Abstract The techniques of homology cloning and anchored PCR were used to clone the Hsp90 gene from black tiger shrimp. The full length cDNA of black tiger shrimp Hsp90 (btsHsp90) contained a 5 0 untranslated region (UTR) of 72 bp, an ORF (open reading frame) of 2160 bp encoding a polypeptide of 720 amino acids with an estimated molecular mass of 83kDa and a 3 0 UTR of 288 bp. The sequence of the coding region showed 90 and 84% homology with that of theChiromantes haematocheir and Homo sapiens, respectively. Conserved signature sequences of Hsp90 gene family were found in the btsHsp90 deduced amino acid sequence. The temporal expressions of Hsp90 gene were constitutively in the black tiger shrimp tissues including liver, ovary, muscle, brain stomach, and heart, and their levels were markedly enhanced after 30min heat treatment at 37C. In ovarian maturation stages, the expression of btsHsp90 was strongest in the second stage, weaker in the fourth and first stage
Mol Biol Rep (2009) 36:127–134 DOI 10.1007/s11033-007-9160-9 Molecular cloning and expression analysis of a heat shock protein (Hsp90) gene from black tiger shrimp (Penaeus monodon) Shigui Jiang Æ Lihua Qiu Æ Falin Zhou Æ Jianhua Huang Æ Yihui Guo Æ Keng Yang Received: 19 August 2007 / Accepted: 28 September 2007 / Published online: 13 October 2007 Ó Springer Science+Business Media B.V 2007 Abstract The techniques of homology cloning and anchored PCR were used to clone the Hsp90 gene from black tiger shrimp The full length cDNA of black tiger shrimp Hsp90 (btsHsp90) contained a 50 untranslated region (UTR) of 72 bp, an ORF (open reading frame) of 2160 bp encoding a polypeptide of 720 amino acids with an estimated molecular mass of 83-kDa and a 30 UTR of 288 bp The sequence of the coding region showed 90 and 84% homology with that of the Chiromantes haematocheir and Homo sapiens, respectively Conserved signature sequences of Hsp90 gene family were found in the btsHsp90 deduced amino acid sequence The temporal expressions of Hsp90 gene were constitutively in the black tiger shrimp tissues including liver, ovary, muscle, brain stomach, and heart, and their levels were markedly enhanced after 30-min heat treatment at 37°C In ovarian maturation stages, the expression of btsHsp90 was strongest in the second stage, weaker in the fourth and first stage Keywords Cloning Á Hsp90 Á RT expression Á Black tiger shrimp (Penaeus monodon) Introduction Animals are capable of producing proteins in response to environmental changes such as temperature elevation [1], exposure to oxidative stress [2], and myocardial ischemia [3, 4] These proteins are highly conserved among various S Jiang (&) Á L Qiu Á F Zhou Á J Huang Á Y Guo Á K Yang Biotechnology and Aquiculture Laboratory, The South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, 231 Xingangxi Road, Guangzhou 510300, P.R China e-mail: Jingsg@zlen.com organisms and collectively termed heat shock proteins (Hsps) According to their apparent molecular weights and degrees of homology, Hsps are classified into several families, Hsp90s (83–99 kDa), Hsp70s (68–80 kDa), Hsp60s, and the small Hsps (25–28 kDa) [1] Heat shock protein 90 (Hsp 90) is one of the most abundant cytosolic proteins in eukaryotes, amounting to approximately 1% of soluble protein in some cells even in the absence of stress [5] Reported roles for Hsp90 family members include protein chaperoning protect cells against stress [6], oncogenic transformation [7, 8], cell cycle control [9] and antigen presentation [10] It possesses the ability to refold denatured proteins into proper conformations [11], associates with steroid hormone receptors and maintains them in a non-functional state until hormone binding [12, 13] Hsp90 also interacts with other nuclear or cytoplasmic proteins, including transforming or regulatory tyrosine kinases, some serine/threonine kinases, transcription factors, cytoskeletal proteins, calmodulin, and bc subunits of G proteins [9, 14–18] The deletion of Hsp90 is lethal for eukaryotic cells [5, 19, 20] Under non-stress conditions, Hsp90 has been shown to play a key role in many cellular processes and most of its identified cellular