Identification Of Virus Infecting A Tractylodes Macrocephala Koidz,Pinellia Ternata Breit And Rehmannia Glutinosa Libosch By Small RNA Deep Sequencing

Shanxi is a province with abundant Chinese herbal medicine.In recent years,with the large-scale cultivation of Chinese herbal medicine,viral diseases have become increasingly serious year by year,affecting yield and quality of Chinese medicinal materials.Identification of viral pathogens and biological characteristics are the basis and key of the prevention and control of virus disease.Due to the poor research of Chinese medicinal Inaterials viruses,detection the viruses are very difficult by traditional methods.Deep sequencing technology has provided a great opportunity for the identification of plant viral diseases.This technology has broken through the limitations of traditional virus detection methods and has the advantage of virus information-independent,which can simultaneously detect the known and unknown,high titer and low titer of all of the DNA viruses,RNA viruses and viroids in plant samples,and has might discover some new viruses.In this study,we detected the viral pathogens in Atractylodes macrocephala Koidz,Pinellia ternata Breit and Rehmannia glutinosa Libosch from Shanxi Province by small RNA deep sequencing technology.(1)The samples of Atractylodes mnacrocephala Koidz with typical chlorotic mosaic and curl symptoms were infected by Broad bean wilt virus 2(BBWV 2),Cucumber mosaic virus(CMV)and Pecan mosaic-associated virus(PMaV).To the best of our knowledge,this is the first report of PMaV on Atractylodes macrocephala Koidz.The complete genome sequence of the strain of Pecan mosaic-associated virus(PMaV-Am)was cloned.PMaV-Am genome is 9310 nucleotides(nt)in length,and possesses a typical organization characteristic of the genus Potyvirus,which has a large open reading frame(ORF)encoding a putative polyprotein with 2976 amino acids.Phylogenetic analysis based on the amino-acid sequences between PMaV-Am and other representative Potyviridae viruses showed that PMaV-Am is clustered together with Potyvirus viruses and most closely related to PMaV-LA.(2)The samples of Pinellia ternata Breit with typical mosaic and curl symptoms were infected by Soybean mosaic virus(SMV),Cucumber mosaic virus(CMV),Dasheen mosaic virus(DsMV)and Konjac mosaic virus(KoMV).The complete genome sequence of SMV-SXBX was cloned and determined to be 9735 nt in length.Sequence comparative analysis showed that SMV-SXBX shared high homology with SMV isolates from Pinellia ternata Breit and shared low homology with other SMV isolates from Soybean.Phylogenetic analysis based on the amino-acid sequences suggested that SMV-SXBX is clustered together with SMV isolates from Pinellia ternata Breit,whereas less relation with SMV isolates from Soybean.(3)The samples of Rehmannia glutinosa Libosch with typical mosaic and curl symptoms were infected by Rehmannia mosaic virus(ReMV)and Youcai mosaic virus(YoMV).The coat protein(CP)gene of ReMV and YoMV were cloned and inserted into the vector pMal-c2X.Then the prokaryotic expression vector pMal-c2X-ReMV CP and pMal-c2X-YoMV CP were transformed into the Escherichia coli BL21,and the 60 kDa recombinant CP proteins were expressed after induction with IPTG.These proteins can be used as an antigen to prepare antiserum for viruses.The analysis of size distribution,5'-terminal nucleotide preference,polarity distribution and hotspot of virus-derived small interfering RNAs(vsiRNAs)are helpful to understand the production of vsiRNAs and its role in antiviral in defense.These results provide guidelines on studying of interaction between virus and host and designing antiviral strategies using host RNA silencing defense systems.