Molecular cloning, mRNA expression, and characterization of heat shock protein 10 gene from humphead snapper Lutjanus sanguineus

Heat shock protein 10 (HSP10) gene of humphead snapper (Lutjanus sanguineus), designated as ByHSP10, was cloned by rapid amplification of cDNA ends (RACE) techniques with the primers designed from the known EST sequence identified from the subtracted cDNA library of the head kidney of humphead snapper. Sequence analysis showed the full length cDNA of ByHSP10 was 529 bp, containing a 5′ terminal untranslated region (UTR) of 51 bp, a 3′ terminal UTR of 181 bp, and an open reading frame (ORF) of 297 bp encoding a polypeptide of 99 amino acids. Based on the deduced amino acid sequence, the theoretical molecular mass of ByHSP10 was calculated to be 10.92 kDa with an isoelectric point of 9.46. Moreover, chaperonins hsp10/cpn10 signature was found in the amino acids sequence of ByHSP10 by PredictProtein. BLAST analysis revealed that the amino acids of ByHSP10 had the highest homology of 88% compared with other HSP10s. Fluorescent real-time quantitative RT-PCR was used to examine the expression of ByHSP10 gene in eight kinds of tissues of humphead snapper after the challenge with Vibrio harveyi. There was a clear time-dependent expression pattern of ByHSP10 in head kidney, spleen and thymus after bacteria challenge. The expression of mRNA reached the maximum level at the time point of 9 h, 6 h and 24 h, respectively and then returned to control level in 36 h. The up-regulated mRNA expression of ByHSP10 in humphead snapper after bacteria challenge indicated that the HSP10 gene was inducible and might be involved in immune response. A phylogenetic tree was constructed based on the ORF nucleotide sequences of HSP10 for 30 species. The relatonships among them were generally in agreement with the traditional taxonomy which suggested that HSP10 genes could aid in the system classification research.     

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.     

凡纳滨对虾MT基因序列及其在卵巢发育和低温胁迫中的表达分析

在低温处理仔虾全长cDNA文库的筛选测序中, 获得凡纳滨对虾(Litopenaeus vannmei)金属硫蛋白基因全长cDNA序列, 该序列含有425个碱基, 包含177 bp开放阅读框, 上游98 bp的非编码区及下游150 bp 的非编码区, 编码58个氨基酸, 其中半胱氨酸含量丰富, 富含金属硫蛋白典型的Cys-X(1-3)-Cys 结构。多序列比对表明, 凡纳滨对虾MT蛋白序列与美洲螯龙虾(Homarus americanus)MT蛋白序列具最高同源性72.4%。Real-time PCR结果表明, 凡纳滨对虾MT基因在卵巢组织中呈优势表达, 在不同发育期的卵巢中的表达量都很高, 在低温处理凡纳滨对虾肝胰腺组织中上调表达。实验所得结果为研究凡纳滨对虾金属硫蛋白基因在生殖发育和低温应激中的功能提供了参考。       

Cloning, characterization and expression analysis of a novel CXC chemokine from turbot (Scophthalmus maximus)

Chemokines represent a superfamily of chemotactic cytokines playing an important role in leucocyte chemotaxis. Here we report a novel turbot CXC chemokine screened from a turbot spleen cDNA library. The complete cDNA of the turbot CXC chemokine contains an 81bp 5' UTR, a 414bp open reading frame (ORF) encoding 137 amino acids and a 449bp 3' UTR. Four exons and three introns are identified in the turbot CXC chemokine gene. Phylogenetic analysis showed that the turbot CXC chemokine clustered apart from all other CXC chemokines. RT-PCR demonstrated that turbot CXC chemokine was expressed highly in spleen and head kidney. During the early stages of embryo development after fertilization, it appears that low expression level of turbot CXC chemokine was firstly observed at somites stage. Interestingly, the turbot chemokine was highly and rapidly (5h) induced in liver, spleen and head kidney of turbot after challenge with Vibrio anguillarum. Furthermore, the expression of CXC chemokine was also dramatically increased after challenge in turbot embryonic cells (TECs). These results indicated that the turbot CXC chemokine played an important role in turbot immune response.     

