Construction And Detection Of Infectious Clones Of Soybean Mosaic Virus

Glycine max(L.)Merr,an annual herb of the genus Leguminosae,is the world's major oil crop,rich in soluble fiber,saponin,proteins and fat and other nutrients beneficial to the human body.Pinellia ternata Breit is a perennial herb of the genus Araceae,which is a traditional Chinese medicinal material,rich in Pinellia starch,alkaloids,?-sitosterol,Pinellia protein,amino acid and organic acids.However,in the growth and development of soybean and Pinellia ternata,it is affected by various pests and diseases,which seriously affects its yield and quality.Soybean mosaic virus(SMV)is the main viral disease that damages soybean yield and quality.The host range is very narrow,mainly legumes,but some researchers have found that SMV can infect the Pinellia ternate,a medicinal plant of Araceae.At present,the research on SMV is mainly devoted to the study of soybean plants and resistance genes,and there are few reports on the breakthrough of host range.Therefore,in this study,Shanxi soybean and Pinellia ternata were used as research materials,and ds RNA,RT-PCR and infecious cloning techniques were used to study them.The main experimental results were as follows:1.Using ds RNA technology to detect soybean leaves showing typical mosaic symptoms,it was found that susceptible soybeans were infected by SMV.Then,using the strategy of segmental cloning,the SMV-SX was amplified by RT-PCR,and the whole genome was 9587 nt.Analysis of the genomic structure revealed that SMV-SX has the structural characteristics of a typical Potyvirus.It encodes a polyprotein consisting of 3067 amino acids and is cleaved to 11 mature proteins,P1,HC-Pro,P3,P3N-PIPO,6K1,CI,6K2,NIa-VPg,NIa-Pro,NIb and CP,respectively.It is speculated that the enzyme cleavage sites are Y/S,G/G,Q/A,Q/G,Q/S,E/S,E/S,Q/A,and Q/S,respectively.Furthermore,the sequence homology and similarity of SMV-SX and SMV isolates from other sources were analyzed.The results showed that SMV-SX had the highest similarity with SMV isolate XFQ008(SMV-XFQ008)from soybean,and the nucleotide and amino acid identity were 97.03% and 98.08%,respectively.Nucleotide phylogenetic tree analysis also showed that SMV-SX and XFQ008(SMV-XFQ008)were clustered together,with the closest relationship.2.Gibson rapid assembly technology was used to construct an infectious cloning shuttle plasmid of SMV,which contains the recognition starting sites in E.coli and Agrobacterium,and can be expressed in both bacteria at the same time.This method not only makes up for the time-consuming and laborious shortcomings of traditional methods for constructing infectious clones,but also avoids the toxicity of special genomic sequences of Potyvirus virus to E.coli.3.The nucleotide and amino acid sequence similarities of SMV-SX and SMV-SXBX genomes were compared and analyzed.It was found that among the SMV genomes of Shanxi soybean and Pinellia ternata,the lowest similarity was P1 protein,the amino acid similarity was only 23.03%,followed by P3 and CP protein,and the amino acid similarity was 83.00% and 84.04%,respectively.Because the CP gene of SMV genome is related to host symptoms,the SMV-SX genome and the CP gene of SMV-SXBX were fused by fusion PCR technology to construct an mutant infective clone of SMV-CP.The results are expected to lay a theoretical foundation for identifying the pathogenic factors of SMV infection in Pinellia ternata.