Acta Entomologica SinicaDecember 201154 12 1445 1451

ISSN 0454-6296

1
3
4
2
3

2
1*
2*

1 271018 2
100101 3 650201 4 100193

Acyrthosiphon pisum

13 14 4
14 6 3 CXXC
9 scaffold PCR

DNA

Q966
A
0454-6296 2011 12-1445-07

Cloning and expression profiling of a saliva protein family at different

developmental stages in Acyrthosiphon pisum Hemiptera Aphididae
ZHANG Yan1 WU Guo-Xing3 GUO Kun4 WANG Wei2 DING Xu-Po3 SONG Xi-Ming2 XU YongYu1* CUI Feng2* 1 College of Plant Protection Shandong Agricultural University Tai an
Shandong 271018China 2 State Key Laboratory of Integrated Management of Pest Insects Rodents
Institute of Zoology Chinese Academy of Sciences Beijing 100101 China 3 College of Plant
ProtectionYunnan Agricultural UniversityKunming 650201China 4 Institute of Medicinal Plant
DevelopmentChinese Academy of Medical SciencesBeijing 100193China
Abstract The pea aphidAcyrthosiphon pisumis an important piercing-sucking pest The secreted
saliva plays roles in feeding host plants and virus transmission In order to explore the function of saliva
proteinswe cloned an unknown protein family that is highly expressed in salivary glands of the pea
aphid The family consists of 13 geneswhich encode 14 proteinsand four of them are highly expressed
in salivary glands The family is unique to aphids and enriched in cysteine Fourteen cysteines are
conserved6 of which form three CXXC domains Genes of this family have no introns and are distributed
in 9 scaffolds of the aphid genome Semi-quantitative reverse transcription PCR showed that there was no
stage-specific expression for this family We speculate that the expression of this family may be tissuespecific and this protein family probably has the functions of oxidoreductases or DNA methyltransferases
Key words Acyrthosiphon pisum protein family saliva proteins developmental stage gene cloning
gene expression

2006

KSCX2-EW-N-05 201103022
1984 E-mail mili05@ 163 com
*
Corresponding authorsE-mail xuyy@ sdau edu cn cuif@ ioz ac cn
Received 2011-07-12 Accepted 2011-09-28

1446

Acta Entomologica Sinica

54

Eurygaster integriceps 1

23

1246 8 d 123 4
2006

Kazzazi et al2005
Mayetiola destructor Say

10 mg 5
80

RNeasy Mini Qiagen

Promega EZNA Gel Extration

Chen et al2004 2006

Kit Axygen pGEM-T easy Vector

Promega Escherichia coli DH 5

Tiangen
1 2 RNA cDNA

2008
Acyrthosiphon pisum

RNeasy Mini
RNA 80 2 mL RNase-

The
International Aphid Genomics Consortium2010
EST Carolan et
al2011

EST 4
Carolan et al2011

9

semi-quantitative reverse
transcription and polymerase chain reactionSemi-QRTPCR

A pisum Pisum
sativum 21 1 RH

1 1

60% 5% 16L 8D 40 cm 40
cm 40 cm Vicia faba

free 600 L Buffer

RLT 6 L -
5 min 13 000 r / min 5 min

DNase RNA 30
L10
Buffer 5 LDNase 1 LNuclease-free

water 14 L 37 20 30 min
5 L DNase Inactive Reagent
2 3 2 min 10 000 g
5 min RNase-free

cDNA 1 g RNA
cDNA Promega

1 3

UTR ORF PCR 1 cDNA

PCR 50 L
2+
10 LA PCR Buffer with Mg 8 L
dNTP 2 5 mmol / L 5 L 10 mmol / L
1 2 cDNA 1 LLA Taq DNA

5 U / L 0 5 L 33 5 L
94 5 min 94 30 s55
56 30 s72 40 s 1 min 35
72 5 min

12

1
Table 1

1447

PCR

Primers and products of PCR amplification

5' 3'

