| Virus disease symptoms such as yellowing,flower and leaf shrinkage have appeared on tomato,lilac and dahlia plants in Beijing,which has affected their own value to varying degrees.In this paper,the viruses infecting tomato,lilac and dahlia plants in Beijing were identified by small RNA deep sequencing technology,the types of viruses were identified,and some viral infectious clones were constructed,so as to provide a basis for the prevention and control of related plant diseases.1.Tomato chlorosis virus(ToCV),Tomato mosaic virus(ToMV)and a new satellite RNA virus(Sat RNA)were detected on tomato by small RNA deep sequencing technology.? Specific primers were designed to amplify the complete sequence of ToCV and analyze its genome structure and enzyme digestion sites.The pCB301 vector was selected.The infectious clone pCB301-ToCV was successfully constructed by seamless cloning method and transferred into agrobacterium tumefaciens to infiltrate tobacco and tomato.ToCV was detected in the system leaves after 7 days of tobacco inoculation,and only in the inoculated leaves after 15 days of tomato inoculation.The pCB301-ToCV can infect tobacco,but not tomatoes and has no symptoms in tobacco.? Specific primers were designed to amplify the whole sequence of Sat RNA and analyze its genome structure and enzyme digestion sites.The pCB301 vector was selected.The infectious clone pCB-Sat RNA was successfully constructed by double enzyme digestion method and transferred into agrobacterium tumefaciens to infect tobacco and tomato.Sat RNA was detected 7 days after tobacco inoculation and 15 days after tomato inoculation,but sat RNA was not detected.? Because Sat RNA virus cannot replicate and move by itself,it completely depends on the auxiliary virus for replication and coating.Therefore,it infects tobacco and tomato together with pCB301-ToCV to study whether the pCB-Sat RNA is a Sat RNA virus of ToCV.Through the experiment,it is concluded that the Sat RNA has nothing to do with ToCV.2.Lilac yellow leaf curl virus(LYLCV)was a new virus found in our laboratory and has obtained the whole sequence.On this basis,we analyzed the genome structure and enzyme digestion sites of LYLCV,selected the pCAMBIA1300 vector,and successfully constructed the pCAMBIA1300 LYLCV infectious clone in two parts by double enzyme digestion method,and transferred it into agrobacterium tumefaciens to infiltrate tobacco,after 7 days,LYLCV was detected in the inoculated leaves and system leaves of tobacco,and there were no obvious symptoms in tobacco.3.A new virus named dahlia stunt-associated virus(DsaV)and Cucumber mosaic virus(CMV),Dahlia common mosaic virus(DCMV)were detected by small RNA deep sequencing.? Specific primers were designed to amplify the whole sequence of DCMV-BJ.Through NCBI website comparison and ICTV website genome description,DCMV-BJ belongs to Caulimoviridae Caulimovirius,and its genome is a double stranded DNA virus with a total length of 7949 bp.The virus encodes motion protein,aphid transmission factor protein,DNA binding protein,capsid protein,polymerase poly protein and inclusion body protein.Through similarity comparison and phylogenetic tree construction,it is judged that DCMV-BJ is a highly similar isolate of DCMV-NZ.? Specific primers were designed to amplify the whole sequence of DsaV.Through NCBI website comparison and ICTV website genome description,DsaV belongs to Secoviridae,Comovirinae,Fabavirus and its genome is two sense single stranded RNA.RNA1 5560 bp and RNA2 3460 bp.RNA1 encodes co-factor,helicase,viral protein genome-linked,proteinase and RNA dependent RNA polymerase;RNA2 encodes movement protein,large coat protein and small coat protein.Through similarity comparison and phylogenetic tree construction,it is judged that DsaV is a new member of Fabavirus. |
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