Ribose-phosphate pyrophosphokinase 1 (PRPS1) associated with deltamethrin resistance in Culex pipiens pallens

Abstract

Ribose-phosphate pyrophosphokinase 1 (PRPS1) was identified and isolated as a differentially expressed gene between deltamethrin-susceptible (DS) and deltamethrin-resistant (DR) Culex pipiens pallens and Aedes albopictus C6/36 cell line through microarray and 2D-Gel. An open reading frame of PRPS1 cloned from C. pipiens pallens has 1,011 bp and encodes for a 336 amino acids protein which shares high homology with Culex quinquefasciatus. Real-time polymerase chain reaction was used to determine the transcript expression level of PRPS1 in DS and DR strains. The expression levels of PRPS1 were higher in DR laboratory strains and natural population JXZ-DR, JXZ-LDR. PRPS1 was also detected and expressed at all developmental stages of C. pipiens pallens and increased expression level in DR3 strain than DS strain in the third and fourth instar larvae, female and male stages. In addition, to further investigate the role of PRPS1 in deltamethrin resistance, PRPS1 was transiently expressed in A. albopictus C6/36 cells and detected by western blotting. Cells transfected with PRPS1 had an increased resistance to deltamethrin compared with control cells. These results suggested that the increased expression level of PRPS1 may play roles in the regulation of deltamethrin resistance.

Fig. 1

The nucleotide and deduced amino acid sequence of Culex pipiens pallens PRPS1. The deduced amino acid sequence is presented below the nucleotide sequence in single letter code. The putative polyadenylation signals of the 3′-untranslated region are underlined by red line. The initial code “ATG” and the termination code “TAA” are also boxed. N-terminal domain is frequently found to the ribose-phosphate pyrophosphokinase is underlined by black line. Phosphoribosyltransferase (PRT)-type I domain which catalyzes the displacement of the alpha-1′-pyrophosphate of 5-phosphoribosyl-alpha1-pyrpphosphate (PRPP) by a nitrogen-containing nucleophile is underlined by a green line

Fig. 2

a Amino acid sequence alignment of Culex pipiens pallens PRPS1. Asterisks indicate identical amino acid, and dots indicate similar amino acids. b Phylogenetic relationships of PRPS1 among C. pipiens pallens and some other species. Species name and GenBank Accession No.: C. pipiens pallens, JQ478426; Aedes aegypti, XP_001648571.1; Culex quinquefasciatus, XP_001846292.1; Acromyrmex echinatior, EGI65763.1; Harpegnathos saltator, EFN85070.1; Tribolium castaneum, EFA00168.1; Glossina morsitans morsitans, ADD19462.1; Drosophila mojavensis, XP_002007996.1

Fig. 3

Relative mRNA expression levels of Culex pipiens pallens PRPS1. a PRPS1 relative expression levels in DS strain and DR1, DR2, and DR3 strains. b PRPS1 transcripts at different developmental stages. c PRPS1 relative expression levels in JXZ-DS strain and JXZ-DR strain. d PRPS1 relative expression levels in JXZ-LDS strain and JXZ-LDR strain. Results are normalized against the internal control actin. Results are expressed as “mean ±SD” of three independent experiments. *P<0.05

Fig. 4

a Western blot analysis of PRPS1 expression in transiently transfected cells. 1, C6/36-control (vector control); 2, C6/36-PRPS1. Tubulin was used as an internal control. Three independent experiments were repeated. b Overexpression of PRPS1 enhances deltamethrin resistance in C6/36 cells. Transiently transfected cells C6/36-control and C6/36-PRPS1 were treated with deltamethrin at the indicated concentrations, and cell viability was measured after 48 h treatment. The percentage of viable cells is shown relative to the 0-μg/ml concentration. Results are expressed as mean±SD, and three independent experiments were done. *P<0.05 compared to C6/36-control