Silencing Of Cytochrome P450in Sitobion Avenae And Myzuspersicae Through RNA Interference

Sitobion avenae and Myzus persicae were major pests of a variety of commercial crops,can bring significant economic losses about agricultural production, cytochrome P450geneis a class of widely exist in many organisms, the heme iron-binding protein genesuperfamily. Cytochrome P450involved in metabolizing drug of organisms, synthetizingimportant hormones and other metabolic pathways, it closely related to many life activitiesof organisms. In recent years, the plant-mediated RNAi technology had become one of thehotspots of the crop insect genetic engineering. Cytochrome P450gene fragment ofSitobion avenae and Myzus persicae were cloned by PCR technology, cytochrome P450gene conserved sequence dsRNA of Sitobion avenae and Myzus persicae were synthesizedby using double-stranded RNA synthesis kit, the expression level of cytochrome P450geneof aphid that fed by dsRNA, was analyzed by RT-PCR, to research whether cytochromeP450gene of Sitobion avenae and Myzus persicae can be silenced by using RNAitechnology. So as to achieve the purpose of controlling the pest’s hazards, provide the basisfor the use of RNAi technology to create the system of resisting of aphid of wheat andvegetables.In this study, the following experimental results were obtained:1: Cytochrome P450gene fragments were cloned from cDNA of Sitobion avenae andMyzus persicae, the homology of cloned fragments was90.1%. Cytochrome P450genecDNA sequence informations and amino acid sequence informations of Sitobion avenae andMyzus persicae were obtained after sequencing, and choosing13kinds of insects whoseamino acid sequences of cytochrome P450were similar and constructing a phylogenetictree.2: It was found that with the increasing of the the feeding P450dsRNA concentrationof Sitobion avenae and Myzus persicae, the mortality rise over time, the highest mortalitycan be up to75.56%and64.44%, presenting significant difference compared with controlgroup in the statistical. While as GFP（Green Fluorescent Protein) dsRNA concentrationincreasing, the mortality of wheat aphid and green peach aphid didn’t increased comparedwith the control group, and didn’t show significant differences in the statistical. Both aphids showed retarded after8d of feeding P450dsRNA, the same situation were not observed inaphids fed no dsRNA and GFP dsRNA.3: Real-time PCR results show that the design of fluorescent quantitative primerspecificity were well, they can be used for quantitative PCR experiments. By processingand analysizing the data produced by fluorescence quantitative instrument, we found thatafter feeding2,4,6,8d (the dsRNA concentration of experimental group is7.5ng·μl-1), theexpression level of cytochrome P450gene of Sitobion avenae and Myzus persicaedecreased continuously with the extension of feeding time, indicating that the cytochromeP450dsRNA of the conserved sequences of the wheat aphid and green peach aphid fed thecorresponding aphids in vivo, can significantly reduced the level of expression of thecytochrome P450gene and cause the silencing of the purpose of gene and the death ofaphids.