Research On Resistance Mechanisms Of Pyridaben Mediated By P450 Gene In Phyllotreta Striolata

Phyllotreta striolata is one of the most serious pests of cruciferous vegetable, which is widely distributed in South China, mainly by Guangdong, Guangxi, Fujian and other places. Because of increasing dose of chemical insecticide and spraying frequency, the levels of the insecticide resistance of this pest are increasing. As well as, it is difficult to control this pest. In addition, with the characteristics of diffusion, resistance, and the overlapping armor, this pest outbreak is a serious constraint to the development of cruciferous vegetable industry. P.striolata is becoming more and more serious. Additional resistance mechanisms will be found as genes related to the insecticide resistance, is applied to further examples of resistance. However, the insectcide resistance mechanism of P.striolata has not been published in the literature. Thus, by studying the molecular mechanisms of the insecticide resistance mediated by P450 gene, it would help us to find new prevention and treatment strategies of P.striolata.Based on the previous transcriptome database of P.striolata, we obtained 50 the cytochrome P450 c DNA sequences of P.striolata fragment. It provided effective data for the molecular resistance mechanism mediated by P450. By homology comparison, these candidate genes are divided into the different family such as CYP4, CYP6, CYP9, CYP306, CYP314, CYP315, CYP334, CYP345, CYP349, CYP353. Using rapid-amplification of c DNA ends（RACE） and molecular cloning technology, we lengthened the coding sequences and a total of 19 full-length P450 genes were obtained. Bioinformatics and phylogenic analysis of P. striolata P450 gene were carried out. And Real-time quantitative PCR methods was employed to assess the expression levels of P450 genes in different resistant strains. The results show as follows: 1) Identification and sequence analysis of P450 genes of P.striolataThe full-length c DNAs of P450 are 408-1704 bp nucleic acid and encoding 135-567 amino acids. The sequence Clustal W alignment analysis show that P450 were six conserved domains, including consensus sequence（FXXGXXXCXG） heme-binding loop and helix K conserved absolutely E（W） XXR elements. Signal peptide prediction shows that Pstr₄7 contains seven single peptide composed of a signal peptide encoded by the gene sequence of 1-21 amino acid. The transmembrane structure prediction shows that there is a transmembrane domain such as Pstr₆, Pstr₂6, Pstr₃4, Pstr₃6, Pstr₄1, Pstr₄5, Pstr₄8. And Pstr₄4 have two transmembrane domains.The phylogenic analysis showed that all P450 of P.striolata we studied here were divided into CYP4, CYP6, CYP9 and CYP3 subfamily according to the function definitions. Pstr₂6, Pstr₄5, Pstr₃6, Pstr₃4, Pstr₄1, Pstr₅ were clustered into one clade, which are shorter genetic distance from the CYP6BQ; Pstr₁7, Pstr₄4 CYP9Z4 were clustered into one clade, and the genetic distance is the nearest; Pstr₅0, Pstr₄7, Pstr₆ were clustered into one clade, also the CYP4 Q is close with them in genetic distance. Other genes clustered into a branch, which belong to CYP4, CYP6, CYP9 and CYP3 family. Among them, the P450 gene is closely related with the resistance is mainly CYP4, CYP6 family. 2) The differential expression levels of P.striolata P450 gene of the different resistantstrains.First, the ability of P.striolata to resiste Pyridaben amount of lethal agents LC50 =394μg/m L and LC10=10μg/m L are determined by the immersion method. The c DNA templates of susceptible and resistant strains were constructed, and the expression levels of P450 genes P450 genes in different resistant strains were investigated by Real-time quantitative PCR methods.The results showed that it is different expression levels of 19 P450 genes in different strain, and their patterns of expression is also different with the level of resistance increasing. The relative expression of 12 P450 genes（Pstr₅、Pstr₆、Pstr₇、Pstr₁0、Pstr₁6、Pstr₂6、Pstr₃4、Pstr₄1、Pstr₄5、Pstr₄7、Pstr₄8、Pstr₅0） is increasing in different resistant strains. Pstr₃6 remains the same in different resistant strains. Meanwhile,Five P450 genes（Pstr₁7, Pstr₂9、Pstr₃9, Pstr₄4, Pstr₄6） is decreaseing in P.striolata with the resistance level increasing. The study shows that the resistance of P.striolata to pyridaben could be involve in multiple P450 gene interaction.