Cloning Of A Novel Ribosomal Protein S4 Gene From Culex Pipiens Pallens And Preliminary Characterization Of Its Role In Deltamethrin Resistance

【Objective】To acquire the full length cDNA of ribosomal protein S4(RPS4) gene from Cx. pipiens pallens. To further research the relationship between RPS4 gene and insecticide resistance.【Methods】A pair of special primers were designed according to EST sequence(GenBank accession no. BE247804). The full length cDNA of RPS4 was amplified by PCR with the special primers and the vetor primers(SP6 or T7). The ORF(open reading frame) sequence of RPS4 was cloned by RT-PCR, sequenced and analyzed with bioinformatic softwares. Transcript expression levels of RPS4 in different developmental stages of the deltamethrin-susceptible and-resistant strains of Cx. pipiens pallens were determined by real-time PCR. The prokaryotic and insect expression plasmids of RPS4 were constructed by gene re-construction methods. The recombinant insect expression vectors were transfected into Aedes albopictus C6/36 cells. Stable-transfected strains were established by screening culture with blasticidin, and the transcript expression of the RPS4 was identified by RT-PCR. The viability of the transfected cells and untransfected ones to deltamethrin at different concentrations was detected by ~3H-TdR incorporation test.【Results】A sequence of 1002 bp including an ORF of 789 bp was gotten(GenBank accession no. DQ647667). Alignment of the deduced amino acid sequence with RPS4 from some species of Diptera revealed sequence identities of 96％with Ae. aegypti RPS4, 91％with Anopheles gambiae RPS4 and 78％with Drosophila RPS4. RPS4 is expressed at all developmental stages. Meanwhile, in pupae and adults, RPS4 is overexpressed in the deltamethrin-resistant strain than-susceptible strain. The expressed plasmids pET-32a(+)/RPS4 and pIB/VS-His/RPS4 were re-constructed successfully. In our study, stable expression of RPS4 in the mosquito C6/36 cells did not confer moderate deltamethrin resistantce (P＞0.05).【Conclusion】The ribosomal protein S4 gene from Cx. pipiens pallens was cloned successfully. RPS4 is overexpressed in the deltamethrin-resistant strain than in the deltamethrin-susceptible strain of Cx. pipiens pallens. In our study, we did not observe the protection of C6/36 cells from deltamethrin resulted from stable expression of RPS4.