Soybean Mosaic Virus Pinellia Strain Encoding The P1 And 6k1 Protein And The Host Plant Protein Interaction Preliminary Study

Pinellia ternate belongs to the family Araceae and is an important traditional Chinese medicinal herb. In recent years, we have isolated a virus related to soybean mosaic virus (SMV) in Pinellia ternate from Zhejiang Academy of Agricultural Science (ZAAS) , Hangzhou, Zhejiang province. This is the first report that SMV infects Araceae plant. SMV Pinellia ternate isolate (SMV-P) has lower identity to SMV in the P1 gene region, but it has about 60% identity to dasheen mosaic virus (DsMV) in the same region. We have collected 14 Pinellia ternate samples from different regions in China, and found that 6 Pinellia ternate samples were infected by SMV-P. They were from ZAAS, Shanghai Botanical Garden, Shanghai city, Wuhan Botanical Garden, Hubei province, Huazhong Agricultural University, Hubei province, Yuzhou, Henan province and Sheyang county, Jiangsu province. Sequences of P1 gene region of 6 SMV-P isolates were determined and there was no significant difference between the sequences and they shared higher identity to DsMV (about 60%), but lower identity to SMV (nucleotide 38.3%-45.2%, amino acid 23.7%-31.9%). It shows that this phenomenone exists generally in the genome of SMV Pinellia ternate isolates, and implicates that SMV-P probably is a recombinant product of SMV and DsMV.In this study, The SMV-P P1 and 6K1 gene were cloned into prokaryotic expression vector pGEX-3X. They were highly expressed in E.coli BL21 (DE3) pLysE and the antisera against the proteins were raised in mouse. The SMV-P P1 antiserum was used in the subsequent experiment and the 6K1 antiserum was used to label the 6K1 protein in leaf of Pinellia ternate infected with SMV-P.The recombinant plasmid was constructed by ligating the P1 gene of SMV-P into pGBKT7, and then transformed into yeast AH109. Western blot analysis revealed that the P1 was expressed as a fusion protein with BD in AH 109. The plasmid pGBKT7-(SMV-P) P1 has no toxicity to yeast strain AH 109 and is unable to activate transcription of reporter genes by itself, therefore it can serve as a bait plasmid of yeast two-hybrid system.The total RNA was isolated from Pinellia ternate with Trizol. The first-strand cDNA was synthesized with SMART technology, and the dscDNA was further synthesized with 5' and 3' PCR primer by long distance PCR. The yeast strain Y187 was transformed with dscDNA and pGADT7-Rec in order to construct the cDNA library. The library consists of 2.1 ×10~6 independent clones, and the library titer is 3.1×10 cfu/ml. The library can be used to screen.AH109/pGBKT7-(SMV-P) PI was mated with the pretransformed Pinelliaternate cDNA library yeast strain Y187, and putative positive clones were obtained by nutritional selection screening and X-a-gal assay. Four putative positive clones were obtained and their plasmids were isolated. The AD library inserts were amplified by PCR and the PCR result showed that the inserted fragments were about 600 bp. These putative positive clones were used to further analysis by using a modified two-hybrid system. At last, these positive clones were confirmed and the sequences were determined. The sequences were submitted to the GenBank and the result showed that the 4 sequences were identical. They all encode the N-terminal region of Rieske Fe/S protein of Cytochrome b6/f complex in photosynthesis electron chain and the sequences have more than 83% indentity to the corresponding region of nicotiana tabacunu spinaciaoleraceax oryza sativa and solanum tuberosum etc.SMV-P 6K1 antiserum was used for localization of the protein in the infected plant cells by immuno-gold labeling. The result shows that the 6K1 protein locates near the cell wall specially and it further confirms the presumption that 6K1 protein may have membrane anchoring function. In addition, it also suggests that the 6K1 protein may have function in the virus movement.