Molecular Cloning And Characterization Of A P-Glycoprotein From The Abamectin-resistant Diamondback Moth, Plutella Xylostella (Lepidoptera:Plutellidae)

Diamondback moth (DBM), Plutella xylostella (L.)(Lepidoptera: Plutellidae), is a major pest of cruciferous plants worldwide. Macrocyclic lactones such as abamectin are major control agents for DBM control. However, widespread resistance to abamectin poses a serious threat to the management of DBM. P-glycoprotein (Pgp), a member of the ABC transporter superfamily, plays a crucial role in the removal of amphiphilic xenobiotics, suggesting a mechanism for drug resistance in target organisms. In this study, Abamectin-resistant strain was selected in laboratory and P-glycoprotein gene was cloned and characterized from P. xylostella. The differences of sequence and transcriptional expression levels in susceptible and resistant strains were detected. All of these work, with the addition of RNAi, were used to understand the role of Pgp in the resistance to abamectin in DBM.1. Laboratory selection of abamectin resistance in P. xylostellaThe resistant strain (ABM-R) was selected continuously with formulated abamectin. Compared to the relative susceptible strain (ABM-S), the resistance ratio was217.68-fold. The LC50value of ABM-R was5.442mg/L, while LC50of ABM-S was0.0025mg/L.2. Clone and characterization of P-glycoprotein in P. xylostellaThe full-length cDNA sequence of a Pgp gene from P. xylostella was obtained and named as PxPgpl. It has a3774bp open reading frame (ORF), a133bp5’-untranslated region (UTR) containing a TATA box, and a258bp3’-UTR containing a32bp poly-A tail. The PxPgpl cDNA encodes a1257-amino acid peptide with a molecular weight of137.775kDa and an isoelectric point of5.71. The deduced protein has two distinct sections, which mirror each other and is comprised of a transmembrane domain (TMD) containing multiple transmembrane regions and a nucleotide-binding domain (NBD). The TMDs and NBDs were arranged in the N-to C-terminus order of TMD-NBD-TMD-NBD, which is the classical domain architecture of a full ABC transporter. PxPgpl was constantly expressed during the entire life cycle of P. xylostella and the expression level increased continually during the larval stage. PxPgpl in adult males were significantly higher than that in other developmental stages, which were7.82,4.33,4.94and4.35times higher than that in the third-instar larvae, fourth-instar larvae, prepupae and adult females, respectively. In addition, PxPgpl was highly expressed in midgut, malpighian tubules and testes. the relative expression level of PxPgpl were highest in the midgut, which was about11.83-,9.37-, and7.24-fold higher than that in the malpighian tubules, testes and carcass, respectively. Spatial distribution of the protein may participate in the transportation, absorption and metabolism of xenobiotics.3. The differences of sequence and transcriptional expression level of PxPgpl in susceptible and resistant strainsAlthough the full-length sequences of PxPgpl in the resistant and susceptible strains differed at some nucleotide sites, no consistent difference were identified. Elevated expression of PxPgpl was observed in P. xylostella strains after they were exposed to the abamectin treatment. In addition, the constitutive expressions of PxPgpl were significantly higher in laboratory-selected and field-collected resistant strains in comparison to their susceptible counterpart. PxPgpl expression levels of ABM-R and ZJ were9.51-and3.51-fold higher than that in the susceptible strain, respectively. Therefore, PxPgpl is possibly participate in the transportation of abamectin and contributes to resistance in P. xylostella.4. Primary work of RNA interferenceIn order to identify the function of PxPgpl, RNAi was used. Primers of dsRNA for NBD2region and TM6region were designed and selected according to the sequence and characterizations of the PxPgpl.