Cloning, Characterization and Bioinformatic Analysis of the Gene Encoding the Larval Serum Protein 2 in Diapause of the Onion Maggot, Delia Antiqua

doi:10.4236/eng.2013.510B100

Paper Menu >>

Journal Menu >>

Engineering, 2013, 5, 487-490

http://dx.doi.org/10.4236/eng.2013.510B100 Published Online October 2013 (http://www.scirp.org/journal/eng)

Cloning, Characterization and Bioinformatic Analysis of

the Gene Encoding the Larval Serum Protein 2 in Diapause

of the Onion Maggot, Delia Antiqua

Jingjing Xu, Bin Chen*, Zhengbo He, Youjin Hao

Institute of Entomology and Molecular Biology, College of Life Sciences, Chongqing Normal University,

Shapingba, Chongqing, China

Email: *[email protected].

Received 2013

ABSTRACT

The full-length cDNA encoding Larval serum protein 2 (LSp-2) in the onion maggot, Delia antiqua, was cloned and

sequenced by rapid ampliﬁcation of cDNA ends methods. The result showed that the cDNA was 2203 bp long and the

open reading frame (ORF) of 2106 bp encoded 701 amino acid with a calculated molecular weight of 80.5 kDa and an

isoelectric point of 5.87. The onion maggot LSp-2 shows highest homology (83%) to that of Calliphora vicina at amino

acid level. Its signal peptides, domains and structures were predicted and analyzed by using bioinformatic methods. The

amino acid sequence of LSP-2 suggests that it would be a typical hexamerin.

Keywords: Delia Antiqua; Diapause; Larval Serum Protein 2; Cloning ; Mo lecular Characterization; Bioinformatic

Analysis

1. Introduction

Larval serum protein 2 (LSP-2) belongs to a superfamily

of hexameric hemolymph proteins that have been found

in all insect species investigated [1]. In holometabolous

insects, hexamerins are thought to act mainly as storage

proteins that provide energy and amino acids during me-

tamorphosis [2,3]. Expression of LSP-2 is restricted to

the fat body cells, where it reabsorbs proteins and other

macromolecules that have accumulated in the haemo-

lymph during the larval feeding period [5,6]. Roughly

half of the cell population survives metamorphosis, indi-

cating a specific degree of differentiation during postem-

bryonic life. Diapause is a developmental strategy wide-

spread among insects and their arthropod relatives. The

onion maggot (Delia antiqua), a serious pest of onion

(Alliumcepa), passing over winters and/or summers as

diapausing pupae, is an excellent model for diapause

research [4]. LSP-2 as a kind of storage proteins is be-

lieved to provide with a means for the feeding insect to

build reserves of amino acids for use in protein synthesis

and energy metabolism during diapause stages.

2. Materials and Methods

2.1. Insects

The colony of D. antiqua was reared on an artificial diet

at 25˚C with a 16L:8D cycle and relativ e humidity 50% -

70%. Larvae were maintained at 25˚C with a 16L:8D

photocycle to induce SD and 16˚C with 12L:12D to in-

duce WD.

2.2. RNA Extraction and RT-PCR

Total RNAs were extracted with Trizol® reagent (Invi-

trogen Co., USA) from the pupae according to the man-

ufacturer’s instructions. The ﬁrst -strand cDNA was syn-

thesized from 1 µg of total RNA in 20 µl reaction mix-

ture prepared with RevertAidTM First Strand cDNA Syn-

thesis Kit (Fermentas). One microliter of the reaction

mixture was a dded to 25 µl of PCR reaction system, PCR

ampliﬁcation was performed using one speciﬁc primers

ku6contig11F (5’-CCACTGGCTTGACTCGCATG-3’)

and ku399R (5’-GCGGGCTCCAAAGTATTCA-3’) (Fig-

ure 1) based on known EST sequences in SSH cDNA

library conservative sequences with the following reac-

tion conditions: 1 min at 94˚C, 30 S at 55˚C, 30 S at 72˚C

with 35 cycles, then 10 min at 72˚C.

2.3. RACE Amplification

Speciﬁc primers were synthesized for 5’ and 3’RACE

based on the cDNA sequences obtained from internal

ampliﬁcation. For 5’ and 3’RACE, the cDNA was syn-

thesized according to the manufacturer’s protocol

*Corresponding a uthor.

J. J. XU ET AL.

488

(SMARTer ™ R AC E cD N A Ampliﬁcation Kit, Clontech).

