Cloning， expression and characterization of tRNAisopentenyltransferase genes (tRNA-ipt) from paulownia witches'-broom phytoplasma

Abstract:

Abstract:［Objective］To identify the tRNA-ipt gene of phytoplasmas and analyze the relationship between tRNA-ipt and synthesis of cytokinin as well as pathogenesis in phytoplasmas.［Methods］The paulownia witches'-broom phytoplasma (PaWB) tRNA-ipt gene was expressed in E.coli and specific antibody was prepared．Then the growth curve and cytokinin contents of E.coli with PaWB tRNA-ipt were measured by photodensitometry and ELISA respectively． ［Results］The length of tRNA-ipt genes from PaWB as well as mulberry dwarf，periwinkle virescence and Chinaberry witches'-broom phytoplasmas were 876 bp．All these genes encoded the proteins consisting of 291 amino acids．They contained and indentical motif (GPTASGKT) at N-terminal region related to the ATP or GTP binding sites．Four phytoplasma tRNAIPTs shared the 99.1－99.5%，amino acid sequence indentity with each other，shared 95.4－99.3% sequence identity with the same group phytoplasmas，whereas the less than 70% identity with 16SrX apple proliferation and 16SrXII Australia grapevine yellows phytoplasmas．The expression of the tRNA-IPT protein and localization in the phloem in phytoplasma-infected paulownia were confirmed by Western blotting and immunofluorescence detection． The determination of growth curve suggested that the growth rate increase of E.coli was affected by the transformation of exogenous tRNA-ipt gene，which might be associated with the cytokinin accumulation.［Conclusion］This protein was assumed to be involved in the cytokinin synthesis in phytoplasmas as well as other bacteria，which may play an important role in pathogenic processes of phytoplasmas and symptom development.

Key words:Keywords: tRNA-isopentenyltransferase，sequence and function analysis，expression in E.coli，growth curve