Molecular cloning of a phosphotriesterase-related protein gene of silkworm and its expression analysis in the silkworm infected with Bombyx mori cytoplasmic polyhedrosis virus

doi:10.4236/as.2011.24052

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Vol.2, No.4, 406-412 (2011)

doi:10.4236/as.2011.24052

Agricultural Scienc es

Molecular cloning of a phosphotriesterase-related

protein gene of silkworm and its expression analysis

in the silkworm infected with Bombyx mori cytoplasmic

polyhedrosis virus

Xiu Wang1, Kun Gao2,3, Ping Wu2,3, Guangxing Qin2,3, Ting Liu2,3, Xijie Guo2,3*

1College of Biotechnology and Chemical Engineering, Jiangsu University of Science and Technology, Zhenjiang, China;

2Sericultural Research Institute, Jiangsu University of Science and Technology, Zhenjiang, China;

3Sericultural Research Institute, Chinese Academy of Agricultural Sciences, Zhenjiang, China; *Corresponding Author: guoxijie@126.com

Received 9 September 2011; revised 20 October 2011; accepted 30 October 2011.

ABSTRACT

Bombyx mori cytoplasmic polyhedrosis virus is

one of the major viral p athogens for the silkworm.

The immune response of silkworm to the virus

infection is obscure. A phosphotriesterase-re-

lated protein gene of silkworm, Bombyx mori

(BmPTERP) was found in our previous mi-

croarry analysis of the midgut infected with the

virus. In the present study, we cloned and ana-

lyzed the full-length cDNA of BmPTERP gene by

means of rapid amplification of complementary

DNA ends (RACE) and bioinformatic analy sis for

exploring its functions in interaction between

the silkworm and the virus. The nucleotide se-

quence of the gene is 1349-bp and contains a

131 bp 5’UTR and a 165 bp 3’UTR. The 1053 bp

open reading frame encodes a 350 amino acid

protein. The deduced protein contains specific

hits of phosphotriesterase-related proteins and

belongs to the amidohydrolase superfamily. RT-

PCR analysis revealed that BmPTERP gene was

expressed in all the tissues tested, including

midgut, hemocyte, gonad, fat body and silk gland.

Real-time quantitative polymerase chain reac-

tion analysis indicated that the relative tran-

script of BmPTERP gene in the infected midgut

was 19.32 fold lower than that in normal midgut

at 72 hours post inoculation.

Keywords: Silkworm; Cytoplasmic Polyhedrosis

Virus; Phosphotriesterase-Related Protein; Gene

1. INTRODUCTION

Phosphotriesterase-related protein (also called phos-

photriesterase homology protein, PHP) is a member of

amidohydrolase superfamily and exhibits higher sequence

identity, and high sequence similarity to phosphotries-

terase (PTE) [1]. PTE is a group of bacterial enzyme that

catalyzes the hydrolysis of a wide range of organophos-

phate triesters including organophosphate insecticides

and chemical nerve agents [2]. PTE exists as a homodimer

with one active site per monomer. The active site is lo-

cated next to a binuclear metal center, at the C-terminal

end of a TIM alpha-beta barrel motif and contains tow

zinc ions in native enzyme. However, these ions can be

replaced with other metals such as cobalt, cadmium,

nickel or manganese and the enzyme still remains active.

The hydrolysis reaction of PTE is sufficient to utilize the

nucleophilicity of the bridging hydroxide according to

theoretical study of the phosphotriesterase reaction mecha-

nism [2].

The PTE has attracted much interest in recent years

because of their potential ability in the decontamination

of hazardous organophosphate compounds [3]. The most

efficient PTEs have been identified from several micro-

bial species, such as Pseudomonas diminuta, Sulfolobus

solfataricus and Flavobacterium [4]. The directed evolu-

tion research of PTE resulted in improvement in func-

tional expression and enzymatic activity [5,6]. Data col-

lected showed that PTE was evolved from the family of

phosphotriesterase-related proteins [7-9]. The research

on its structure and expression would elucidate the evo-

lutionary story of PTEs. To date, the phosphotriesterase-

related proteins have been isolated from E. coil and

some other organisms and grouped into a single family

[7,9]. Although considerably homologous in sequence

and in the structure of the bimetal catalytic site, phos-

photriesterase-related proteins differs from PTE and ex-

hibits no phosphotriesterase activity [1,7]. But, subse-

quent studies reported a weak esterase activity and PTE

activity in an E. coli PHP mutant [1].

