The Research Of RNAi Effect On The Resistibility To Aphis Gossypii

RNA interference RNA interference (RNAi) is the specific downregulation of gene expression by double-stranded RNA (dsRNA), which is cleaved into siRNAs with the size of-22bp by dsRNA-specific endonucleases referred to as Dicers. SiRNA is assembled into an RNA-induced silencing complex (RISC) in conjunction with the Argonaute protein, giving the rise to the degradation of a target mRNA. The potential of using RNAi effects to protect plants against insects by downregulating essential gene functions in the herbivore, thus resulting in its death, has been recognized since the discovery of RNAi. The RNAi method to resist aphid meets the need of new strategy to prevent aphid as the disadvantages of traditional method protection against insects have been on the rise. The method of RNAi would be effective, safty and more durable, which gives it preferential right in protecting insects.Mao identified a cytochrome P450 gene, CYP6AE14, which is highly expressed in the insect midgut. Tobacco and Arabidopsis plants were engineered to produce dsRNAs directed against the bollworm CYP6AE14 gene. When plant material of both species was fed to larvae, effective repression of the endogenous CYP6AE14 transcript was observed. Baum's research demonstrated that transgenic corn was engineered to express dsRNA directed against V-ATPase A can effectively suppress the expression endogenous mRNA.The key to the application of RNAi technology to give resistance to herbivorous insects is: (i) identification of a suitable and effective insect target; (ii) dsRNA delivery, which includes in planta expression of dsRNA and delivery of sufficient amounts of intact dsRNA for uptake by the insect. In this study, we select V-ATPase A, Cathepin B, Ribosomal protein L18,vesicle-fusing ATPase as the target of dsRNA. Addtionally, because the feeding of aphis is Piercing-sucking type, in which aphid sucking nutrition from plant using mouthpiece piercing into the phloem of plants, we choose the bidirectional promoter RolC expressing sepecifically in the phloem of plant to express dsRNA. And considering the package role of the coat protein of virus ToMV, the other end of bidirectional promoter was connected with CP gene sequence, expecting the role of CP can effect on dsRNA, of which the circular structure is the original assembly sequence (OAS) of ToMV.5 RNAi vectors aimed to selected gene were constructed concluding the bidirectional promoter RolC expressing sepecifically in the phloem, in the two end of which dsRNA and CP gene are expressed. The RNAi vectors successfully constructed were transferred into wild type Arabidopsis mediated by Agrobacterium. The transgenic plants were identified by PCR analysis. Besides, the expression of dsRNA in transgenic plants were detected by RT-PCR and Northern blot. And the efficiency of protection against aphid was evaluated by counting the number of aphid after the aphid were inoculated onto the transgenic plant.The five successfully constructed RNAi vectors were triumphantly transferred into wild type Arabidopsis. The identification by PCR showed the vectors were integrated into DNA of Arabidopsis. RT-PCR and Northern analysis displayed there are expression of dsRNA in transgenic plant. The number of aphid cultivated on the transgenic plant was not clearly reduced, which maybe is due to the damage to the aphid in the experiment.