| Turnip mosaic virus (TuMV), an important member of Potyvirus belonging tofamily Potyviridae. TuMV has a wide range of host plants, possesses anexceptionally broad host rang and infects more than300species of156genera,especially the family Brassicaceae. It is also the most widely spread and the mostdestructive of viruses for brassica plants, which can cause significant economiclosses to agriculture.TuMV is a single-stranded, positive-sense RNA molecule of about10000ntwith a genome contains a large open reading frame (ORF). P3is an importantprotein encoded by TuMV. Compared with other proteins, P3is highly variable, wehave known less about its structure and function. So far it is known that, P3is amembrane protein and plays an important role in the process of TuMV infecting thehosts, which probably involves pathogenicity, virus movement, replication, symptomformation and host resistance, etc. In this study, the molecular variability of TuMVp3genes was analysed, and P3antiserum with high specificity was prepared. Wehave studied the the interaction between P3protein and AtSWEET1protein inArabidopsis thaliana.In this study,22samples with typical symptoms of viral diseases were collectedfrom infected cruciferous vegetables in Changchun, Jinlin Province. The p3geneswere cloned by RT-PCR, and all the p3genes nucleotide sequences were identified.Characteristics of p3genes nucleotide sequences were analysed initially, and21representative TuMV isolates p3genes nucleotide sequences in the GenBank werealigned. The rusults showed that22p3genes nucleotide sequences coloned havehigh consistence, but shared nucleotide identities of68.8%-99.8%with other21TuMV isolates available in the GenBank, they have diversity from different regions.In the phylogenetic tree constructed with the complete nucleotide sequences of thep3genes, the43TuMV isolates were divided into five groups: basal-BR, basal-B,Asian-BR, world-B and OMs (found recently), and the22TuMV isolates characterized in this resarch belonged to group basal-BR. They shared nucleotideidentities of68.8%-70.0%with four TuMV isolates in the OMs, which have fargenetic relationship. The ratios of non-synonymous and synonymous substitutionscertified the grouping result is reasonable and credible. Predicted the transmembranedomain of TuMV P3protein by TMHMM2.0, P3is a membrane protein thatcontains two transmembrane domains.The N-terminnal663nucleotides of p3gene in isolate JCR06was cloned intoexpression vector pET-28a(+) and transferred into E. coli BL21(DE3) pLysS.SDS-PAGE showed that the p3gene was expressed as a28kDa fusion protein withIPTG induction. Rabbit was immunized with purified fusion protein to obtain theantiserum with high specificity. Double immunodiffusion assay show that immuneprecipitation line appeared between purified fusion protein and antiserum dilutedfour times. The titer was1:2048determined by indirect ELISA test to detect TuMVin radish. Western blotting showed that the prepared antiserum could specificallybind to fusion protein.The p3gene of isolate JCR06was cloned into bait vector pGBKT7, andscreened a host AtSWEET1protein from the A. thaliana cDNA library by yeast twohybrid fusion method. Finally, we verified the result by using the method of yeasttwo hybrid cotransformation. AtSWEET1is also a membrane protein that containsseven transmembrane domains predicted by TMHMM2.0. AtSWEET1gene wascloned into plant expression vector pCG-1301, analyzed its subcellular localization.The result showed that AtSWEET1located at cell membrane, the same as prediction.On this basis, made use of BiFC to verify the interaction between P3andAtSWEET1, the result showed that they had interaction in plants, and the site islocated in the cell membrane, which predicted that two proteins are all the membraneproteins. This study will provide mechanism of interaction between P3andAtSWEET1and the theoretical foundation for research the functions andmechanisms of P3protein in the pathogenicity of TuMV. |
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