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.     

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。     

朱砂叶螨热激蛋白HSP70基因TCHSP70-4的克隆与表达

为了研究朱砂叶螨Tetranychus cinnabarinus(Boisduval)热激蛋白HSP70的表达与其适应高温和低温胁迫的关系,我们利用物种的同源性及RACE 技术,获得朱砂叶螨热激蛋白HSP70基因1个,命名为TCHSP70-4（GenBank 登录号为EU977182）。该基因全长2 182 bp,包含1 959 bp的开放阅读框,编码653个氨基酸,理论分子量为70.9 kDa,等电点为5.4,含有HSP70家族高度保守的基序。运用real-time PCR分析冷激（4℃）和热激（40℃）1 h后TCHSP70-4在朱砂叶螨体内的表达量。结果显示冷激后TCHSP70-4表达量明显下降,而热激后TCHSP70-4表达量却明显上升。这些结果一方面表明该基因属于诱导型HSP70基因,另一方面揭示了朱砂叶螨分别受到冷和热胁迫后体内TCHSP70-4的表达及所起的保护作用是不同的。     

二化螟热休克蛋白70基因的克隆及热胁迫下的表达分析

热休克蛋白70是已知热休克蛋白家族中最重要的一种, 它在细胞内的大量表达可以明显改善细胞的生存能力, 提高对环境胁迫的耐受性。为探讨热胁迫对二化螟Chilo suppressalis幼虫热休克蛋白70表达的影响, 采用RT-PCR及RACE技术从二化螟血淋巴细胞中克隆了热休克蛋白70基因全长cDNA序列。该基因全长2 102 bp, 开放阅读框 (open reading frame, ORF)为1 959 bp, 编码652个氨基酸; 5′非编码区（untranslated region, UTR)为81 bp, 3′UTR为62 bp。从该基因推导的氨基酸序列与其他昆虫的同源序列比较有很高的相似性（73%~97%)。实时定量PCR显示二化螟HSP70基因能被热胁迫诱导表达, 幼虫血淋巴细胞的HSP70基因在36℃时表达量最高。流式细胞术研究发现HSP70在蛋白质水平上的表达变化与在mRNA水平上高度一致, 说明二化螟HSP70基因在转录及翻译水平上受到热应激的调节。     

Characterization and SNP variation analysis of a HSP70 gene from miiuy croaker and its expression as related to bacterial challenge and heat shock

Heat shock proteins (HSPs) play crucial roles in the immune response of vertebrates. In order to study immune defense mechanism of heat shock protein gene in miiuy croaker (Miichthys miiuy), a cDNA encoding heat shock protein 70 (designated Mimi-HSP70) gene was cloned from miiuy croaker. The cDNA was 2195?bp in length, consisting of an open reading frame (ORF) of 1917?bp encoding a polypeptide of 638 amino acids with estimated molecular mass of 70.3?kDa and theoretical isoelectric point of 5.55. Genomic DNA structure analysis revealed that the Mimi-HSP70 gene contain no introns in coding region and four SNPs with 373?C/T, 789?G/A, 1005?C/T, and 1185?G/A were detected by direct sequencing of 20 samples from six different populations. BLAST analysis, structure comparison and phylogenetic analysis indicated that Mimi-HSP70 should be an inducible cytosolic member of the HSP70 family. The deduced amino acid sequence of Mimi-HSP70 had 82.4%-92.2% identity with those of vertebrate. A real-time quantitative RT-PCR demonstrated that the HSP70 gene was ubiquitously expressed in ten normal tissues. Under different temperature shock stress, the expression of Mimi-HSP70 gene in miiuy croaker increased at first and then decreased with the rise of temperature, finally, reached a maximum level in liver, spleen and kidney tissues. Infection of miiuy croaker with Vibrio anguillarum resulted in significant changes expression of Mimi-HSP70 gene in the immune-related tissues. These results indicated that expression analysis of Mimi-HSP70 gene provide theoretical basis to further study the mechanism of anti-adverseness in the miiuy croaker.     

