Cloning And Expression Of Gene Encoding Gqα-protein In The England Grain Aphid Sitobion Avenae

The aphid is not only the primary pest of crops in variable zone but one of the most important pest to global agriculture also. For insects, olfaction plays an essential role in processing chemical signals from the environment, leading to the detection of food, reproductive partners, oviposition sites, host, prey or predators. Elucidation of the olfaction signalling will provide the theoretical foundation for the development of the effective attractant and deterrent, and the fresh ideas and approaches for potent control of Aphid.In this paper, a Gqαgene was cloned by RACE techniques and PCR from the England grain aphid, Sitobion avenae. further, the recombinant protein V5-His6Gqαwere obtained by eukaryotic expression after an exhaustive analysis on both the cDNA sequence and amino sequence. The main results are as follows:Gqαwas cloned for the first time from the England grain aphid(Sitobion avenae). Based on conserved homologous amino acid sequences of Gq proteinαsubunit in several animals, a pair of degenerate primers were designed to amplify the gene from the England grain aphid, Sitobion avenae. by RT-PCR and (3′/5′)-RACE techniques, A Gqαwas obtained from the alate adult aphids. The open reading-frame is 1062bp, encoding 352 amino acid residues with calculated molecular weight of 40.8kDa. Both the nucleic acidsequence and amino acid sequence were deposited in GenBank with the accession number, EF638906 and ABR37295. The deduced amino acid sequence of Gqαshared high identity (≥82.17%) with reported Gqαfrom other insects and even vertebrates, and with the typical characteristics of Gqαprotein.In order to explore the function of Gqαgene, a eukaryotic expressional system (Baculovirus Expression Vector System, BEVS) had been constructed by TOPO and Gateway techniques. After the Recombinant reaction between the entry clone vector and Gateway-adapted Baculovirus DNA from AcMNPV, the construct recombinant viruses containing V5-His6Gqαwere transfected into the insect cell line of Tn. After collecting the infected cell , the recombinent expression products were detected by SDS-PAGE and confirmed by Western blotting analysis, which showed that the molecule weight of V5-His6Gqαprotein was about 42 kDa. The result showed that the system of recombinant baculovirus and Tn could expressed Gqαprotein successfully.These achievements provide substaintial foundation for further research of G proteins and G-protein signaling pathways in England grain aphid (Sitobion avenae). It also has important theoretic and practical significance on going deep into interaction between insect and host plant, into pest-resistant mechanism of plant and exploring new control method.