X. Wang et al. / Agricultural Sciences 2 (2011) 406-412

407407

Bombyx mori cytoplasmic polyhedrosis virus (Bm-

CPV), which belongs to the genus Cypovirus in the fam-

ily Reoviridae, is a significant virus to the silkworm,

often causing severe economic damages to the sericul-

tural industry [10,11]. Unfortunately, to date, the im-

mune mechanism of silkworm to CPV infection remains

obscure. In our previous studies, a differentially expressed

gene similar to the genes of phosphotriesterase-related

proteins was identified in the CPV-infected silkworm by

using microarry analysis [12]. In the present study, we

first cloned the gene of phosphotriesterase-related pro-

tein from silkworm, Bombyx mori (BmPTERP) by means

of RACE method. Furthermore, the expression patterns

of BmPTERP in midgut and some other tissues after

infection with BmCPV were analyzed by quantitative

real-time PCR. The results provided useful information

for further study of the immune mechanism of silkworm

to virus infection.

2. MATERIALS AND METHODS

2.1. Silkworm Strain

The silkworm strain p50 was provided by the National

Silkworm Genetic Resource Preservation Center of

Chinese Academy of Agricultural Science. The larvae of

silkworm were reared with mulberry leaves at standard

temperature of 25˚C and under a photoperiod of 12 h

light and 12 h dark up to the fourth molting for virus

inoculation.

2.2. Virus Inoculation

BmCPV was suspended in disinfected distilled water

to a concentration of 108 polyhedra per mL. One mL

viral suspension was spread totally on 10 pieces of mul-

berry leaves which were nearly 15 cm2 each. 25 newly

molted fifth instar larvae were fed on it. The control lar-

vae were treated with the same amount of mulberry

leaves spread with distilled water. The infection does

was calculated as 4 × 105 polyhedra per larva.

2.3. Collection of Midgut and Other Tissues

Midgut of BmCPV-infected and control larvae were

collected at 24, 48 and 72 h post-inoculation by dissect-

ing the larvae on ice. The midgut were quickly washed

in diethylpyrocarbonate (DEPC)-treated water to remove

the attached leaf pieces and then immediately frozen in

liquid nitrogen. At the same time, other tissues namely

gonad, silk gland, hemocyte and fat body were also col-

lected at 48 h post inoculation and frozen in liquid ni-

trogen before being stored at –80˚C. The same tissues of

five larvae for midgut and of ten larvae for gonad, silk

gland, hemocyte and fat body at each time point were

mixed for RNA extraction and following experiments.

2.4. Isolation of Total RNA

Total RNA was extracted respectively from midgut,

gonad, silk gland, hemocyte and fat body collected at

different time point post inoculation by using Trizol re-

agent (Invitrogen, Carlsbad, CA, US) and subjected to

DNase I treatment according to the manufacturer’s pro-

tocol. The concentration of total RNA was determined

by using a Biophotometer (Eppendorf, Hamburg, Ger-

many) to measure the absorbance at 260 nm and 280 nm.

RNAs with the A260:A280 value of 1.9 to 2.0 were

stored at –80˚C and used for further study.

2.5. Rapid Amplification of cDNA Ends

(RACE)

Full-length cDNA of BmPTERP gene was synthesized

using 2 μg total RNA of midgut as a template with

SMARTTM RACE cDNA Amplification Kit (Clontech).

