Construction And Detection Of Infectious Clones Of Soybean Mosaic Virus Isolates From Pinellia Ternata And Atractylodes Macrocephala

Soybean Mosaic virus(SMV),a typical member of Potyviridae.The natural host range of SMV is very narrow,mainly infects soybeans.In recent years,researchers have found that SMV can also infects the Pinellia ternate and Compositae Atractylodes macrocephala.In order to study the gene function of the virus and better understand the infection mechanism of SMV breaking through the host range,in this study,the full-length genome of the SMV Atractylodes macrocephala isolates was amplified by RT-PCR combined with RACE technology using Atractylodes macrocephala and Pinellia ternate as research materials.Full-length infecious clones of the SMV-SX mutant and SMV-SXBX genome were constructed by Gibson assembly technology.The main experimental results were as follows:1.Based on the whole genome sequence of Shanxi Pinellia ternate SMV isolate(SMV-SXBX)previously obtained in the laboratory,infectious cloning shuttle plasmid of SMV was constructed by using Gibson rapid assembly technology,which contains the recognition starting sites in E.coli and Agrobacterium,and can be expressed in both bacteria.We amplified the whole genome of the virus in two segments and then connected the whole genome to the p Green II-35 S vector carrying the 35 S promoter to obtain the recombinant plasmid p Green II-35S-SXBX.After inoculation with Agrobacterium tumefaciens healthy Pinellia ternate,the symptoms of Pinellia ternate leaves were consistent with those of the wild type SMV,which was confirmed as SMV by RT-PCR.2.The use of RT-PCR technology and Rapid Amplification c DNA End(RACE)of typical SMV Atractylodes macrocephala isolates(SMV-Am)genome c DNA cloning and sequence determination,full length c DNA contains 9587 nt encoding 3067 amino acid composition of protein polymers,analyzes its genome structure,found the SMV-Am has a typical Potyvirus genus structure characteristics of the members of the virus.Eleven mature proteins,P1,HC-Pro,P3,6K1,CI,6K2,NIa-VPg,NIa-Pro,NIb and CP,were obtained after cleavage and code shift mutation.The restriction sites were speculated to be Y/S,G/G,Q/A,Q/G,Q/S,E/S,E/S,Q/A,and Q/S,respectively.Meanwhile,the structural and functional analysis of genomic nucleotide sequences and their derived amino acid sequences showed that SMV-Am had the highest similarity with SMV-Liaoning,with the similarity of nucleotide and amino acid being96.25% and 98.86% respectively.Nucleotide phylogenetic tree analysis also showed that SMV-Am and SMV-Liaoning,a Soybean SMV isolate,were isolated into a cluster with the closest genetic relationship.3.Infectious cloning of Shanxi Soybean SMV isolate(SMV-SX)was successfully constructed in the laboratory.To SMV-SX and SMV-Am nucleotide and amino acid sequence similarity comparison,found that the infection of Soybeans and Atractylodes macrocephala SMV genome structure,similarity of the lowest protein for P1 protein,amino acid similarity was 94.82%,the fusion PCR technology is adopted to SMV-SX and SMV-Am P1 protein in the genome to replace the key differences between area,get a SMV-SX mutant clone.The aim of this study is to establish the basis for the construction of the mutant's infectious clone and the search for the pathogenic factors for SMV to break through the host range of legume and infect the Compositae plant Atractylodes macrocephala.