targets are signal transducers whose conformational instability is relevant to their roles as molecular switches Under stress conditions, Hsp90 prevents irreversible aggregations of proteins Hsp90 is a participant in the heat shock (stress) response of the cell, a response which is widely recognized and accepted as a major weapon in the cell’s armamentarium for protection against and recovery from environmental insult, both physical and chemical [6, 21–23] With the development of the technique of gene cloning, molecular techniques have recently enabled the identification of Hsp90 genes from the invertebrates, such as: Metapenaeus ensis (GenBank accession No EF470346), 123 128 Mol Biol Rep (2009) 36:127–134 Litopenaeus setiferus (GenBank accession No BE846722), Chiromantes haematocheir (GenBank accession No AY528900), Bemisia tabaci (GenBank accession No DQ093381), Ceratitis capitata (GenBank accession No CAJ28987), Locusta migratoria (GenBank accession No AY445913) In the Fenneropenaeus chinensis, partial sequence of Hsp90 was cloned using SSH and found the gene was up-regulated in the hepatopancreas during WSSV infection [24] In this report, we describe the cloning, sequencing, and expressions of the 83-kDa Hsp90 gene from the black tiger shrimp (Penaeus monodon) The main objectives of this study are (1) to clone the full length cDNA of Hsp90 from black tiger shrimp and compare it to other known Hsp90 genes to prove the existence of Hsp90 in black tiger shrimp, (2) to investigate the expression pattern of Hsp90 gene in the tissues in defending environmental temperature change, (3) to detect if the expression has difference during the three important ovarian maturation stages primarily because the Hsp90 gene could strongly expressed in the ovary without stimulation Table Oligonucleotide primers used in the experiments Primer name (50 ? 30 ) Nucleotide sequence Fe TGATTGGACAGTTYGGTGT Re TACAGYTTGATTTGTTCTT F1 GCCGACAAGGTGACCGTAGT R1 TGTTCTTCTGCTTGCGGTTC R2 TCCTCCCAGTCGTTGGTCAG Oligo-dT adaptor GGCCACGCGACTAGTAC(T)16 Adaptor GGCCACGCGACTAGTAC Oligo-dG GGGGGGGGGGGGGGGH b-Actin F TTGCTACATCGCCCTTGACT b-Actin R TGTGGACGGTTTCCTGAATA F R CCACGAGGATTCCACCAACC CCTTCGTCACCGAGACAAGC reagent following the protocol of the manufacturer, and resuspended in DEPC-treated water and stored at –80°C Synthesis of the cDNA first strand Materials and methods Shrimp About 40 appear healthy black tiger shrimps (P monodon) with fresh weight of about 60–300 g each were purchased from Sanya, Hainan province, P R China The shrimp were cultivated in the aerated seawater (salinity 30) for days at 24–25°C Then they were used as the examined materials in the following examination (1) (2) Gene cloning: The shrimps were cultivated without heat stimulation prior to the RNA was isolated from the ovary Expression: Three shrimp (fresh weight about 200 g) were cultivated for 30 at 37°C prior to the RNA was isolated from the tissues including hepatopancreas, ovary (belong to the yolky stage, [25]), muscle, brain stomach, heart Three appear healthy shrimp cultivated at 24–25°C were used as the control In each ovarian maturation stages, three shrimp without heat stimulation were selected which were classified according to the report of Huang [25] before the RNA was isolated from ovary O1: primordial germ cell stage; O2: chromatin nucleolus stage; O4: yolky stage Total RNA isolation Total RNA was isolated from the examined tissues (weight 50 mg) of the shrimps using Trizol (Invitrogen, Japan) 123 cDNA was synthesized from lg of total RNA by Moloney Murine Leukemia Virus reverse transcriptase (M-MLV, Promega, USA) at 42°C for 50 with oligodT-adaptor primer (Table 1) following the protocol of the manufacturer The cDNA was used as the template for PCR reactions in gene cloning and expression analysis Gene cloning and sequencing Initially, PCR was performed using the cDNA prepared above as template, with the degenerated primers of Fe and Re (Table 1) designed according to the conserved regions of other known Hsp90 gene sequences (such as Xenopus Laevis, Danio rerio, Homo sapiens, Chlamy farreri), in order to obtain the partial fragment of Hsp90 gene from the shrimp