Expression of the Tribolium castaneum （Coleoptera： Tenebrionidae） hsp83 gene and its relation to oogenesis during ovarian maturation

Heat shock proteins (HSP) can protect organisms and cells from thermal damage. In this study, we cloned the full length cDNA encoding the HSP83 protein (the homologue of HSP90) of Tribolium castaneum (red flour beetle). The isolated cDNA contains the full coding sequence, a partial 5′ untranslated region of 55 bp and the complete 3′ untranslated region. We found the hsp83 gene is located on chromosome 5 of the T. castaneum genome. The predicted HSP83 protein sequence has a high similarity (on average 86.77%) with that of other insect species. The expression of the hsp83 gene in the whole body and in the ovary could be induced with heat stress (40°C for 1 h) in newly hatched (within 3 h post emergence) and mature (10 days post emergence) beetles. Under normal conditions, the hsp83 expression in the ovary is about 3-fold higher than in the whole body at both stages. No significant difference in hsp83 expression was observed between the two ovarian developmental stages regardless if the beetles were treated with heat shock or not. The expression of the HSP83 protein in the whole body could also be induced with heat stress in newly hatched and mature beetles. However, in the ovary, HSP83 was only expressed in the follicle cells of mature beetles and not in newly hatched beetles, regardless if the beetles were treated with heat shock or not. Furthermore, the females were not able to produce mature oocytes after knock-down of the hsp83 expression by injecting dsRNA. These results suggest that the HSP83 protein is involved in protection against heat stress and could be involved in oogenesis during ovarian maturation of T. castaneum.     

Molecular cloning and expression of two HSP70 genes in the Wuchang bream (Megalobrama amblycephala Yih)

Two complementary deoxyribonucleic acid (cDNA) clones encoding heat shock cognate 70 (HSC70) and inducible heat shock protein 70 (HSP70) were isolated from the liver of Wuchang bream (Megalobrama amblycephala Y.) using RT-PCR and rapid amplification of cDNA ends (RACE). They were named Ma-HSC70 and Ma-HSP70, respectively. The cDNAs were 2336 and 2224 bp in length [not including poly (A)] and contained 1950 and 1932 bp open reading frames (ORFs), respectively. The ORFs encoded proteins of 649 and 643 amino acids with predicted molecular weights of 71.24 and 70.52 kDa, and theoretical isoelectric points of 5.25 and 5.30, respectively. Genomic DNA structure analysis revealed that Ma-HSC70 gene contained seven introns with all introns conforming to the GT/AG rule whereas Ma-HSP70 gene did not contain any intron in the coding region. Amino acid sequence analysis indicated that both Ma-HSC70 and Ma-HSP70 contained three signature sequences of HSP70 family, two partial overlapping bipartite nuclear localization signal sequences (NLS) and cytoplasmic characteristic motif (EEVD). Homology analysis revealed that Ma-HSC70 shared more than 93.0% identity with the known HSC70s of other vertebrates, while Ma-HSP70 shared more than 85.0% identity with the known HSP70s of other vertebrates, and Ma-HSC70 and Ma-HSP70 shared 86.5% identity. Bioinformatics analysis indicated that the proteins encoded by Ma-HSC70 and Ma-HSP70 genes were hydrophilic, rich in B cells antigenic sites, without any signal peptide or transmembrane region. The two proteins also contained many protein kinase C phosphorylation sites, N-myristoylation sites, casein kinase II phosphorylation sites, and N-glycosylation sites, predicting that they could play essential roles in protein folding, translocation, intracellular localization, signal transduction and regulation. The predominant secondary structures of the two proteins were α-helix and random coil. Fluorescent real-time quantitative RT-PCR was used to study the effects of heat shock (34 °C), crowding stress (100 g L^?1) and challenge with bacteria Aeromonas hydrophila on the mRNA expression of the two HSP70s in Wuchang bream liver. The results indicated that, during 24 h stress, Ma-HSC70 mRNA expression decreased at first and then rose to the level before stress under heat shock and crowding stress, but Ma-HSP70 mRNA expression increased at first and then decreased under heat stress, and appeared to increase continuously under crowding stress. After bacterial challenge, the mRNA levels of both Ma-HSC70 and Ma-HSP70 increased at first and then decreased. The cloning and expression analysis of the two HSP70s provide theoretical basis to further study the mechanism of anti-adverseness and expression characteristics under stress conditions of Wuchang bream.     