5’ and 3’-RACE ampliﬁcation was performed on 2.5 ml

of 3’-re ady-cDNA with UPM and GSPF. The reaction

mixture was subjected to 30 thermal cycles that consisted

of 94˚C, 1 min; 65˚C, 1 min; 72˚C, 1 mi n.

2.4. Cloning and Sequencing

The ampliﬁed DNAs were separated on a 1.5% agarose

gel and puriﬁed using DNA Gel Extraction Kit. DNA

fragments were cloned according to the manufacturer’s

protocol (TOPO TA Cloning Kit for Sequencing). Mul-

tiple sequencing reactions were run using both M13F and

M13R primers.

2.5. Phylogenetic Analysis

The program MEGA5.0 was used for the phylogenetic

tree reconstructions, and Neighbor-joining tree were in-

ferred with the program.

3. Results

3.1. Cloning of Onion Maggot LSP-2 cDNA

The tools provided by the ExPASy Molecular Biology

Server of the Swiss Institute of Bioinformatics (http://

www.expasy.org) were used for the analyses of DNA

and amino acid sequences. Onion maggot LSP-2 cDNA

contains a 2106 bp open reading frame (ORF) encoding a

701 amino acid precursor protein. The ﬁrst 21 amino

acids are predicted for the sequence of a signal peptide

using SignalP Version 3.0 software (http://www.cbs.dtu.

dk/ser vic es/Sign alP). The 5’ untranslated region upstream

of the transcription start code (ATG) is about 23 nucleo-

tides. The ORF is terminated by a TAA stop code that is

followed by a 74bp 3’ untranslated region.

3.2. Bioinformatics Analysis

As determined by the use of protparam software (http://

web.expasy.org/protparam) the deduced amino acid se-

quence of 680 residues has a molecular mass of 80.5 kDa

and a pI of 5.87. Onion maggot LSP-2 was most similar

to LSP-2 from Drosophila melanogaster and Calliphora

vicina. Using the SMART software (http://smart.embl-

heidelberg.de) found domains (Figure 1). Putative gly-

cosylation and phosphorylation sites as well as the con-

served motifs ADKDFLXKQK (position 27; Gordadze et

al., 1999) and TMMRDPMFY (position 480; PROSITE,

Bairoch et al., 1997), found in several insect hexame-

rins, were identified in the LSP-2 amino acid sequence

[7].

The position of secondary-structure elements was de-

duced using UniProt (http://www.uniprot.org) (Figure

2).

Name Begin End E-value

Pfa m: Hemocyanin_N 31 156 6.80E-32

Pfa m: Hemocyanin_M 160 421 1.30E-53

Pfa m: Hemocyanin_C 427 694 1.30E-89

Figure 1. Predicted domain of LSP-2.

Figure 2. Protein secondary-structure of onion maggot

LSP-2.

J. J. XU ET AL.

489

The position of tertiary-structure elements was de-

duced using UniProt (http://www.uniprot.org) (Figure

3).

3.3. Phylogenetic Analysis

The multiple-sequence alignment of 6 LSP-2s was ana-

lyzed by the neighbor-joining method. The tree is rooted

by the LSP-2 of Bomyx mori (Figure 4).

4. Discussion

Here we cloned and characterized the cDNA sequence of

the Lsp-2 gene and examined the structure of its protein

Except the 21 amino acid signal peptide, translation of

the LSP-2 ORF yields a polypeptide of 80.5 kDa. During

diapause LSP-2 synthesized in the fat body and secreted

into the hemolymph is markedly elevated [8-11]. During

diapause and metamorphosis LSP-2 may serve as an

amino acid store for development of other adult struc-

tures. Thus, it is interesting to study the fate of LSP-2

Figure 3. Protein tertiary-structure of the on ion maggot LS P-2.

Figure 4. Phylogenetic analysis by NJ trees based on the

amino acids sequences of LSP-2s. Branch lengths are pro-

portional to the numbers of amino acid substitutions. Gen-

Bank accession number was: Calliphora vicina (AAC

24157.1), Musca domestica (AAP13346.1), Drosophila mela-

nogaster (NP524816.1), Culex qui nquefa sciatu s (EDS32906.1),

Bombyx mori (BAA02093.1).

during metamorphosis and post-diapause development

respectively in order to fully understand the exact func-

tion of thi s protei n during.