The obtained PCR products were separated by 1.2% agarose gel, and then purified by PCR purification kit The purified PCR product was ligated with the PMD20-T vector (Takara, Japan), and transformed into the competent Escherichia coli cells The recombinants were identified through blue–white color selection and screened with M13 forward and reverse primers Three of the positive clones were sequenced on an ABI3730 Automated Sequencer (Applied Biosystem) Sequences generated were analyzed for similarity with other known sequences using the BLAST programs (http://www.ncbi.nim.nih.gov/) Having isolated a partial Hsp90 sequence, the 50 and ends of mRNA were obtained by rapid amplification of cDNA ends (RACE) methods, using gene-specific primers Mol Biol Rep (2009) 36:127–134 129 Fig The Black tiger shrimp Hsp90 gene sequence Hsp90 family c signature motif sequence was highlighted; the spark showed the stop code The polyadenylation signal sequence (AATAAA or AATAAT) is underlined, the RNA instability motif were highlighted and underlined, GxxGxG motif is in box Potential phosphorylation sites are underlined shown in Table In 30 RACE–PCR, PCR reaction was performed with primer F1 and adaptor primer (Table 1) In 50 RACE–PCR, the first strand cDNA obtained was tailed with poly (C) at the 50 ends using terminal deoxynucleotidyl transferase (TdT, Takara, Japan) PCR was performed initially with primer R1 and Oligo-dG, followed by seminested PCR with R2 and Oligo-dG The PCR products were gel-purified, sequenced, and the resulted sequences were subjected to analyze Generated sequences were analyzed for similarity with other known sequences using the BLAST program (http:// www.ncbi.nlm.nih.gov/BLAST/) Multiple sequence alignments were performed using the CLUSTAL W program at the European Bioinformatics Institute (http://www ebi.ac.uk) Analyses of the deduced amino acid sequences utilized the programs PSORT (Kenta Nakai, National Insitute Basic Biology), Scan Prosite (EXPASy Molecular Biology Server) and Predict Protein (EMBL-Heidelberg) The phylogenetic tree was constructed by the neighborjoining (NJ) method using using the programs of CLUSTAL X1.83 [26] and MEGA3.1 [27] Expression studies Reverse transcription PCR was used to study the temporal expressions of Hsp90 in black tiger shrimp Gene specific primers F and R, which gave rise to a product of 293 bp, were used in RT-PCR to detect the temporal expression of the Hsp90 gene in black tiger shrimp The products were cloned, sequenced and confirmed to be the correct form of Hsp90 gene Primer b-actin F and b-actin R were used in the RT-PCR to amplify a 220 bp fragment of black tiger shrimp b-actin gene (GenBank accession No EF087977) as a positive control to verify the successful transcription and to calibrate the cDNA template for correspond samples The products were cloned, sequenced, and confirmed to be the correct form of b-actin gene Results Cloning and sequence of btsHsp90 gene Three overlapping products were obtained by RT-PCR amplification (Fig 1), which comprised the full-length 123 130 Mol Biol Rep (2009) 36:127–134 btsHsp90 cDNA The sequence consisted of 2523 nucleotides including a 2160 bp single open reading frame (ORF), a 72 bp 50 untranslated region (50 UTR) and a 288 bp 30 UTR In the 30 UTR, there were two RNA instability motifs (ATTTA), a 24 bp poly (A) tail and two polyadenylation signal which located 40 and 20 bp, respectively, upstream of the poly (A)+ tail The ORF encoded a 720 amino acids precursor peptide with a molecular weight about 83 kDa, and theoretical point of 4.9 The complete nucleotide sequence of btsHsp90 cDNA and the deduced amino acid sequence are shown in Fig The software search yielded several obvious sequence motifs or domains In the deduced amino acids, there are five Hsp90 family signature motifs and a GxxGxG motif essential for ATP binding showed in the Fig 1; a Histidine kinase-like ATPases (HATPase-c) domain from aa33 to aa187; N-glycosylation sites: NSSDaa44–aa47, NKTKaa279– aa282 , NISRaa385–aa388, NTSKaa428–aa431; two coiled coil: 212 Lys –Val248, Leu537–Asp565 (they were not shown in the Fig 1) Homology analysis The deduced amino