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)。     

Cloning and expression analysis of hsp70 gene from freshwater planarian Dugesia japonica

Heat shock protein 70 (HSP70) is an important member of the heat shock protein family. It plays a key role in the process of protecting cells by facilitating the folding of nascent peptides as well as the cellular stress response. We isolated and sequenced a full-length HSP70 cDNA from planarian Dugesia japonica (designated Djhsp70) using rapid amplification of cDNA ends (RACE). Djhsp70 cDNA is highly homologous to other reported hsp70 family genes, and the deduced amino acid sequence shares several eukaryotic HSP70 family motifs. The length of the Djhsp70 ORF from planarian genomic DNA and cDNA was identical, which indicated the absence of introns in the Djhsp70 gene. Semi-quantitative RT-PCR was employed to detect the expression levels of Djhsp70 in response to stressors. Our results indicated that Djhsp70 was an inducible gene, expressed in response to temperature changes, amputation and starvation. Interestingly, the phylogenetic analysis of DjHSP70 supports the idea that planarians belong to a new phylogenetic position — Lophotrochozoans. This is the first molecular analysis of a heat shock protein gene in planarians.     

大螟HSC70基因克隆及表达模式分析

【目的】近年来,大螟Sesamia inferens (Walker)对水稻的为害逐渐加重并成为水稻的重要害虫之一。随着全球气候变暖,大螟的分布区域也在逐渐向北延伸。HSP70家族作为分子伴侣参与生物生长发育并对外界刺激产生响应,对生物功能蛋白质的正确折叠及其转运有着重要意义。本研究旨在明确HSP70家族HSC70基因在大螟不同组织、不同发育阶段以及低温胁迫下的表达差异,初步探讨大螟对环境适应的分子机理。【方法】应用RT-PCR及RACE技术从大螟5龄幼虫中克隆得到HSC70基因;进行基因组验证,得到其基因组序列,分析内含子的位置及大小;应用实时定量PCR技术分析大螟HSC70基因的表达模式。【结果】大螟HSC70基因长2 160 bp,命名为Sihsc70（GenBank登录号：KJ639908）,开放阅读框长1 962 bp,编码653个氨基酸,推测分子量为71.6 kDa。其氨基酸序列中含有3个HSP70家族保守序列,在C-末端存在细胞质定位信号,说明大螟HSC70是细胞质热激蛋白家族成员。大螟HSC70基因组序列长度为3 522 bp（GenBank登录号：KJ639909）,含有2个内含子,长度分别为685 bp（位于编码区上游）和803 bp（位于编码区内）。在大螟5龄幼虫的不同组织中Sihsc70表达量差异不显著（P＞0.05）,其中在中肠、后肠和体壁中的表达量较高,在唾腺中的表达量最低;在大螟不同发育阶段中,Sihsc70的表达量在雌成虫最低,较高的3个阶段依次为卵、2龄幼虫和5龄幼虫,分别为雌成虫表达量的6.33,3.21和1.86倍;相对于对照组（27℃）,低温胁迫对大螟5龄幼虫HSC70基因表达的影响差异不显著（P＞0.05）。【结论】结果说明,大螟HSC70基因在不同发育阶段和幼虫不同组织中具有不同的表达水平,而低温胁迫不能诱导该基因大量表达。     

Seasonal variation in expression pattern of genes under HSP70

Heat shock protein 70 (HSP70) is one of the most abundant and best characterized heat shock protein family that consists of highly conserved stress proteins, expressed in response to stress, and plays crucial roles in environmental stress tolerance and adaptation. The present study was conducted to identify major types of genes under the HSP70 family and to quantify their expression pattern in heat- and cold-adapted Indian goats (Capra hircus) with respect to different seasons. Five HSP70 gene homologues to HSPA8, HSPA6, HSPA1A, HSPA1L, and HSPA2 were identified by gene-specific primers. The cDNA sequences showed high similarity to other mammals, and proteins have an estimated molecular weight of around 70 kDa. The expression of HSP70 genes was observed during summer and winter. During summer, the higher expression of HSPA8, HSPA6, and HSPA1A was observed, whereas the expression levels of HSPA1L and HSPA2 were found to be lower. It was also observed that the expression of HSPA1A and HSPA8 was higher during winter in both heat- and cold-adapted goats but downregulates in case of other HSPs. Therefore, both heat and cold stress induced the overexpression of HSP70 genes. An interesting finding that emerged from the study is the higher expression of HSP70 genes in cold-adapted goats during summer and in heat-adapted goats during winter. Altogether, the results indicate that the expression pattern of HSP70 genes is species- and breed-specific, most likely due to variations in thermal tolerance and adaptation to different climatic conditions.     

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.