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Cloning and eukaryotic expression of olfaction-related Gqα-protein gene in English grain aphid (Sitobion avenae)

Published online by Cambridge University Press:  30 October 2009

Fan Jia ,
Chen Ju-Lian ,
Cheng Deng-Fa  and
Sun Jing-Rui
Fan Jia
Affiliation:
State Key Laboratory for Biology of Plant Diseases and Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China
Chen Ju-Lian*
Affiliation:
State Key Laboratory for Biology of Plant Diseases and Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China
Cheng Deng-Fa
Affiliation:
State Key Laboratory for Biology of Plant Diseases and Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China
Sun Jing-Rui
Affiliation:
State Key Laboratory for Biology of Plant Diseases and Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China
*
*Corresponding author. E-mail: jlchen@ippcaas.cn or cjl2000@hotmail.com
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Abstract

Based on conserved homologous amino-acid sequences of the Gq protein α subunit in arthropods, a pair of degenerate primers were designed to amplify the gene from the English grain aphid (Sitobion avenae), using reverse transcriptase polymerase chain reaction (RT-PCR) and (3′/5′)-rapid amplification of cDNA ends (3′/5′ RACE) techniques. A Gqα protein was obtained from the alate adult aphids. The open reading-frame was 1062 bp, encoding 352 amino-acid residues with a calculated molecular weight of 40.8 kDa. The cDNA sequence was deposited in GenBank with accession no. EF638906. The deduced amino-acid sequence of Gqα shared a high identity (≥82.17%) with reported Gqα from other insects and even vertebrates, and had the typical characteristics of Gqα protein. In order to explore the function of the Gqα gene, a eukaryotic expressional system (baculovirus expression vector system, BEVS) was constructed by TOPO and Gateway techniques. After the recombinant reaction occurring between pUC-Gqα and the Gateway-adapted baculovirus DNA from Autographa californica nuclear polyhedrosis virus (AcMNPV), the construct recombinant viruses containing V5-His6Gqα were transfected singly into the insect cell line of Tn-5B1-4. After collecting the infected cell, detection was conducted by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting. The result showed that the system comprising recombinant baculovirus and Tn could express Gqα protein successfully.

Keywords

English grain aphidprotein related with olfactoryGqα proteinGqα gene cloningbaculovirus expression vector system (BEVS)
Type
Research Papers
Information
Chinese Journal of Agricultural Biotechnology , Volume 6 , Issue 2 , August 2009 , pp. 111 - 117
Copyright
Copyright © China Agricultural University 2009

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