acid sequence of the btsHsp90 shows very high homology with that of the other invertebrates: C haematocheir (90% Identity, E = 0), L migratoria (85% Identity, E = 0), B tabaci (80% Identity, E = 0); even with the mammalians: Mus musculus (84% Identity, E = 2e – 102), H sapiens (84% Identity, E = 6e – 60) (Table 2) Multiple sequence alignments show the high conserved with the other species Hsp90 It shows that the different potential btsHsp90 motifs are in conserved positions (Fig 2) and it indicated that btsHsp90 should have the similar functions with the other animals Hsp90 gene Based on the nucleotide acid sequence of Hsp90 genes, a phylogenetic tree was constructed by using the programs of CLUSTAL X1.83 and MEGA3.1 (Fig 3) All the vertebrate’ Hsp90 genes and invertebrate’ Hsp90 genes were Table Homology of Hsp90 protein of black tiger shrimp with other known Hsp90 amino acid Expression studies A product of 293 bp of expected size was amplified from most of the examined tissues including hepatopancreas, ovary, muscle, brain, stomach, and heart The sequences of the resulting RT-PCR products were identical to the Hsp90 cDNA sequences which indicated the mRNA expression could be detected by RT-PCR The expression of the btsHsp90 was observed in the most of the examined tissues, but the expression level varied significantly among the tissues There was a high level in ovary and hepatopancreas, lower in brain, stomach and heart, while lowest in muscle After stimulated with heat treatment, the Hsp90 expression level was enhanced, especially in the brain, stomach and heart (Fig 4) The Hsp90 expression in the ovary was found to be different in the ovarian maturation stages by RT-PCR analysis The expression level in the second stage (O2) is the highest among the three stages, and it is higher in the fourth stage (O4) than in the first stage (O1) (Fig 5) Discussion The Hsp90 family is a group of abundantly expressed and highly conserved molecular chaperones whose exact function is presently undefined They recognize and regulate the activity of several intracellular substrates and also operate in the absence of stress [11] In the present study, we cloned full length of Hsp90 gene from the black tiger shrimp (P monodon) using the Score (bits) Chiromantes haematocheir Bemisia tabaci Identity (%) E-value Accession number 721 91 AY528900 1011 80 AAZ17403 Ceratitis capitata 999 81 CAJ28987 Locusta migratoria 694 85 AY445913 Spodoptera frugiperda 685 84 2e – 100 AF254880 Salmo salar 989 78 AAD30275 Xenopus Laevis 123 clustered together and formed a group, respectively In the tree, the black tiger shrimp shows the closest relationship with the C haematocheir, the result is similar with the result of the BLAST So the relationships displayed in the phylogenic tree were corresponded to their classification position 1013 80 AAV41061 Gallus gallus 685 84 8e – 102 P11501 Mus musculus 683 84 2e – 102 BC094024 Homo sapiens 682 84 6e – 60 AJ890083 Mol Biol Rep (2009) 36:127–134 131 Fig Multiple alignments of black tiger shrimp Hsp90 with other known Hsp90 amino acids sequences aligned by the CLUSTAL W program Identical and similar sites were shown with sparks (*) and dots ( or : ), respectively; Residues involved in hydrogen bonding with geldanamycin are highlighted GxxGxG motif is indicated by overline Hsp90 signature sequences were in the box Organism and GenBank database accession nos for sequence are: Xenopus Laevis (AAV41061), Bemisia tabaci (AAZ17403), Ceratitis capitata (CAJ28987), Salmo salar (AAD30275), Chiromantes haematocheir (AY528900), Locusta migratoria (AY445913), Gallus gallus (P11501), Mus musculus (BC094024), Homo sapiens (AJ890083), Spodoptera frugiperda (AF254880), Penaeus monodon (unsubmitted) technique of homology and RACE (GenBank accession No ZF015589) In 30 UTR, there are two repeats of the sequence ATTTA which known to decrease both the stability and translation efficiency of an mRNA [28, 29] There are two polyadenylation signal sequences, one is AATAAA, same as most animals, the other is AATAAT which is same as Oomycete (Achlya ambisexualis) [30] The reason why there are two polyadenylation signal sequences we not