Molecular Variability Of Turnip Mosaic Virus And The Function Of P3 Protein In The Interaction Between Virus And Host

Turnip mosaic virus(Tu MV) is an important member of Potyvirus belonging to family Potyviridae. It has a wide range of host plants, possesses an exceptionally broad host rang and infects more than 300 species of 156 genera, no one can infect the Brassica but Tu MV. At present, Tu MV has become one of the most five serious viruses in the world that hazards vegetables, being ranked second only to Cucumber mosaic virus, especially the family Brassicaceae. It had an effect on yields and quality of field crops.Studying the molecular variability and the interaction between virus and host of Tu MV, it can not only make us more clearly about molecular mechanisms of evolution of Tu MV to provide a theoretical basis for breeding disease-resistant germplasm, but also enrich our knowledge of the molecular biology to provide a reference for understanding the pathogenesis of Tu MV. We sequenced genome of 3 Tu MV isolates and cp of 18 Tu MV isolates, and analyzed them; we constructed an infectious c DNA clone of LWLB belong to basal-BR of Tu MV, and analyzed the function of P3 protein in the interaction between virus and host by site-directed mutagenesis. The results was divided into four parts:(1)We determined the complete genome sequences of three Tu MV basal-BR isolates infecting radish from Shandong and Jilin Provinces in China. Three isolates were named CCLB, LWLB, WFLB14 respectively. Their genomes were all composed of 9833 nucleotides, excluding the 3’-terminal poly(A) tail, and the homology was 99.2-99.5%. By the phylogenetic tree analysis, Tu MV isolates were clustered to five lineages corresponding to basal-B, basal-BR, Asian-BR, world-B and OMs, and three Tu MV isolates in this study were all in basal-BR group. Recombination analysis showed that the three isolates in this study had no ‘clear’ recombination. Genetic diversity analyses indicated that the PIPO gene was under the highest selection pressure and the selection pressure on P3N-PIPO and p3 was almost the same. Using MEGA6.06 software to analysis the genetic distance in the group of basal-BR that the three isolates belonging to, the results showed that the genetic distance was 0.080 ± 0.001. We also had analyzed the molecular variability of cp gene in this study. By the phylogenetic tree analysis, the 18 Tu MV isolates that we sequenced showed that they were all located in basal-BR, excluding isolates of CCJSL10, GRJCJ09, RRJCJ09 and CCJCJ09, which were located in world-B. They had no ‘clear’ recombination also.(2)We had constructed an infectious c DNA clone of LWLB. This research mainly included three part:(1)The selection of the sites of intron. We selected three sites which located in the C-terminal of p3, N-terminal and C-terminal of CI to prevent the recombination.(2)We had stitched the gene sequences of 5’-UTR and 35 S together by over-lap PCR.(3)We had segmented the whole genome to stitch the binary vector of p Cambia0390, and the genome had been successfully connected to the carrier of p Cambia0390 by the verification of PCR and analysis by restriction enzyme. Unfortunately, the infectious c DNA clone infected Nicotiana benthamiana by transformed into Agrobacterium EHA105, and there was no symptom of diseases.(3)Studies by our group had shown that there had an interaction between P3 and At SWEET1. And on this basis, we verified the fact that interaction in Nicotiana benthamiana by Bimolecular Fluorescence Complementation technology in this study. Besides, in order to prove the interaction between P3 and other 16 members of SWEET in Arabidopsis thaliana, we verified the interaction between P3 and At SWEET4 and At SWEET15 by the experiment of yeast two-hybrid.(4)According to available reports, P3 protein has an important function in viral replication, determination of pathogenicity, formation of symptom, host resistance, viral movement and so on. In order to define the important amino acid of the P3 and Tu MV,which can analysis the mechanism of P3 protein in the role of the interaction between virus and host, we had selected eight amino acid sites which were located in the 62, 67, 123, 188, 299 and 300 amino acid of P3 and termination of gene expression of p3 in 530 th bases and the 555 th base to terminate PIPO gene expression to construct mutant bait vector. The results showed that all mutations had an interaction with At SWEET1 excluding the mutation of 299 th amino acids of P3, which suggested that 299 th amino acid of P3 was the key amino acid to involve the interaction between virus and host.