Identification Of Virus From Three Chinese Herbal Medicines And Preliminary Construction Of MVCV Alcea Rosea Strain Infectious Clone

In recent years,with the country’s emphasis on and support for the Chinese medicinal material system,the scale of Chinese medicinal material cultivation has continued to expand,but viral diseases have followed,and the output and quality of Chinese medicinal materials have deteriorated,which has seriously restricted the artificial cultivation and economic benefits of Chinese medicinal materials.At present,the research on virus disease of traditional Chinese medicine plants is relatively weak.Therefore,it is of great significance to identify and identify the viral pathogens and evolutionary sources of diseased plants in time.In this study,the viruses of Chrysanthemum morifolium Ramat.in Zhejiang Tongxiang,Daturae Flos and Isatidis Radix in Shanxi were identified,and MVCV Alcea rosea Strain infectious clones were constructed.The experimental results are as follows:1.The Chrysanthemum morifolium Ramat.which shows typical chlorotic and shrinking symptoms,was infected by Chrysanthemum virus B(CVB)and Chrysanthemum virus R(CVR).The complete genome sequence of the strain of Chrysanthemum virus R(CVR-TX)was amplified,and the total length of the CVR-TX genome was 8872 nt.It has 6 open reading frame(ORF),encoding 6polyproteins.Identity analysis and phylogenetic tree were constructed based on the full length of the genome,replicase and CP proteins.The results showed that CVR-TX was far related to the members of Carlavirus,and was closest to CVR-BJ.(2)The Daturae Flos,which shows typical chlorotic and shrinking symptoms,was infected by Broad bean wilt virus 2(BBWV2).This is the first report of BBWV2 on Daturae Flos.The complete genome sequence of BBWV2-DF was cloned.The full length of BBWV2-DF RNA1 genome is 5985 nt,and the full length of RNA2 genome is 3600 nt.Further analysis of the sequence similarity between BBWV2-DF and BBWV2 members from other sources,the results showed that BBWV2-DF RNA1 is more homologous to BBWV2 isolate from Atractylodes macrocephala,and RNA2 is similar to BBWV2 isolate from Gynura procumbens.The amino acid phylogenetic tree showed that BBWV2-DF RNA1 and RNA2 were clustered with BBWV-SN,and the relationship was closest.(3)Isatidis Radix with typical yellowing and mottled symptoms was infected by Turnip mosaic virus(TuMV)and BBWV2.To the best of our knowledge,this is the first report of TuMV on Isatidis Radix.The complete genome sequence of TuMV-BLG was cloned and we obtained the full length of TuMV-BLG is 9833 nt.It has an ORF and encodes a polyprotein consisting of 3166 amino acids.After cleavage could obtained proteins: Pl,HC-Pro,P3,P3N-PIPO,6KI,CI,6K2,VPg,NIa,NIb and CP,and the specific digestion sites are Y/S,G/G,Q/A,Q/T,Q/N,E/A,E/S,Y/A,Q/A.Through comparison,it was found that TuMV-BLG is similar to TuMV isolates from other sources,but it is far away from each other.Among them,it is the farthest from the Japanese radish isolate JPN1.(4)The MVCV and p Green II-35S shuttle plasmids were ligated by Gibson assembly technology to construct MVCV Alcea rosea Strain infectious clones.The construction of infectious clones of MVCV can effectively avoid its toxic effect on E.coli,facilitate the smooth development of research on the pathogenic mechanism,control strategies of MVCV and Alcea rosea virus disease.