know now and there is no any report about it 123 132 Fig Phylogenetic tree show the relationship among the full-length black tiger shrimp Hsp90 amino acids sequence with other representative Hsp90 sequences The sequences were aligned by CLUSTAL W program and the phylogenetic tree was constructed by neighbor-joining methods using MEGA version 3.1 Mol Biol Rep (2009) 36:127–134 82 89 Bemisia tabaci Locusta migratoria 90 Spodoptera frugiperda Ceratitis capitata Chiromantes haematocheir 100 100 Penaeus monodon Xenopus Laevis Salmo salar Gallus gallus 99 Mus musculus 100 100 Homo sapiens Fig RT-PCR analysis of Hsp90 expression in various tissues of black tiger shrimp RTC reverse transcription negative control Hep, hepatopancreas; Ov, ovary; Mu, muscle; Br, brain; St, stomach; He, heart Fig RT-PCR analysis of Hsp90 expression in ovarian maturation stages O1: primordial germ cell stage; O2: chromatin nucleolus stage; O4: yolky stage RTC reverse transcription negative control The result of the homology analysis with other known Hsp90 genes revealed that btsHsp90 showed high homology with Hsp90s of the C haematocheir over 90%, whereas a lower homology was observed with mammals such as H sapiens and M musculus of 84% approximately [31, 32] The results of the blast indicated that the E-values were lower than 0.005 [33], so the clones was the homological gene of Hsp90 Five typical Hsp90 family signature motifs could also be found in the predicted protein of this sequence, we could think that the clones should be the member of Hsp90 family and had similar primary structure with other known Hsp90 The sequence of btsHsp90 contained a highly hydrophobic and acidic C-terminal end, but no potential Nterminal signal sequence as expected for a secreted protein The glutamine-rich sequence (TQTQDQ) or the sequence PEETQTQDQPME at the amino terminus of the mammals 123 Hsp90 is phosphorylated by the dsDNA-activated protein kinase [34] Lacking both amino-terminal threonines, btsHsp90 cannot be phosphorylated Together with Hsp70 and Hsp60, Hsp90 helps newly synthesized proteins to fold and modulate the transcription factors and protein kinases [19, 35] The amino terminal domain of btsHsp90 showed high homology with other Hsp90s and contained a GxxGxG motif essential for ATP binding [36] The motif also overlaps with the GA binding motif [37] The presence of the EEVD motif at the C-terminal end suggests the cytosolic localization of btsHsp90 [38, 39] And the functional motif sequences all locate in the conserved domains (Fig 2) The structure analysis suggests that the btsHsp90 should have similar function with the other animals’ Hsp90 gene Similar to Hsp70, the Hsp90 is also a molecular chaperone, which is conserved among all living organisms to protect cells against stress [11, 40] In the present study, tissue-specific differences in levels of constitutive Hsp90 were observed The highest expression levels were in ovary not hepatopancreas, the lowest in muscle Intermediate levels were detected in hepatopancreas, brain, heart, and stomach Our findings are almost in agreement with those found in rabbits [41] and porcine [42, 43] which the highest level is in testis Mammalian Hsp90 are expressed at basal levels under unstressful conditions; various stresses increase the expression to different degrees [42] In black tiger shrimp, heat treatment could induce the Hsp90 expression level in the tissues But the highest also was in the ovary Now we Mol Biol Rep (2009) 36:127–134 not really understand the reason why the highest expression level was in the ovary not in the immune organ, and there was any report about it The result indicated that btsHsp90 was constitutive and inducible expressed and could play a critical role in defending the circumstantial temperature elevation The levels of constitutive Hsp90 in ovarian maturation stages were different When the ovary began to mature, the expression level was the highest But the expression levels were lower in the other two stages So the Hsp90 expression level could change during the ovarian maturation stages From the result we deduced that the Hsp90 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