Specific primers for 5’RACE and 3’RACE were de-

signed based on BmPTERP gene cDNA fragment re-

vealed in our previous study. The primer for 5’RACE

was 5’-TTACAACTGCCTCCTTCGCGGCCTGA-3’ and

that for 3’RACE was 5’-CGGCTGCGGTGTCAGTTT-

CCATCCTC-3’. 5’-RACE was performed in a reaction

system of 25 μL containing 17.25 μL PCR-Grade water,

2.5 μL 10 × Advantage 2 PCR buffer, 0.5 μL dNTP Mix

(10 μM), 0.5 μL 50 × Advantage 2 Polymerase Mix, 1.25

μL 5’-RACE-Ready cDNA, 2.5 μL UPM (10×), 0.5 μL

5’-specific primer (10 μM) and as the following proce-

dures: 32 cycles of 95˚C for 30 s, 68˚C for 3 min. 3’

RACE was performed in a reaction system of 25 μL

containing 17.25 μL PCR-Grade water, 2.5 μL 10 × Ad-

vantage 2 PCR buffer, 0.5 μL dNTP Mix (10 μM), 0.5

μL 50 × Advantage 2 Polymerase Mix, 1.25 μL 5’-

RACE-Ready cDNA, 2.5 μL UPM (10×), 0.5 μL 3’-

specific primer (10 μM) with the same procedures. The

PCR products were examined by electrophoresis in 1%

agarose gel and the fragment sizes were determined rela-

tive to marker DNA. The appropriate band was purified,

cloned into pGEM-T Easy vector and sequenced by

Sangom Biotech Co. Ltd. (Shanghai).

2.6. Sequence Anal ysis, Multiple Sequence

Alignment and Phylogenetic Analysis

The sequences were searched in GenBank with

BLASTx for comparative analysis and assembled with

the obtained fragments. The sequences were analyzed

using the BLAST algorithm at NCBI (http://www.ncbi.

nlm.gov/blastn). The gene structure was predicted with

Gene Structure Display Server (GSDS, http://gsds.cbi.

pku.edu.cn). The deduced amino acid sequence of Bm-

X. Wang et al. / Agricultural Sciences 2 (2011) 406-412

408

PTERP was analyzed with Protparam software (www.

expasy.ch/tools/protparam.html) and ProtScale software

(http://www.expasy.org/tools/protscale.html). The signal

peptide was predicted with the SignalP 3.0 (http://www.

cbs.dtu.dk/services/SignalP/). Multiple protein sequences

were aligned using the MegAlign program by CLUSTAL

W method in DNASTAR software package. A phyloge-

netic tree was constructed by MEGA 4.0 software.

2.7. RT-PCR

Total RNA extracted as described above from differ-

ent tissues of fifth instar silkworm, including hemocyte,

silk gland, fat body, gonad and midgut were used as

template to synthesize cDNA using the Prime ScriptTM

RT Reagent Kit (TaKaRa). The PCR reaction was per-

formed as the procedures with denaturation for 2 min at

94˚C, 30 cycles of 30 s at 94˚C, 30 s at 54˚C and 10 min

at 72˚C.

2.8. Quantitative Real-Time PCR of

BmPTERP Gene

Total RNAs extracted respectively from the midgut of

BmCPV-infected and control larvae at 24, 48 and 72 h

were adjusted with diethylpyrocarbonate (DEPC) H2O to

a concentration of 500 ng/μL. 1000 ng of each of the

total RNAs was reverse transcribed in 20 μL of reaction

system containing 4 μL 5 × PrimeScript buffer (for Real

time), 1 μL PrimerScript RT Enzyme Mix I, 1 μL Oligo

dT Primer (50 μM), 1 μL Random 6 mers (100 μM), 2

μL Total RNA, 11 μL RNase Free dH2O using the Prime

ScriptTM RT Reagent Kit (TaKaRa). Quantitative real-

time PCR was performed using 1 μL of diluted first-

strand cDNA (1/10) in each 25 μL reaction volume ac-

cording to the manufacturer’s instructions for the SYBR

Premix Ex TaqTM (TaKaRa). Specific primers for Bm-

PTERP gene and β-actin were designed by Primer Pre-

mier 5.0 software (Primer Premier, Palo Alto, CA, US).

For BmPTERP gene, the forward primer was 5’-ATTTA-

GACCGAACCCTACTTG-3’ and the reverse primer was

5’-TCGTGGGACATTAACACTTT-3’ and for β-actin,

they were 5’-AATGGCTCCGGTATGTGC-3’ and 5’-

TTGCTCTGTGCCTCGTCT-3’ respectively. Reactions

were run in triplicate for the same pooled samples on an

Opticon lightcycler (BioRad, Hercules, CA, US) using

the following thermal cycling parameters: 95˚C for 10 s,

40 cycles of 95˚C for 5 s, 60˚C for 20 s, 72˚C for 5 s.

