Dsrna Mediated Tobacco Resistance To Virus Disease(TMV,CMV,PVY) And Target Spot Disease(Rhizoctonia Solani)

With the development of molecular biology technology,RNA interference(RNAi)has become a new effective method to control plant diseases.In this study,virus-induced gene silencing(VIGS)was used to screen the most effective targets to suppress TMV,CMV,PVY and tobacco target spot disease(Rhizoctonia solani)respectively.Then,dsRNA simultaneously targeting the most effective target of these three viruses and dsRNA targeting the effective target of tobacco target spot disease pathogen were transcribed in vitro and applied in tobacco plants.The inhibition effect of these three viruses and tobacco target spot disease was detected by RT-qPCR.The main research results were as follows:1.Using multiple nucleic acid sequences of TMV,CMV and PVY as silencing targets,VIGS vectors were constructed and their silencing effects were detected.In this study,we selected sequences of p126,p183,MP,CP,3'UTR of TMV,sequences of 1a,2a,3'UTR of CMV and HC-pro,NIb,CI,CP genes of PVY as silencing targets to construct recombinant expression vectors.The recombinant expression vector was introduced into tobacco at 4-6 leaf stage by Agrobacterium tumefaciens infiltration and VIGS was induced.TMV,CMV and PVY were inoculated respectively.After 10 days of inoculation,RNA accumulation of TMV,CMV and PVY were detected by RT-qPCR.The results showed that p126,2a and HC-pro are the most effective target genes to inhibit TMV,CMV and PVY,respectively.VIGS vector of the most effective target nucleic acid fragment fusion of TMV,CMV and PVY and detection of its silencing effect.Three fragments of p126(140bp),2a(125bp)and HC-pro(133bp)were integrated to TRV vector by homologous recombination technique,and the recombinant expression vector pTRV-3FF was constructed.TRV-3FF was introduced into tobacco through Agrobacterium tumefaciens,and theRNA accumulation of TMV,CMV and PVY were detected by RT-qPCR.The results show that TRV-3FF can inhibit TMV,CMV and PVY at the same time,with inhibition rates of 38.11%,28.85% and 37.86%,respectively.2.VIGS construction and silencing effect detection of Rhizoctonia Solani.TRV-endo PGs has the best inhibition effect on tobacco target spot.Two virulence related genes RPMK1 and endo PGs of Rhizoctonia Solani were selected as targets to construct VIGS silencing expression vectors pTRV-RPMK1 and pTRV-endo PGs.RT-qPCR was used to detect the silencing inhibition of TRV-RPMK1 and TRV-endo PGs on tobacco target spot on tobacco.The inhibition rates of TRV-RPMK1 and TRV-endo PGs were 44.95% and 51.38% respectively.The inhibition rate of TRV-endo PGs was higher than that of TRV-RPMK1.3.Expression of dsRNA and preliminary analysis of its resistance to disease.dsRNA-3FF targeting TMV-p126,CMV-2a,PVY-HC-pro,dsRNA-RPMK1 and dsRNA-endo PGs targeting RPMK1 and endo PGs genes of tobacco target spot disease pathogen were transcribed in vitro by a T7RNAi Transcription Kit.The results showed that dsRNA-3FF could reduce the accumulation of TMV,CMV and PVY simultaneously,among which the inhibition rate of TMV was 37.5%,and the inhibition effect was the strongest.dsRNA-RPMK1 and dsRNA-endo PGs inhibited the growth of tobacco target spot fungus mycelium at 24 h,48 h and 72 h.dsRNA-RPMK1 and dsRNA-endo PGs had the best inhibition on the growth of tobacco target spot fungus mycelium at 48 h,the inhibition rates were 44.03% and 50.49%,respectively.Exogenous dsRNA-RPMK1 and dsRNA-endo PGs were also applied to tobacco leaves in vitro.The results showed that the lesion diameter of tobacco leaves treated with dsRNA-RPMK1 and dsRNA-endo PGs was significantly smaller than that of the control group.RT-qPCR showed that both dsRNA-RPMK1 and dsRNA-endo PGs have inhibitory effect on Rhizoctonia Solani,the inhibition rates were 33.94% and 43.75%.dsRNA-endo PGs have stronger inhibitory effect on Rhizoctonia Solani than dsRNA-RPMK1.This study provides a new solution for the complex infection of virus and the control of tobacco target spot.