Molecular cloning of interleukin 1beta from rainbow trout Oncorhynchus mykiss reveals no evidence of an ice cut site.

The complete coding sequence of rainbow trout IL-1beta has been obtained. The gene contains a short 5' UTR (97 bp), a 780 bp open reading frame and a 466 bp 3' UTR, which includes a polyadenylation signal, 7 ATTTA motifs and an 18 bp poly A tail. The predicted amino acid sequence (260 amino acids) contains 3 potential glycosylation sites, with a predicted molecular weight of 29 kDa, and shows between 49 and 56% amino acid similarity to mammalian IL-1betas and 57% similarity to carp IL-1beta. Greatest homology was apparent within the secondary structure of the gene, with few of the amino acids known to bind to the IL-1 receptor being conserved. No ICE cut site was apparent but multiple alignment with mammalian sequences allowed a putative mature peptide of 166 amino acids to be identified, in which Ala(95)would be the amino terminus. Northern blot analysis showed that whilst no IL-1beta expression was detectable in head kidney leukocytes immediately after isolation, expression could be induced by stimulation with LPS for 4 h in culture. Similarly, with isolated head kidney macrophages expression was significantly increased following stimulation with LPS.     

Molecular cloning, characterization and expression of a novel serine proteinase inhibitor gene in bay scallops (Argopecten irradians, Lamarck 1819)

Serine protease inhibitors, critical regulators of endogenous proteases, are found in all multicellular organisms and play crucial roles in host physiological and immunological effector mechanisms. The first mollusk serine proteinase inhibitor (designated AISPI) cDNA was obtained from the bay scallop Argopecten irradians by randomly sequencing a whole tissue cDNA library and rapid amplification of cDNA ends (RACE). The full-length cDNA of the scallop serine protease inhibitor was 1020 bp, consisting of a 5'-terminal untranslated region (UTR) of 39 bp, a 3'-terminal UTR of 147 bp with a canonical polyadenylation signal sequence AATAAA and a poly(A) tail, and an open reading frame of 834 bp. The AISPI cDNA encoded a polypeptide of 278 amino acids with a putative signal peptide of 22 amino acids and a mature protein of 256 amino acids. The deduced amino-acid sequence of AISPI contained six tandem and homologous domains similar to that of Kazal-type serine protease inhibitors, including the conserved sequence C-X(7)-C-X(6)-Y-X(3)-C-X(2,3)-C and six cysteine residues responsible for the formation of disulfide bridges, indicating that the AISPI protein from bay scallop should be a member of the Kazal-type serine protease inhibitor family. The temporal expression of AISPI was measured by semi-quantitative RT-PCR after injury or bacterial challenge. After the adductor muscle was wounded or injected with Vibrio anguillarum, the expression of AISPI mRNA in hemolymph was up-regulated and reached the maximum level at 8 and 16 h, respectively, and then progressively dropped back to the original level. The results indicated that AISPI could play an important role in injury healing and immune response in mollusks as it could be induced by injury and bacterial challenge.     

朱砂叶螨HSP90基因克隆及原核表达

采用RT-PCR及RACE技术克隆朱砂叶螨Tetranychus cinnabarinus的热激蛋白90(HSP90)基因, 并进行序列分析, 得到一条长2 595 bp的cDNA序列, 该序列开放阅读框（open reading frame, ORF）为2 169 bp, 编码722个氨基酸, 分子量约为83.45 kDa, 理论等电点为4.81, 3′非编码区（untranslated region, UTR）为249 bp, 5′UTR为177 bp。通过Antheprot分析发现5个HSP90家族的签名序列及胞质HSP90特征序列MEEVD。同源性分析表明, 朱砂叶螨HSP90编码区核苷酸序列和其他已知的HSP90, 尤其是节肢动物昆虫的HSP90, 具有很高的相似性。将鉴定正确的原核重组表达质粒pET43a-TcHSP90, 转化大肠杆菌Escherichia coli BL21(origami) 进行原核表达, 应用SDS-PAGE和Western blotting技术分离并检测融合蛋白, 结果表明构建的原核表达质粒可以在宿主菌中稳定、正确表达。朱砂叶螨TcHSP90基因的克隆、原核表达, 为进一步研究HSP90的性质和功能的研究提供有用的实验材料。     

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.     