Following amplification, melting curves were constructed.

Data were analyzed and normalized relative to β-actin

transcript levels by Opticon Monitor Analysis software

(MJ Research, Waltham, MA, US). The relative quanti-

tative method was used to evaluate the differential ex-

pression of BmPTERP gene [13]. Ct for amplified target

product of BmPTERP gene and internal control β-actin

was determined for each sample to normalize the differ-

ences in the amount of template and the efficiency of

RT-PCR (ΔCt = Cttarget − Ctβ-actin). The RNA of the normal

silkworm larvae was used as calibrator and the ΔCt for

each sample was subtracted from the ΔCt of the calibra-

tor to calculate the difference ΔΔCt. 2−ΔΔCt was used to

calculate the relative expression level of BmPTERP gene.

Relative expression level is expressed as average ± SE.

For other tissues, the quantitative real-time PCR of

BmPTERP gene were performed with the same proce-

dure described above but using the total RNA from a

specific tissue as template.

3. RESULTS

3.1. Occurrence of Infection

Inoculation of BmCPV with the concentration of 4 ×

105 polyhedra per larva to the fifth instar silkworm

caused infection and disease to all the larvae. It was con-

firmed by the appearance of white wrinkles on the mid-

gut as typical symptoms at approximately 72 h post-

inoculation and the observation of polyhedra under a

microscope.

3.2. cDNA Cloning and Sequence Analysis

of BmPTERP Gene

The full length cDNA of the phosphotriesterase-re-

lated protein gene of the silkworm, Bombyx mori (Bm-

PTERP gene) was cloned by RACE method and then

sequenced. The obtained full length cDNA of BmPTERP

gene is 1349-bp, containing a 131-bp 5’untranslated

region (UTR), a 165-bp 3’untranslated region and a

1053-bp open reading frame (ORF). The nucleotide se-

quence of the cloned gene has been deposited in Gen-

Bank with the accession number HQ391899. The ORF

which locates in the region from 132-bp to 1184-bp en-

codes a putative protein of 350 amino acids. A putative

polyadenylation signal AATAAA was detected in the

3’UTR 14-bp upstream from the poly(A) tail.

For analysis of the gene structure, the cloned sequence

of BmPTERP gene was aligned with the genome se-

quence of Bombyx mori by Blastn. It was found that the

full cDNA was completely contained in the genome se-

quence of Bombyx mori (GenBank accession number:

BABH01012392.1) . The result by GSDS (Genes struc-

ture display server) showed that the gene sequence of

BmPTERP gene contains six exons and five introns

(Figure 1). Each exons-intron boundary comforms to the

“GT-AG” rule.

3.3. Characteristic and Phylogenetic

Analysis of BmPTERP

Analysis by using Protparam software revealed that

X. Wang et al. / Agricultural Sciences 2 (2011) 406-412

409409

3.5. Expression Analysis of BmPTERP Gene

in Normal and CPV-Infected Midgut of

Silkworm

the molecular weight of the putative BmPTERP was

39.03-KDa and isoelectric point 5.72. Analysis of the

amino acid sequence with ProtScale software indicated

that BmPTERP has stronger hydrophilicity. No typical

signal peptide was predicted by SignalP 3.0, which sug-

gested that BmPTERP may be a soluble protein. Ho-

mology analysis of the protein indicated that BmPTERP

shares 69% identity to the phosphotriesterase-related

protein of Helicoverpa zea (ADK73626.1) and 54%

identity to the phosphotriesterase-related protein of Apis

mellifera (XP_395159.2). A phylogenetic tree was con-

structed (Figure 2), showing that BmPTERP is clustered

well with phosphotriesterase-related protein from Heli-

coverpa zea.

BmCPV infects the midgut and multiplies mainly in

the columnar cells of the midgut of silkworm larvae.

