Determination Of Complete Genomic Sequence Of Soybean Mosaic Virus And Interaction Analysis Between Soybean EIF4E/EIFISO4E Gene And SMV VPG Gene

Soybean mosaic virus (SMV) is a plant pathogenic virus that infects many commercially important plants worldwide, including soybean, and impairs their production and quality. Determination and analysis of the complete genomic sequence of SMV strains will provide insight into variation in its structure and genes and help to identify mechanisms involved in infection and virulence. In addition, analysis of the complete sequence of the SMV population will provide useful information on gene stability and evolution. The disease reactivity of three strain and isolates were verified on10differential soybean cultivars and the genomes of theirs were sequenced. These sequences were compared with the complete genome sequences of all SMVs to identify molecular differences among strains and to explore the association between virus genome sequence and its virulence and geographical distribution. VPg, a protein of genus Potyvirus, can interacts with both the translation initiation factor4E (eIF4E) and its isoforms eIFiso4E of their host. It can affect the interaction with VPg and acquire resistance by mutations on critical point of eIF4E or eIFiso4E gene. In this study, SMV strains SC3and SC15were used to evaluate the resistance of soybean materials to SMV. We sift out excellent resistance material and detected the polymorphism of eIF4E and eIFiso4E gene. In order to dig out broad-spectrum resistance genes of the soybean and wide the sources of resistance genes, Yeast two-hybrid system was used to study the interaction relationships between eIF4EleIFiso4E and VPg genes. The main results are as follows.1. The disease reactivity of three strain and isolates (SC3,6202-2, and6067-1) were verified on10differential soybean cultivars and their genomes were sequenced by RT-PCR, the5’-RACE and3’-RACE method. The complete genomic sequences of three SMV strains and isolates were compared with the sequences of54other published SMV strains and isolates for analysis. The genomic nucleotide diversity analysis results showed that the5’UTR, the PI N-terminus300-bp segment and the third protein (P3) C-terminus200-bp segment exhibited the highest variability in the whole genome, while the nuclear inclusion a protein (NIa-Pro), nuclear inclusion b protein (Nib),6K2and coat protein (CP) genes had lowest polymorphism. Tajima Neutrality Test suggested that each gene of SMV was under negative selection pressure to varying degrees, and the5’UTR, P1and helper component-proteinase (HC-Pro) genes may be under greater selection pressure. The Fu and Li test showed that5’UTR, VPg and P1gene had been influenced by negative selection pressure. Fu and Li’s also indicated that the rate of mutation in SMV may have increased more recently. Phylogenetic analyses indicated that sequence variation in the cytoplasmic inclusion (CI) cistron of virus was correlated closely with the level of virulence and with the source of the virus isolate.2. SMV SC3was low virulence strain and SMV SC15(6067-1isolate) had high virulence. The resistance identification of208soybean material from different parts to SMV SC3and SC15strains was completed in this study.17soybean materials resistant to the two strains were filtered out and56materials were resistant to one strain, the rest of all the material were infected by the two SMV strains. The eIF4E and eIFiso4E gene CDS sequences of susceptible varieties Nannong1138-2as well as the17resistant materials were determined. Sequence analysis found that eIFiso4E gene CDS sequence is very conservative and no polymorphism was detected. Compared to Nannong1138-2, there were8eIF4E gene mutations in the17resistant materials, of which three were nonsense mutations and5were missense mutations. There are three amino acid changes in the missense mutation.3. To study the interaction between SMV VPg gene and eIF4E/eIFiso4E genes. Yeast two-hybrid system was employed to construct bait vectors containing VPg gene, and activation vectors containing eIF4E/eIFiso4E genes respectively and these vectors were verified through self-activation and toxicity testing. Two-hybrid screening results show that: SMV VPg gene interacted with both eIF4E and eIFiso4E genes of susceptible cultivar Nannong1138-2and the eIF4E genes of resistant materials2,11,29and172were not able to interact with and SMV VPg. Results indicated that the change of eIF4E amino acid sequence affected the interactions between VPg and eIF4E gene and this is likely to be the reason that the materials are resistant to SC3and SC15strains.