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.     

血红密孔菌(Pycnoporussanguineus)漆酶基因的克隆与序列分析

为克隆血红密孔菌 (Pycnoporussanguineus)漆酶基因 ,根据真菌漆酶氨基酸序列保守区设计了 1对简并引物 .以血红密孔菌基因组DNA为模板 ,PCR扩增出长 12 2 7bp的漆酶基因片段 .以此序列为基础 ,通过 5′及 3′RACE技术克隆出漆酶全长cDNA序列 ,序列长为 190 2bp ,其 5′端和 3′端非编码区长分别为 5 1bp和 2 97bp ,开放阅读框长 15 5 4bp ,编码 5 18个氨基酸的蛋白 .该蛋白具有 4个铜离子结合区域 ,预测其相对分子量为 5 6 313 2 ,等电点为 5 5 9,其氨基酸序列与Pycnoporuscinnabarinus漆酶 (lcc3 2 )的同源性最高 ,为 96 % .以该cDNA编码区的两端序列为引物 ,PCR扩增得到漆酶的长度为 2 15 4bp的全长DNA序列 ,序列中包括 10个内含子序列 ,长为 5 2～ 70bp     

猪I-FABP基因的分子克隆与组织特异性表达分析

小肠型脂肪酸结合蛋白对长链脂肪酸具有高度的亲和力,参与脂肪酸的吸收和细胞内转运。利用cDNA末端快速扩增（RACE）技术并结合同源克隆策略,克隆到了编码猪小肠型脂肪酸结合蛋白基因（I-FABP）的全长cDNA序列（GenBank接受号：AY960624）,并对系统发育关系等进行了生物信息学分析。猪I-FABP基因的cDNA序列全长614 bp,其中包括399bp的开放式读码框（ORF）,43bp的5’末端非编码区（5’URT）和172bp的3’末端非编码区（3’URT）,编码132个氨基酸残基蛋白,在氨基酸水半上与其他物种的I-FABP具有高度的同源性。以邻接法（Neigbor-Joining,NJ）所构建的系统发育关系表明,猪I-FABP与其他物种的,I-FABP属于同一类群,且与人的遗传距离最近。Northern杂交和半定量RT—PCR分析发现,猪I-FABP在猪体组织中出现约620bp大小的转录本,且在猪体组织中广泛存在,但在小肠组织中表达量最为丰富。     

Erk2 in ovarian development of green mud crab Scylla paramamosain

We identified extracellular signal-regulated kinase 2 (erk2) from green mud crab, Scylla paramamosain, in this article. It was originally identified from an expressed sequence tag fragment from a normalized gonadal cDNA library. 5' Rapid amplification of cDNA end (RACE) technique was used to obtain the 5' untranslated region (UTR). The full-length cDNA of Sp-erk2 is 1516 bp, including a 5'-terminal UTR of 19 bp, an open-reading frame of 1098 bp, and a 3'-terminal UTR of 399 bp. The translated protein is 365 amino acids in length with a predicted molecular weight of 42 kDa, which is the same as other species. It is the first time that the expression of Sp-erk2 in different stages of ovary development of crustacean was analyzed, and the result showed that the expression of Sp-erk2 increased gradually with ovarian development, with a peak in the mature phase. In situ hybridization histochemistry was used to clarify the detail of expression. Positive signals illustrated that Sp-erk2 mRNA is present in follicular cells when the ovary is in early stages, and in both follicular cells and oocytes when it is in mature phases. All above suggest that Sp-erk2 is important for ovarian development.