Therefore, the differential expression of BmPTERP gene

in midgut of both CPV-infected and normal silkworm

larvae at different time points 24, 48 and 72 h post in-

oculation was analyzed in details. Representative ampli-

fication plots of real-time PCR were used to differentiate

transcript level in normal and CPV-infected midget of

silkworm. The results showed that the transcript level of

BmPTERP gene has significant difference between the

midgut of CPV-infected and normal larva at 72 h point,

while at 24, 48 h, its transcript level has no significant

difference. The Ct values and the standard deviations of

A (β-action transcript level in BmCPV infected midgets

at 72 h point), B (β-action transcript level in normal

midgut at 72 h point), C (PTERPBm transcript level in

normal midgut at 72 h point) and D (PTERPBm tran-

script level in BmCPV infected midgut at 72 h point)

were 19.16 ± 0.11, 20.3 ± 0.13, 24.3 ± 0.31, 27.49 ± 0.23,

respectively. The qPCR distinguished that the expression

of BmPTERP gene was obviously down-regulated in the

BmCPV-infected silkworm at 72 h post inoculation. Its

relative expression in the infected midgut was calculated

to be approximately 19.32 fold lower than that in normal

midgut at 72 hours post inoculation (Figure 5).

3.4. Expression Analysis of BmPTERP Gene

in Different Tissues

RT-PCR analysis of the RNA from different tissues of

fifth instar larvae of silkworm revealed that the gene of

BmPTERP was expressed in all the five tested tissues,

namely midgut, fat body, silk gland, hemocyte and go-

nad (Figure 3). The highest transcript level was found in

fat body and the lowest transcript level in silk gland as

compared to other tissues. Furthermore, the gene showed

differential expression in the tissues of silkworm in-

fected with BmCPV as compared to the normal ones. At

48 hours post inoculation, significant difference in the

gene expression was found in gonads, while almost no

changes in the hemocyte (Figure 4).

Figure 1. Genomic structure of BmPTERP gene. The gene contains six exons and five introns. The se-

quences of BABH01012392.1 were downloaded according to the result of alignment. On the basis of

cDNA sequence of BmPTERP gene and downloaded sequence of BABH01012392.1, the gene structure

of BmPTERP gene was analyzed by gene structure display server, GSDS (http://gsds.cbi.pku.edu.cn/).

Exon1, 145bp(4149-4293); exon2, 252bp(4515-4766); exon3, 258bp(5684-5941); exon4, 165bp(6988-

7152); exon5, 218bp(8045-8262); exon6, 279bp(8658-8936).

HQ 391899 B ombyx mori

A DK 73626. 1 Heli coverpa z ea

X P 395159. 2 Api s mellifera

X P 003488340. 1 Bom bus i m pat i ens

X P 001953466. 1 Dros ophi l a ananas sae

X P 002096927.1 Dros ophi l a yakuba

NP 731339.1 Drosophi l a m el anogaster

100

0.05

Figure 2. Phylogenetic tree of BmPTERP gene of silkworm and homologous sequences from other in-

sects. This was constructed by MEGA 4.0.

Openly accessible at

X. Wang et al. / Agricultural Sciences 2 (2011) 406-412

410

Figure 3. Expression analysis of BmPTERP gene in five dif-

ferent tissues. The BmPTERP gene was expressed in all the

five tested tissues. β-actin in corresponding tissue were dis-

played in the bottom panel as internal control.

Figure 4. Relative expression analysis of BmPTERP gene in

different tissues between the BmCPV-infected and normal

silkworm at 48 hours post inoculation. The blank and filled

columns indicate normal and CPV-infected silkworm respect-

tively. Error bars represent standard deviation of triplicate ex-

periments for the same pooled sample.

4. DISCUSSION

Bombyx mori cytoplasmic polyhedrosis virus (Bm-

CPV) belongs to the genus Cypovirus in the family Reo-

viridae. BmCPV infects the midgut epithelium and mul-

tiplies in the cytoplasm of columnar cells and forms in-

clusion bodies which occlude virus particles. The virus

contains segmented, double-stranded RNA as the ge-

nome [14,15]. However, the molecular mechanism of

CPV infection in the silkworm is poorly understood. The

purpose of this study is to provide significant informa-

tion for exploring the molecular mechanism of CPV in-

fection to the silkworm.