bp

ORF bp

GenBank

Primer sequence

Product size

ORF length

GenBank accession number of product

F AATCACAGCAATAATACACATAC
R ATAATTTGCATACCACTGTCAC
F GGTTAGTCAATCAGCTCCGT
R TAATTTGCGTACCACTACCAC
F GAGTCAGTCAATCAGCTCCG
R AACTGATCCCAGCCACTATC
F GAGTCAGTCAATCAGCTCCG
R CCTTCGTGCATTCGTTGTC
F ACATATATATAAATCCAGTCGAC
R TCGGAAGGGCATACCACTGC
F TTGTACGGATCCGACGAAGA
R ATTATAATCTCACGGCGGATG
F GCGATCTACTGAAGAGCAGT
R CAAATATGGTACACGTAGTCCA
F CCACCGAGAAGCAGCAGTTT
R TGCACATTTTCAACGCGTCC
F CTGCAAGCGAACGACCAAGC
R TAGTCTACCTACCACTGCTG
F GTACTGCATATTATTATAGGTAAG
R AAACGGTGAACGTGCTGGC
F ATCGACTTCGACACTGCAAG
R GTGCTGGCTGGCACCGTAC
F GCTCGCATTGTCATATTGGTT
R GAGGGTCGTACCTGGTTTCG
F CCACGACCACGTGGTTACGT
R GTACGGAGCTTACTTGCTCC
F TCATTCACGGAACAACTTCG
R TATACCTAATCTCACAACGGC
F TCGTTACCCTCGGAAAGTC
R GTTGGCATAAGGTGGTTGT
ORF Open reading frame

773

399

XP_003244694

819

399

XP_003243676

352

387 *

BAH 70645

314

321 *

NP_001156276

731

384

XP_003240468

176

393 *

XP_003244691

590

390

JN092371

350

393 *

XP_003242394

595

393

XP_003244696

454

390

JN092373

505

372

JN092374

225

186 *

JN092375

868

354

JN092372

381

384 *

XP_003242391

108

408

NM_001126221 L27

Complete open reading frame according to the genome of

Acyrthosiphon pisum

PCR EZNA Gel

Extraction Kit pGEM-T easy

5 U / L 0 2 L 50 L

Vector DH5

94 5 min
94 30 s55 /56 30 s72 1 min

3
1 4

Semi-QRT-PCR

0 25 mL EP 10
LA PCR Buffer with Mg2 + 5 LdNTP 2 5 mmol /
L 4 L 10 mmol / L 1 L
10 mmol / L 1 LcDNA 1 LLA Taq DNA

35 72 5 min
L27 55 30 s 29

PCR 1% 2% 400 bp

 Acta Entomologica Sinica

1448

1 5

BLAST
CLUSTALW http / / www ebi ac uk /

54

61 117
132
60% 92%
29% 89% GenBank

Tools / msa / clustalw2 /

MEGA 4 0 Neighbor-Joining

1 GenBank EST
EST Aphis gossypii

method SignalP 3 0 Server

Myzus persicae

JN092375
NP001156276 14

2 1

cDNA

15 14
3 CXXC X

14 13
BAH70645 NP001156276

1 N

22
JN092375

XP003242391
1

1
Fig 1

Alignment of protein sequences of the unknown gene family

Yellow shadow Predicted signal peptides Black shadow 85% Amino acid residues
with identity over 85% * 14 14 conserved cysteines

EST
2 3 EST

3 3
4 3 14
15 2

2 2

4 1

12

1449

9 scaffold
4 scaffold 2 scaffold
111793116358120774 128151
scaffold 7
6
3
2 3

Semi-QRT-PCR

XP_003244694
5
4 XP_003244694 5
5

2
Fig 2

Phylogenetic tree of protein sequences of

the unknown family

MEGA Neighbor Joining

1 000 4
Phylogenetic
tree is built using Neighbor-Joining method in MEGA software 1 000
bootstrap supporting values are shown at branch nodesand the branch
length indicates the evolutionary distance Members of four clusters are
designated with different colors The members that are highly expressed in
salivary glands of aphids are shaded in gray

3
Fig 3

2006

James and Perry2004

Distribution and transcriptional direction of the members of unknown family on the genome of Acyrthosiphon pisum

2 scaffold EQ The colors of family members

are the same as in Fig 2 Arrows indicate the transcriptional direction The number of cysteines in each member is put under the arrow The scaffold
number on genome is initiated with EQ

 Acta Entomologica Sinica

1450

54

Chivers et al1997 DNA

CpG Pradhan et al
2008
DNA

RT-PCR 13

Fig 4

Expression of 13 genes of the unknown family in

Acyrthosiphon pisum at different developmental stages

detected by Semi-QRT-PCR
1 5 1 4 1st to 4th instar nymphs and adult
respectively L27 Inner control

Semi-QRT-PCR

EST

XP_003244694

Semi-QRT-PCR

Cottrez et al1994
2006 RNA cDNA

RT-PCR XP_003244694

Fig 5

Expression of XP_003244694 in Acyrthosiphon pisum at

different developmental stages detected by semi-QRT-PCR

1 5 1 4 1st to 4th instar nymphs and adult
respectively A E 5 Five biological replicates L27
Inner control

14

PCR
Semi-QRT-PCR mRNA
Udvardi et al2008
Semi-QRT-PCR
Freeman et al1999 Marone et al2001

PCR
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