Molecular Cloning And Expression Ananysis Of P450CYP6CY3 Genes From Rhopalosiphum Padi

Cytochrome P450 monooxygenases(P450s) play an important role in metabolism of both the endogenous and exogenous compounds. Rhopalosiphum padi(Hemiptera:Aphidoidea) is a serious pest insect of cereal plants in many countries. Damage caused by R.padi is increasing year by year in many wheat-growing regions of China. The widely use of insecticides caused the resistance of R.padi to different insecticides. Over-expression of the P450 genes regulates high resistance of insects to varies types of insecticidesIn order to reveal the role of P450 s in the resistance of R.padi to insectides including imidacloprid. We cloned CYP6CY3-1 and CYP6CY3-2, and analyzed the expression level of these two genes in the resistant and susceptible strains of R. padi, the expression level of these two genes after the aphids were treated with 4 different insecticides for 24 hours as well as treated with imidacloprid for different time.1. Using the RACE method, we cloned the full length of CYP6CY3-1 and CYP6CY3-2c DNA sequences. The length of the two genes are 1699 bp and 1721 bp, and the two genes encoded 511 and 532 amino acids, respectively.2. Using the Real Time PCR method, we analyzed the expression level of the insecticide resistant strains and susceptible strain. We found that CYP6CY3-1 and CYP6CY3-2 was up-regulated in imidacloprid resistance strain, with 1.6 and 2.5 times of the expression level to the susceptible strain, respectively. The expression level of CYP6CY3-1 and CYP6CY3-2 was not sigificant up-regulated in the isoprocarb, chlorpyrifos, and Lambda-cyhalothrin resistant strains.3. Twenty-four hours after the R. padi was treated with four different insecticides the expression level of CYP6CY3-1 and CYP6CY3-2 was all significantly higher in all the insecticide treatments compared with the control. The expression level of CYP6CY3-1and CYP6CY3-2 in the imidacloprid treatment was 4 times and 5 times of that in the control, respectively, whilst which in the treatment of isoprocarb, chlorpyrifos and Lambda-cyhalothrin was 10 times and 4 times, 13 times and 7 times, and 11 times and4 times of the expression level in the control, respectively.4. The expression level of the CYP6CY3-1 and CYP6CY3-2 gene, 3h, 6h, 9h, 12 h, 24 h and 36 h after R. padi was treated with LC50 imidacloprid was analyzed. It was shown that CYP6CY3-1 gene expression level significantly increased after 3h, 12 h, 24 h and36h with 14 times, 59 times, 7 times and 1.8 times of the expression level in the control,respectively. And the CYP6CY3-2 gene expression level significantly increased after3 h, 12 h, 24 h and 36 h with 3 times, 26 times, 2 times and 1.8 times of the expression level in the control, respectively.