Involvement Of Pxapn2and Pxabcc2in Cry1ac Resistance Of Plutella Xylostella

The diamondback moth Plutella xylostella (L.)(Lepidoptera:Plutellidae), is one of the most serious pests on the cruciferous vegetables. The diamondback moth have short generation time, high fecundity, strong adaptability, serious overlapping generations, long distance migration, so it’s difficult to control them in the field. The diamondback moth developed resistence to at least84kinds of insecticides, including Bt formulation. It was estimated that the annual loss caused by the diamondback moth were about the4-5billion dollars worldwide. The resistance management situation is not optimistic.To be the first insect evolved resistance to Bt toxins in the field, lots of researches have been conducted on the biochemical and molecular mechanisms of Bt resistance in the diamondback moth. In this paper, we studied the mRNA expression of PxAPN2in the fourth instar larvae of the different strains of the diamondback moth, as well as the relationship between the expression levels of PxAPN2and resistance level of Cry1Ac in diamondback moth. At the same time, we cloned ABC transporter protein C2gene (PxABCC2) from the resistant and susceptible strains. The mRNA expression levels of PxABCC2in the fourth instar larvae were compared among different strains. Linkage between the PxABCC2locus and resistance to Cry1Ac was determined with a backcross analysis.1.The relationship between PxAPN2expression levels and Cry1Ac resistance in the ROTH strainWe used quantitative real-time PCR to determine the mRNA expression of PxAPN2in the fourth instar larvae from different strains of the diamondback moth. Compared to the susceptible strain ROTH, the expression levels of PxAPN2were0.95-fold in the control strain SZ and0.51-fold in the Cry1Ac-resistant strain SZBT. After feeding with dsRNA of PxAPN2, the expression of PxAPN2in the third instar larvae of the ROTH strain were reduced by57%and30%, and the corresponding levels of resistance to Cryl Ac were increased by6.9-fold and4.3-fold.2. RNAi-based knock-down of PxAPN2reduced Cry1Ac sensitivity in different strainsExpression levels of PxAPN2in the third instar larvae of ROTH strain fed with dsAPN2were reduced by25to35%compared to the control (CK) fed with dsGFP, and resistance to Cryl Ac were increased by3.4-to3.8-fold. Expression levels of PxAPN2were reduced by31to32%in SZ, and resistance to Cry1Ac was increased by2.6-to3.4-fold. Similarly, when PxAPN2was knocked down by20to23%, resistance to Cry1Ac was increased by2.1-to2.2-fold in the Cryl Ac-resistant strain SZBT. These results confirmed that down-regulation of PxAPN2expression can result in resistance to Cry1Ac in P. xylostella strains with different levels of resistance to Cry1Ac.3. Cloning and mRNA expression levels of PxABCC2gene in different strainsThe full-length cDNA of PxABCC2was cloned and sequenced from three different strains (the Cryl Ac-resistant SZBT, the control SZ, and the susceptible ROTH strain). The full-length cDNA of PxABCC2was3997bp, and the mature protein consists of1332amino acids. No amino acid mutations associated with CrylAc resistance were detected among the three strains. However, PxABCC2showed1.4-1.5-fold overexpressin in the control SZ strain, and3.2-3.4-fold overexpression in the resistant SZBT strain when compared to that of the susceptible ROTH strain.4. Linkage analysis between CrylAc resistance and PxABCC2locusOne genetic marker was isolated when comparing the genomic DNA sequences of PxABCC2between the ROTH and SZBT strains. With the isolated marker, we determined the linkage between CrylAc resistance and PxABCC2locus through a backcross analysis. An incomplete genetic linkage was detected between CrylAc resistance and PxABCC2locus.