5. Acknowledgment

This work was supported by the National Natural Sci-

ence Foundation of China (Nos. 31372265 and 31071968),

Key Scientific and Technological Project of Chongqing

(CSTC-2012GG-YYJSB80002) and the Par -Eu Scholars

Program.

REFERENCES

[1] S. Mousseron-Grall, J. Kejzlarova-Lepesant, T. Burme-

ster and C. Chihara, “Sequence, Structure and Evolution

of the Ecdysone-Inducible Lsp-2 Gene of Drosophila

melanogaster,” Biochemistry, Vol. 245, 1997, pp. 192-

198.

[2] S. O. Zakharkin, A. V. Gordadze and S. E. Korochkina,

“Molecular Cloning and Expression of a Hexamerin

cDNA from the Malaria Mosquito, Anopheles gambiae,”

Biochemistry, Vol. 246, 1997, pp. 719-726.

[3] T. Burmester, C. Antoniewski and J.-A. Lepesant, “Ec-

dysone-Regulation of Synthesis and Processing of Fat

Body Protein 1, the Larval Serum Protein Receptor of

Drosophila melanogaster,” Biochemistry, Vol. 262, 1999.

pp. 49-55.

[4] B. Chen and T. Kayukawa, “DaTrypsin, a Novel Clip-

Domain Serine Proteinase Gene Up-Regulated during

Winter and Summer Diapauses of the Onion Maggot,”

Delia antiqua,” Gene, Vol. 347, 2005, pp. 115-123.

http://dx.doi.org/10.1016/j.gene.2004.12.026

[5] D. A. Hahn1, L. N. James1 and K. R. Milne1, “Life His-

tory Plasticity after Attaining a Dietary Threshold for Re-

production Is Associated with Protein Storage in Flesh

Flies,” NIH Public Access, Vol. 22, No. 6, 2008, pp.

1081-1090.

[6] C. K. Moreira, M. de L. Capurro and M. Walter, “Prima ry

Characterization and Basal Promoter Activity of Two

Hexamerin Genes of Musca domestica,” Journal of Insec t

Science, Vol. 4, 2004, p. 2.

[7] A. S. Cristino and F. M. F. Nunes, “Organization, Evolu-

tion and Transcriptional Proﬁle of Hexamerin Genes of

the Parasitic Wasp Nasonia vitripennis (Hymenoptera:

Pteromalidae),” Insect Molecular Biology, Vol. 19, 2010.

pp. 137-146.

http://dx.doi.org/10.1111/j.1365-2583.2009.00970.x

[8] J. Godlewski, B. Kłudkiewicz and K. Grzelak, “Expres-

sion of Larval Hemolymph Proteins (Lhp) Genes and

Protein Synthesis in the Fat Body of Greater Wax Moth

(Galleria mellonella) Larvae during Diapause,” Journal

of Insect Physiology, Vol. 47, 2001, pp. 759-766.

http://dx.doi.org/10.1016/S0022-1910(01)00050-6

[9] P. Nagamanju, I. A. Hansen and T. Burmester, “Complete

Sequence, Expression and Evolution of Two Members of

the Hexamerin Protein Family during the Larval Devel-

opment of the Rice Moth, Corcyra cephalonica,” Insect

Del. a n tiq ue

Cal. vi c i n a

Mus. d o mestic a

Dr o . melan og a s ter

Cul. q u inq u efasc ia tu s

Bom. mori

100

J. J. XU ET AL.

490

Biochemistry and Molecular Biology, Vol. 33, 2003, pp.

73-80. http://dx.doi.org/10.1016/S0965-1748(02)00178-9

[10] C. A. D. De Korta and A. B. Koopmanschapa, “Nucleo-

tide and Deduced Amino Acid Sequence of a cDNA

Clone Encoding Diapause Protein 1, an Arylphorin-Type

Storage Hexamer of the Colorado Potato Beetle,” Journal

of Insect Physiology, Vol. 40, 1994, pp. 527-535.

http://dx.doi.org/10.1016/0022-1910(94)90126-0

[11] J. Tungjitwitayakul, T. Singtripop, A. Nettagul, Y. Oda,

N. Tatun, T. Sekimoto and S. Sakurai, “Identification,

Characterization, and Developmental Regulation of Two

Storage Proteins in the Bamboo Borer Omphisa fusci-

dentalis,” Journal of Insect Physiology, Vol. 54, 2008, pp.

62-76.