Sequence analysis of <i>R</i>. <i>padi</i> CPR.

<p>(A) Schematic drawing of RpCPR. Membrane anchor, conserved FMN-, FAD- and NADP-binding domains, FAD-binding motif (Arg₄₅₇ x Tyr₄₅₉ Ser₄₅₆), and catalytic residues (Ser₄₆₀, Cys₆₃₃, Asp₆₇₈ and Trp₆₈₀) are shown. (B) Comparison of the deduced amino acid sequence of RpCPR with those of other CPRs deposited into GenBank. The species and accession numbers are as follows: <i>Musca domestica</i> (NP_001273818), <i>Drosophila melanogaster</i> (NP_477158) and <i>Rattus norvegicus</i> (NP_113764).</p

Relative expression of <i>R</i>. <i>padi</i> CPR at different developmental stages.

<p>Relative expression levels of <i>RpCPR</i> at different developmental stages were normalized to those in the adult. Data shown as the mean ± SE; different letters denoted a significant difference among samples (<i>P</i><0.05, one-way ANOVA).</p

Molecular Cloning, Expression Pattern and Polymorphisms of NADPH-Cytochrome P450 Reductase in the Bird Cherry-Oat Aphid <i>Rhopalosiphum padi</i> (L.)

<div><p>NADPH–cytochrome P450 reductase (CPR) plays an important role in the cytochrome P450 (CYP)-mediated metabolism of endogenous and exogenous substrates. CPR has been found to be associated with insecticide metabolism and resistance in many insects. However, information regarding CPR in the bird cherry-oat aphid, <i>Rhopalosiphum padi</i>, is unavailable. In the current study, a full-length cDNA (2,476 bp) of <i>CPR</i> (<i>RpCPR</i>) encoding 681 amino acids was cloned from <i>R</i>. <i>padi</i>. Nucleotide sequence and deduced amino acid sequence analysis showed that RpCPR exhibits characteristics of classical CPRs and shares high identities with those of other insects, especially with the pea aphid, <i>Acyrthosiphon pisum</i>. The mRNA of <i>RpCPR</i> was expressed at all developmental stages, with the highest expression level found in the second instar and the lowest in adult. Expression levels of <i>RpCPR</i> in isoprocarb-resistant and imidacloprid-resistant strains were 3.74- and 3.53-fold higher, respectively, than that of a susceptible strain. <i>RpCPR</i> expression could also be induced by low concentrations (LC₃₀) of isoprocarb and imidacloprid. Moreover, we sequenced the open reading frame (ORF) of <i>RpCPR</i> from 167 field samples collected in 11 geographical populations. Three hundred and thirty-four SNPs were detected, of which, 65 were found in more than two individuals. One hundred and ninety-four missense mutations were present in the amino acid sequence, of which, the P484S mutant had an allele frequency of 35.1%. The present results suggest that <i>RpCPR</i> may play an important role in the P450-mediated insecticide resistance of <i>R</i>. <i>padi</i> to isoprocarb and imidacloprid and possibly other insecticides. Meanwhile, <i>RpCPR</i>maintains high genetic diversity in natural individuals, which provides the possibility of studying potential correlations between variants and certain special physiological characters.</p></div

Primers used for cloning and expression analysis of <i>RpCPR</i> in <i>R</i>. <i>padi</i>.

<p>Primers used for cloning and expression analysis of <i>RpCPR</i> in <i>R</i>. <i>padi</i>.</p

Sampling information and population statistics for <i>R</i>. <i>padi</i> investigated using <i>RpCPR</i>.

<p>Sampling information and population statistics for <i>R</i>. <i>padi</i> investigated using <i>RpCPR</i>.</p

Relative expression levels of <i>RpCPR</i> in the susceptible strain (SS), the isoprocarb-resistant strain (IS-R) and the imidacloprid-resistant strain (IM-R).

<p>The expression level of <i>RpCPR</i> in SS was set to 1. Data shown as the mean ± SE; different letters denoted a significant difference among samples (<i>P</i><0.05, one-way ANOVA).</p

<i>R</i>. <i>padi</i> CPR protein sequence depicting missense mutations or/and polymorphisms.

<p>Mutation found only in one individual is indicated by a hollow triangle while polymorphic variants identified in more than two individuals are indicated by a solid triangle. The digital number after the mutated amino acids indicated the number of the aphid individuals with the mutation.</p

Phylogenetic tree of <i>R</i>. <i>padi</i> CPR with other insect CPRs.

<p>The neighbor-joining tree was generated using MEGA 5.0 software, and the phylogeny was tested by the bootstrap method with 1,000 replications. Bootstrap values >50% are shown. <i>R</i>. <i>padi</i> CPR is indicated by solid circles. The GenBank accession numbers of the sequences used were listed in supporting information <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154633#pone.0154633.s001" target="_blank">S1 Table</a>.</p