| Tetranychus cinnabarinus is a widely pest in the world that can endanger many kinds of crops.The problems of mite resistance has been prominent and people’s attention to food safety,pesticide residues and other issues has increased.Exploring a new prevention method is imperative.RNAi technology has not only been used as an effective tool for gene silencing,but also has received considerable attention as a potential new pest control method in recent years.As a polyphagous pest,its host adaptation mechanism involves a lot of genes that have significant difference expression.This gene play a key role can be screened as RNAi target.In this study,T.cinnabarinus was transferred from cowpea to eggplant.The changes of the gene expression that at different time points after host transfer were detected by RNA-Seq technique.Two differentially expressed genes were screened out.At the same time,seven genes have important influence on the mite life activities(about skin molting,digestion,metabolism,signal transduction,etc.)were also selected as the candidate target genes.Using the leaf-disc feeding method performes the RNAi experiments,and the interference effect was evaluated by eclosion rate,100-heads weight,mortality and other related phenotypes.On this basis,the plant transient expression technique was used to transfer the ds RNA fragments that are candidate target genes into the eggplant seedling.Then detecting and evaluating the control efficiency.The best target genes for control of this mite were screened with comprehensive analysis.And the main findings are as follows:1.Differentially expressed genes were detected by RNA-Seq analysis after transfer the T.cinnabarinus to the eggplant(2 d,50 d).Annotated seven genes that were up-regulated at 2 d and 50 d,and choosed the CPPR,FAP as target genes for further study.2.Through literature analysis,selected three kinds of genes which may be involved in the T.cinnabarinus life activities :(1)Digestive and metabolic related proteins: β-COP,CPR;(2)Molting related protein: Sro,CS,ER;(3)Conserved protein: Actin,EF.3.Cloning and informatics analysis of nine candidate target gene sequences and RNAi effect analysis based on this;Detection of target genes silencing efficiency found that Actin was extremely low,ER,CS genes silencing efficiency were 40-50%,and other genes were greater than 60%.The CPPR,FAP,β-COP and Sro genes had a significant effect on the mites’ life activities.The control group had significant difference after interfering the β-COP in female adult mites at 36 h,the death rate was 57.4% at 108 h,and the death phenotype was feets curled up on the side of this body.The death rate was 28.8% at 96 h and dead in the molting process when RNAi the Sro.4.Using the transient expression of target genes into eggplant seedlings for control efficacy test,the leaf damage index of β-COP,Sro and FAP genes was significantly lower than the blank control group after 15 d.Indicating that the damage of this mites on eggplant plants was effectively inhibited.Furthermore,the growth and development of plants didn’t have significant effect.In conclusion,using two RNAi methods for screening and verification these candidate target genes,then having basically consistent: β-COP,Sro and FAP genes can be used as RNAi target genes for the control of T.cinnabarinus. |
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