Molecular Cloning And Expression Patterns Of Rhopalosiphum Padi ATP-binding Cassette Transporter Genes

The bird cherry-oat aphid, Rhopalosiphum padi(L.)(Hemiptera: Aphididae), is a major piercing-sucking pest insect of wheat crops worldwide. This pest not only causes direct harm by feeding crops directly, but also serves as a vector for the barley yellow dwarf virus(BYDV), which causes severe yield losses every year. Historically, chemical insecticide spraying has been the main method for controlling R. padi. However, R. padi has developed a high degree of resistance to insecticides after many years of their excessive use. Faced with the selection pressure of powerful insecticides, aphids emerged rapid evolution and adaptation through gene mutation, gene amplification, and the changes of metabolic enzymes activity. Previous studies have shown that the increased gene expression level of ABC(ATP-binding cassette) transporters can reduce the effective concentration of intracellular exogenous substances, leading to insensitivity of insects to insecticides. In the current study, the full-length sequences of 14 R. padi ABC transporter genes were cloned, the bioinformatic characteristics and the expression patterns of four important ABC transporter genes in different insecticides resistant and susceptible strains as well as in different development stages and different tissue types of R. padi were investigated. Moreover, the synergy of ABC transporter inhibitor verapamil to pesticides were also studied. The results provide a foundation for further study of the insecticides resistance mechanisms of R. padi.1. Molecular cloning and bioinformatics analysis of R. padi ABC transporter genesRT-PCR and RACE were used to clone the full-length c DNAs of 14 ABC transporter genes from R. padi. The 14 ABC transporter genes were Rhpa ABCA3, Rhpa ABCB6, Rhpa ABCB7, Rhpa ABCB10, Rhpa ABCC1, Rhpa ABCG1, Rhpa ABCG4, Rhpa ABCG4-1, Rhpa ABCG5, Rhpa ABCG9, Rhpa ABCG20, Rhpa ABCG23, Rhpa ABCG23-1 and Rhpa ABCG23-2. Rhpa ABCG4 and Rhpa ABCG4-1 may be the isomers caused by alternative splicing and the same as Rhpa ABCG23, Rhpa ABCG23-1 and Rhpa ABCG23-2. The respective open reading frame lengths of the genes are 5 837, 2 818, 2 337, 1 812, 5 463, 3 074, 2 678, 2 443, 2 437, 2 924, 3 872, 2 383, 2 432 and 2 256 bp, which encode proteins of 1 635, 839, 683, 603, 1 514, 616, 709, 629, 638, 700, 811, 693, 695 and 686 amino acids, respectively. NCBI Blastp indicated that the amino acid sequences of these 14 ABC transporters show high identity to those of the corresponding proteins from Acyrthosiphon pisum and Diuraphis noxia, with more than 90% identifies. Structural analysis showed that all the 14 proteins have the typical structural features of ABC transporter family, which indicated that these 14 genes belong to ABC transporter gene family. ORF finder analysis showed that all the 14 genes have a complete open reading frame. The ABC transporters from R. padi showed near phylogenetic relationship to the respective ABC transporters from A. pisum and D. noxia. The R. padi ABC transporters were located in each of the two major branches, A and G sub-families were located in one branch, and B and C sub-families in another one. The evolutionary relationship of ABCG20 and ABCG23 from the G subfamily was close as they were clustered in a sub-branch. Similarly, the ABCG1 and ABCG4 were clustered in another sub-branch.2. The expression patterns of R. padi ABC transporter genesq RT-PCR was used to characterize the expression of Rhpa ABCC1, Rhpa ABCG9, Rhpa ABCG20 and Rhpa ABCG23 in different strains. The results showed that the expression levels of Rhpa ABCC1, Rhpa ABCG9, Rhpa ABCG20 and Rhpa ABCG23 were different in different strains. The expression of Rhpa ABCC1, Rhpa ABCG9, Rhpa ABCG20 and Rhpa ABCG23 in different developmental stages were detected and analyzed. The results showed that the four genes were expressed at all developmental stages of R. padi, but the expression of different developmental stages was different. To investigate the expression of the multidrug resistance-associated protein Rhpa ABCC1 in different tissues of R. padi, the specific expression of midgut, integument, leg and head tissue from apterous adult females was to detected and analyzed. Rhpa ABCC1 was expressed in all four tissues, with the highest level in the midgut, where its expression was 4.49-fold higher than that in the leg. We treated apterous adult R. padi females with a LC50 concentration(0.934 mg/L) of chlorpyrifos and a LC50 concentration(0.468 mg/L) of imidacloprid over a 2-day period. Then the expression of Rhpa ABCC1 was measured among surviving insects. The results showed that expression levels of Rhpa ABCC1 upregulated after exposure to the two insecticides, and the relative expression level was significantly different between the imidacloprid treatment after 48 h and water treatment from susceptible strain.3. The synergism of ABC transporter inhibitor verapamil to insecticidesTo determine whether insecticide resistance in R. padi was mediated by ABC transporters, we carried out bioassays using insecticides and insecticides in combination with verapamil. In the presence of 500 μM dose of verapamil, the toxicity of chlorpyrifos, isoprocarb, imidacloprid and cyhalothrin increased 2.72-fold, 1.40-fold, 1.64-fold and 1.50-fold, respectively. Verapamil also enhanced the toxicity of chlorpyrifos by a factor of 1.33 in the chlorpyrifos-resistant strain and enhanced the toxicity of imidacloprid by a factor of 1.26 in the imidacloprid-resistant strain.