CDNA Difference And RYR Gene Analysis In Diamide Insecticides Resistant And Susceptible Strains Of Plutella Xylostella

Diamondback moth(Plutella xylostella L.) belongs to the lepidopteran cabbage moths division, it is one of the important pests of cruciferous vegetables in the world. Because of the strong fertility, overlapping generation, and the applying pesticides disorderly in the field, diamondback moth developed high resistance to a variety of insecticides, which caused a big problem for controlling the insect pest. Anthranilic diamides insecticides is a ryanodine receptor(RyR) activator, it could continuous active the intracellular calcium ions releasing in ryanodine receptors, which is the reason why the pesticides could kill the pests. With the anthranilic diamides insecticides application, the insect pest resistance issue increased fast. In order to study the resistance mechanism of anthranilic diamides insecticides in diamondback moth, the improved cDNA representative difference analysis methods(cDNA RDA) was used in this study to analysis the genetic differences between susceptible strain(S) and the insecticide resistance strain in Plutella xylostella. The driver cDNA comes from diamondback moth S strain, while the tester cDNA comes from the chlorantraniliprole resistant strain and the flubendiamide resistant strain, respectively. The results showed that the different cDNA fragments were detected in the above two resistant strains. In addition, the RyR mRNA relative expression and gene mutation site for the resistant strains were investigated. We hope this study would provide some clues for management strategy of anthranilic diamides insecticides. The main results are as follows:1. Using the resistant strain of previous study, the resistant selection was continued with chlorantraniliprole to 30 generations, the LD50 of F30 was 384.68μg/g, with 442.16-fold resistant ratio. The resistance developed rapidly from F19 to F25, and slowly after F25. The flubendiamide resistant strain selection was also carried out, until now 21 generation, the LD50 was 165.98μg/g, with 180.22-fold resistant ratio.2. Using cDNA RDA method, we obtained two discrepancy sequences from chlorant raniliprole and flubendiamide resistance strain, which shows high homology to 28 S rRNA gene. And, one sequence shows homology of 97% to cytochrome P450-like TBP and the other one shows homology of 93% to TcasGA2_TC010626 of hypothetical protei n in Helicoverparmigera.Meanwhile, we also got two unknown sequnences.3. We have cloned and characterized about 1690 bp sequence of PxRyR COOH-terminal of S, F20, BY and ZC, the result of sequence alignment suggested that the mutation site G4946 E was included, however, this site was replaced by Valine(V) in some individuals of the BY and ZC filed population. 4. Real-time quantitative PCR was used to determine the mRNA expression level of RyR in the ZC population, the expression levels of Px RyR in ZC and BY is relative stability, but the LD50 value is gradually decreased, that means the resistant level was decreased in the period of testing. The mRNA expression in flubendiamide-resistant strain were increased with the resistant level increasing, which is 4.28-fold compared with relative S strain, however, mRNA expression gradually decreasing when resistant level decreasing, while no any pesticide applying.