In the present study, the gene of a putative phosphot-

riesterase-related protein was cloned by means of RACE

techniques for the first time from the silkworm, Bombyx

mori and analyzed by bioinformatic method. Sequence

analysis convinced that the gene we cloned encodes for a

putative protein showing sequence identity with the

phosphotriesterase-related protein family and belongs to

the amidohydrolase superfamily. We therefore propose

that the gene be called BmPTERP gene. Homologous

analysis showed that the BmPTERP from silkworm

shared high homologies with other known phosphotri-

esterase-related proteins, especially the highest with that

of Helicoverpa zea. Phylogenetic analysis showed that

the BmPTERP was clustered well with phosphotries-

terase-related protein from Helicoverpa zea, indicating

that these two genes are homologues and might share

similar functions. Quantitative real-time RT-PCR showed

that the relative transcript level of BmPTERP gene in the

infected midgut was 19.32 fold lower than that in normal

midgut at 72 hr post inoculation. The same tendency was

also observed by Wu et al. [12] in microarray analy- sis

of BmCPV-infected midgut of silkowrm. It was clear

that after BmCPV invasion, the expression level of Bm-

PTERP gene in midgut changed correspondingly, sug-

gesting that BmPTERP gene of silkworm might be cor-

related to the interaction between the silkworm host and

BmCPV infection.

Figure 5. Relative expression of BmPTERP gene in BmCPV-

infected and normal midgut of silkworm. The relative expres-

sion of BmPTERP gene in the infected midgut was approxi-

mately 19.32 fold lower than that in normal midgut at 72 hours

post inoculation. Error bars represent standard deviation of

triplicate experiments for the same pooled sample.

X. Wang et al. / Agricultural Sciences 2 (2011) 406-412

411411

Regarding the function of the phosphotriesterase-re-

lated protein, only few reports were available at present.

In mice, phosphotriesterase has hypothetical function

that catalyzes small cytoplasmic molecules which would

prove toxic and protects mice against organophosphate

neurotoxins [16,17]. Attempts to control agricultural and

forest insects and spread of insect-borne diseases, such

as malaria, using organophosphate insecticides are being

frustrated by the development of resistant strains of in-

sects. Some of these achieve resistance by modification

of acetylcholinesterase, others show a phosphotriesterase

activity apparently different from that of bacterial phos-

photriesterases [18]. However, no activity of the phos-

photriesterase-related protein was commonly detected,

except for a weak esterase activity and PTE activity in

an E. coli PHP mutant [1]. Recently, a PHP gene from

the thermophilic bacterium Geobacillus caldoxylosilyti-

cus TK4 was cloned and overexpressed in E. coli [19].

The recombinant protein showed activities with p-ni-

trophenyl acetate and p-nitrophenyl butyrate. This is the

first reported PHP having an extremely pH- and thermo-

stable esterase activity.

Our result in the present study is the first report that a

phosphotriesterase-related protein gene was cloned from

the silkworm. While, its in vivo function still remains

unclear. At present, the only clue comes from its strong

homology to PHPs of bacteria and other organisms, but

there is not yet any experimental proof that BmPETRP is

itself a phosphotriesterase-related protein. As the disease

of silkworm caused by the infection with BmCPV ad-

vanced, the expression of the BmPETRP gene was ob-

viously down-regulated. This might be attributed to the

facts that a series of physiological and pathological

changes takes place due to the infection. Further func-

tional experimental research should be addressed in our

future work. If BmPETRP does turn out to have the ac-

tivities of PHP or phosphotriesterase predicted from its

homology, it would be very interesting to establish

whether insects have homologous genes, whether these

genes might confer resistance to organophosphate pesti-

cides and most importantly whether these genes might

be involved in the interaction between the silkworm and

the BmCPV infecton.

5. ACKNOWLEDGEMENTS

This research was supported by the National Natural Science Foun-

dation of China (Grant No.30972143) and Natural Science Foundation

of Jiangsu Province (Grant No. BK2010353).

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