Promotor Identification Of Satellite DNAβ Associated With Ageratum Yellow Vein Virus Isolate F1

Geminiviruses occur in world wide range and have caused significant yield losses to many crops. In recent years, no geminivirus had been reported to found in Fujian province in China until 2008. The material used in this thesis, which is termed Ageratum yellow vein virus-Fujian isolate F1 (AYVV-[F1]), is the first report of geminivirus found in Fujian. The full length DNA-βmolecular of F1 was cloned into pMD-18T vector by general specific primers. Sequencing result show that the full length of F1 DNA-βis of 1346 nucleotides. Comparison analysis of F1 DNA-βwith other reported DNA-βsequences showed that F1 DNA-βshared 100% identity to Tomato leaf curl virus (TLCV). Further analysis revealed that there is aβC1 ORF encoded in the complemental strand of F1 DNA-β. F1βC1 gene was cloned into pMD-18T vector using specific primers. The expression plasmid pGEX-F1-βC1 was constructed by inserting F1βC1 gene into the expression vector pGEX-4T-3 and was then transformed into E. coli BL21(DE3). The E. coli BL21 cells that harbored the plasmid pGEX-F1-βC1 were induced by IPTG, subsequent SDS-PAGE result shows that F1-βC1 protein was successfully expressed. The partial promoter of F1βC1 ORF was also cloned by specific primers. Promoter elements prediction of ABP segment was done by the online tool PlantCARE, TATA-box, CAAT-box and GC-motif were all founded in this partial promoter, some other plant cis-elements was also being predicted in its promoter sequence.Accroding to the position of promoter cis-acting elements isolated promoter fragments ofβC1 gene,and then were inserted to a plant expression vector, pEGAD,which its CaMV 35S promoter has been knocked out beforehand and has a egfp gene as a reporter gene. The combinant vectors were subsequently transformed into A.tumefaciens EHA105. Further investigations were done to reveal the promoter activity of F1βC1 promoter by transient expression on N. tabacum. The results of western blot analysis and fluorescence microscope assay by transient expression showed that a 173 nt fragment upstream of the translation start site of AYVV F1βC1 ORF had the basic promoter activity,besides,under the upstream of the translation start site existed elements which could enhanced the promoter activity.Tissue expression pattern assay of the stably transformed tobacco plants revealed that F1βC1 promoter was expressed specifically in phloem tissue,was a phloem-specific promoter.