Construction Of AmiRNA Vector For N.Benthamiana RNAi Pathway Genes,Transient Expression And Analysis Of Their Silencing Effects

In order to ensure the normal growth of plants,there are a set of genes involved in resistance against pathogen infection in plants.RNAi(RNA interference)is an important defense response to virus infection in plants.Artificial RNA(amiRNA)is a kind of efficient technology for gene silencing,which is designed by using the principle of biogenesis and working mechanism of natural miRNA,silencing the expression of one or more genes in an organism.Virus induced gene silencing(VIGS)is also a powerful means for plant gene silencing.amiRNA VIGS(MIR VIGS)is a newly developed plant reverse genetics research technology,which has the advantages of both amiRNA and VIGS technology.Nicotiana benthamiana is a widely used organism to study plant-pathogen interactions and can be used as a good host VIGS,as it is sensitive to more than 500 different viruses.To date,almost all of the genes in the plant RNA silencing pathway have been found and functionally verified in Arabidopsis.However,few studies have been reported on the genes involved in the RNA silencing pathway in Nicotiana benthamiana.In this study,we investigated the interference of 11 genes(AGO1a,AGO1b,DCL2,DCL3,AG02,DCL4,DRB4,RNA,HEN1,RDR1,RDR6,SGS3)related to the silencing pathway in Nicotiana benthamiana.Transient expression experiments were performed after the vectors were constructed,and the following conclusions were obtained:1)Thirty two interference vectors in plant virus vector pCVA were constructed using MIR VIGS technology against nine endogenous genes,with silencing efficiency of above 50%;2)amiRNA technology was used to construct eight interference vectors in plant binary expression vector pBI121(Kpn ?),among which silencing efficiency of seven genes was above 45%.The overall efficiency of pBI121-based amiRNA is lower than the viral vector pCVA-based amiRNA;3)Competition was observed between the virus vector pCVA and co-infection other viruses,whereas the plant binary vector pBI121 and co-infected virus did not show competition at low concentration(OD600=0.1);4)When expressed by plant binary vector pBI 121 in transient infection at OD600=0.1,down-regulation of NbAG02 gene compromised plant's resistance against tobacco mosaic virus(pJL24),while down-regulation of NbAGO1a,AGO1b,NbRDRl and NbDCL2 showed no such effects.