Molecular cloning and expression analysis of a heat shock protein (Hsp90) gene from black tiger shrimp (Penaeus monodon)

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

Molecular cloning and expression analysis of a heat shock protein

(Hsp90) gene from black tiger shrimp (Penaeus monodon)

Shigui JiangÆ Lihua Qiu Æ Falin Zhou Æ

Jianhua HuangÆ 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

stron-gest in the second stage, weaker in the fourth and first stage

Keywords Cloning
Hsp90
RT expression

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

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 con-trol [9] and antigen presentation [10] It possesses the ability to refold denatured proteins into proper conforma-tions [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, transcrip-tion 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 insta-bility 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 armamentar-ium 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 identifi-cation of Hsp90 genes from the invertebrates, such as: Metapenaeus ensis (GenBank accession No EF470346),

S Jiang (&)
L Qiu
F Zhou
J Huang
Y Guo
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

DOI 10.1007/s11033-007-9160-9

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

envi-ronmental 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

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

3 days at 24–25°C Then they were used as the examined

materials in the following examination

(1) Gene cloning: The shrimps were cultivated without

heat stimulation prior to the RNA was isolated from

the ovary

(2) Expression: Three shrimp (fresh weight about 200 g)

were cultivated for 30 min at 37°C prior to the RNA

was isolated from the tissues including

hepatopan-creas, 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)

reagent following the protocol of the manufacturer, and resuspended in DEPC-treated water and stored at –80°C

Synthesis of the cDNA first strand

cDNA was synthesized from 2 lg of total RNA by

Moloney Murine Leukemia Virus reverse transcriptase (M-MLV, Promega, USA) at 42°C for 50 min with oligo-dT-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 (Table1) 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

30ends of mRNA were obtained by rapid amplification of cDNA ends (RACE) methods, using gene-specific primers

Table 1 Oligonucleotide primers used in the experiments Primer name (50? 30) Nucleotide sequence

shown in Table1 In 30 RACE–PCR, PCR reaction was

performed with primer F1 and adaptor primer (Table1) In

50 RACE–PCR, the first strand cDNA obtained was tailed

with poly (C) at the 50 ends using terminal

deoxynucleo-tidyl transferase (TdT, Takara, Japan) PCR was performed

initially with primer R1 and Oligo-dG, followed by

semi-nested 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

align-ments 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

Insi-tute Basic Biology), Scan Prosite (EXPASy Molecular

Biology Server) and Predict Protein (EMBL-Heidelberg)

The phylogenetic tree was constructed by the

neighbor-joining (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

con-firmed 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

(Gen-Bank 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

Fig 1 The Black tiger shrimp Hsp90 gene sequence Hsp90 family

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

c

btsHsp90 cDNA The sequence consisted of 2523

nucleo-tides 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.1

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:

Lys212–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)

(Table2) Multiple sequence alignments show the high

conserved with the other species Hsp90 It shows that the

different potential btsHsp90 motifs are in conserved

posi-tions (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

verte-brate’ Hsp90 genes and inverteverte-brate’ Hsp90 genes were

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

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 tis-sues, but the expression level varied significantly among the tissues There was a high level in ovary and hepato-pancreas, 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 regu-late 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

Table 2 Homology of Hsp90

protein of black tiger shrimp

with other known Hsp90 amino

acid

Score (bits) Identity (%) E-value Accession number

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

sta-bility 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 do not know now and there is no any report about it

Fig 2 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)

The result of the homology analysis with other known

Hsp90 genes revealed that btsHsp90 showed high

homol-ogy 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

homo-logical 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

hydro-phobic and acidic C-terminal end, but no potential

N-terminal signal sequence as expected for a secreted protein

The glutamine-rich sequence (TQTQDQ) or the sequence

PEETQTQDQPME at the amino terminus of the mammals

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 over-laps 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 treat-ment could induce the Hsp90 expression level in the tissues But the highest also was in the ovary Now we do

Bemisia tabaci

Locusta migratoria

Spodoptera frugiperda

Ceratitis capitata

Chiromantes haematocheir

Penaeus monodon

Xenopus Laevis

Salmo salar

Gallus gallus

Mus musculus

Homo sapiens

100

100 100

100

99

90

82 89

Fig 3 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

Fig 4 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 5 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

not really understand the reason why the highest

expres-sion 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

expres-sion level could change during the ovarian maturation

stages From the result we deduced that the Hsp90 might

have relationship with the ovarian maturation Certainly

this need further work to verify

Acknowledgments This study was supported by National nature

foundation of China (No 30571447), National ‘‘863’’ Project of

China (No 2003AA603120 ) and Agriculture Department Project of

China(06-05-01B).

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