGTTYS、IFDLGGGTFDVSIL和IVLVGGSTRIPKIQK,以及细胞质特异性调控基序EEVD,C端重复序列GGMP.同源性分析表明,本研究所获双齿围沙蚕HSP70氨基酸序列与已报道的序列相似性高达94%,与其他无脊椎生物的HSP70相似性也高达79%以上.荧光实时定量PCR分析表明,Cu²⁺(0.2～5.0 mg·L^-1)胁迫能够显著诱导沙蚕HSP70 mRNA表达,并于1 d后达到峰值.本研究系统描述了双齿围沙蚕HSP70的分子特性,其可被液态Cu²⁺诱导表达,具备作为环境污染分子生物标记物的潜力.     

Identification of a novel cognate cytosolic Hsp70 gene (MnHsc70-2) from oriental river prawn Macrobrachium nipponense and comparison of its expressions with the first cognate Hsc70 (MnHsc70-1) under different stresses

The 70-kDa family of heat-shock proteins (Hsp70) plays an important role in the host immunity, which is widely expressed in eukaryotic cells as a major chaperone protein. In the present study, the full-length complementary DNA (cDNA) of a second cognate cytosolic Hsp70 family member (MnHsc70-2) was cloned and characterized from Macrobrachium nipponense, which is an economically and nutritionally important crustacean. The cDNA was 2,717 bp, containing an open reading frame (ORF) of 1,950 bp, which encodes a protein of 649 amino acids with a theoretical molecular weight of 71.1 kDa and an isoelectric point of 5.27. Sequence alignment showed that the MnHsc70-2 shared 75–97 % identity with other heat-shock proteins. Compared to the previously identified cognate Hsp70 (MnHsc70-1) in M. nipponense, MnHsc70-2 showed quite different expression profiles under unstressed conditions in all tested tissues, including the hemocytes, heart, hepatopancreas, gill, intestine, nerve, and muscle. The phylogenetic analysis demonstrated that MnHsc70-2 showed the closest relationship with MnHsc70-1. Heat-inducibility assays showed that two isolated messenger RNAs (mRNAs) displayed different expression profiles in both the hepatopancreas and gill tissues. MnHsc70-1 mRNA expression level decreased at first and then increased to the normal level, whereas MnHsc70-2 mRNA level increased at first and then decreased. The expressions of two MnHsc70s showed substantial obvious heat-inducible regulation in both the hepatopancreas and gill. Under bacterial challenge by Aeromonas hydrophila, both MnHsc70-1 and MnHsc70-2 mRNA level was up-regulated moderately. The results suggested that two cognate Hsc70s may play essential functions in mediating responses to heat-shock and bacterial challenge.     

A comparison of heat shock protein genes from cultured cells of the cabbage armyworm, Mamestra brassicae, in response to heavy metals

Heat shock protein (HSP) genes, hsp90, hsp70, hsc70, hsp20.7, and hsp19.7, were cloned and sequenced from cultured cells of the cabbage armyworm, Mamestra brassicae. Analyses of the cDNA sequences revealed open reading frames of 2,151, 1,914, 1,962, 540, and 465 bp in lengths, which encode proteins with calculated molecular weights of 82.5, 69.9, 71.6, 20.7, and 19.7 kDa, respectively. An increased expression was observed in all five genes after exposure to a high temperature. The induction of gene expression was not observed during a low temperature exposure, but was observed when the cells were recovered at ambient temperature. Expression of hsp90, hsp70, and hsp20.7 was induced after exposure to 2 microM of cadmium, while the minimum cadmium concentration for induction of hsp19.7 was 5 microM. The induction of hsp90 expression was somewhat masked by basal levels of expression. Only hsp20.7 expression was induced by exposure to copper. Lead did not induce expression of any of the HSP genes tested. Cadmium-induced up-regulation of hsp70 expression was lasted longer than heat-induced one. These results suggest that hsp70 could be useful to assess the cellular distress or injury induced by cadmium.