Study On The Soybean Proteins Interacting With P3 And P3N-PIPO Proteins Encoded By Soybean Mosaic Virus

Soybean mosaic virus seriously affects the yield and quality of soybeans.It is an important member of the Potyvirus genus.P3 protein encoded by SMV is easy to mutate,and it has important functions on virus replication,determining pathogenicity,and symptom formation.The P3N-PIPO coding region is inside the P3 cistron.P3N-PIPO plays an important role on virus replication and movement.The research of plant virus-host protein interaction not only helps to reveal the pathogenic mechanism of the virus,but also provides a theoretical basis for cultivating virus-resistant soybean varieties.Therefore,in order to further clarify the interaction mechanism between SMV and host,the soybean proteins interacting with P3 and P3N-PIPO proteins were screened and identified,and their bioinformatics characteristics were analyzed.The results of this research are as follows:1.P3 and P3N-PIPO genes were cloned from SMV collected and purified in the field.Bioinformatics analysis of P3 gene found that it contained two transmembrane helical structures,which were predicted to be membrane proteins.P3 gene was used to construct a phylogenetic tree.Evolutionary analysis showed that the homology between JLCC isolate and Liaoning isolate(MK350280.1)reached 99.52%.The homology with Heilongjiang Province isolate(KP710873.1)reached 98.94%.The three isolates all belong to the northeast region and have a certain regional correlation.Because the P3 gene is relatively unconserved,the recombination analysis of P3 gene showed that there was no gene recombination event in the JLCC isolate.Bioinformatics analysis of P3N-PIPO gene sequence showed that it contained a transmembrane domain located at the P3 N terminal and was predicted to be a membrane protein.2.In order to explore the expression of 52 members of soybean SWEET gene family infected by SMV.Real-time PCR results showed that 33 GmSWEET genes were up-regulated and 19 GmSWEET genes were down-regulated.GmSWEET36,which was up-regulated and expressed more than 5 times,was selected,and the gene was cloned for bioinformatics analysis.It was found that it contained 7transmembrane helix structures,which were presumed to be membrane proteins.Phylogenetic analysis showed that the GmSWEET36 obtained in this study and the other 5 soybean SWEET genes formed an evolutionary cluster on the phylogenetic tree.GmSWEET36 protein was transiently expressed in tobacco,and subcellular localization analysis was performed.The results showed that GmSWEET36 protein was localized on the cell membrane.The sucrose content of SMV-infected soybeans was lower than that of uninfected soybeans.The glucose content after 10 days of SMV inoculation was higher than that of uninoculated soybeans,and the glucose content after 15 days of inoculation was lower than that of uninoculated soybeans.3.In order to screen the soybean proteins interacting with P3 protein,the soybean yeast two-hybrid c DNA library was constructed.The soybean light-catching chlorophyll a/b binding protein Gmcab3 interacting with P3 protein was screened using P3 protein as bait protein.Bioinformatics analysis of Gmcab3 gene sequence,the phylogenetic tree shows that the Gmcab3 obtained in this research has the highest homology with the other four soybean Gmcab3 genes,forming an evolutionary cluster.Gmcab3 protein is one of the members of the light-harvesting chlorophyll a/b binding protein family.The members of the family are highly conserved among different species and have high homology among different genera such as Leguminosae and Fagaceae.4.The interaction between soybean GmSWEET36 and Gmcab3 protein and SMV P3 and P3N-PIPO protein was verified by yeast two-hybrid experiment.The results showed that GmSWEET36 and Gmcab3 interact weakly with P3 protein,and strong interaction with P3N-PIPO protein.