Cloning And Characterization Of Rpl22, A Novel Deltamethrin Resistance Associated Gene From Culex Pipiens Pallens

Many new and reemerging mosquito-borne diseases threaten the public health. Chemical control is a major method to manage mosquito-borne diseases. But under the natural selection, excessive and continuous application of insecticides has caused the development of insecticide resistance, which has become the major obstacle to controlling the mosquito-borne diseases. Identification and characterization of insecticide resistance genes will contribute to clarify the mechanism of insecticide resistance and find the target of insecticide resistance management.To investigate the insecticide resistance in mosquitoes, we had employed suppression subtractive hybridization (SSH) and cDNA microarray to identify differentially expressed genes between deltamethrin-susceptible and -resistant strains of Cx. pipiens pallens in our previous experiments, and some deltamethrin resistance related genes were isolated. One of the highly expressed genes in deltamethrin-resistant strain was a RPL22-homolog. In this study, we acquired the full length sequence of RPL22 by 5'-RACE and 3'-RACE methods, detected the differential expression by realtime PCR between deltamethrin-susceptible and -resistant strains of Cx. pipiens pallens and mosquito cells, and explored the relationship between RPL22 and deltamethrin resistance in mosquito cell line by overexpression, RNA interference and inducible higher-expression methods. At the same time, we also detected the mRNA level of CYP6A1 which is an important insecticide resistance gene, and measured the mRNA level of RPL22 in different development stages of the Culex mosquito.RPL22 (GenBank accession no. EF990190) was cloned from Cx. pipiens pallens. An open reading frame (ORF) of 447 bps was found to encode a putative 148 amino acids protein which shares 90% and 80% identity with RPL22 proteins from Aedes aegypti and Anopheles gambiae respectively.Real-time quantitative PCR analysis demonstrated that the transcription level of RPL22 in deltamethrin-resistant strain was 2.57 folds as high as that in deltamethrin-susceptible strain of Cx. pipiens pallens (p<0.001). And the transcription level of RPL22 in deltamethrin-resistant mosquito cells was 1.71 folds as high as that in deltamethrin-susceptible mosquito cells (p<0.001).The expression plasmid of RPL22 was constructed and RPL22 was overexpressed in C6/36 cells. Viability of C6/36-RPL22 cells in the presence of deltamethrin was compared with control cells to observe variance of deltamethrin resistance of these cells. The results showed that C6/36-RPL22 cells are relatively more susceptible to deltamethrin compared with the control. And the mRNA level of CYP6A1was decreased to 41.8% when RPL22 was overexpressed (p<0.001).RPL22 expression in resistant mosquito cells was knocked down by RNA interference, and the results showed that the resistant cell became more susceptible to deltamethrin compared with the control. At the same time, the mRNA level of CYP6A1 was also decreased to 82.5% when RPL22 was down-regulated.To exclude the side-effect of RPL22 overpression, we constructed the inducible expression vector of RPL22 and empty vector. Inducible expression experiment showed that transcription level of RPL22 was 7.9 folds as high as that in the control. And deltamethrin cytotoxicity assay demonstrated that RPL22 could promote the deltamethrin resistance, when the expression level of RPL22 was close to its natural higher expression levels.In this study, we cloned the full length of RPL22, and presented the first evidence that RPL22 confer the deltamethrin resistance in mosquito. And our results provided new evidence for clarifying the molecular mechanisms of insecticide resistance and establishing the new resistance testing methods. It has important theoretical value and potentially practical application .