Comparative analysis of structure, function and genetic variation of upstream sequences adjoining tuf gene in paulownia and jujube witches'-broom phytoplasmas

Abstract:

[Objective] Exploring the difference of structure, function as well as the genetic diversity of upstream sequences adjoining tuf gene in paulownia and jujube witches'-broom phytoplasmas. [Methods] Thermal asymmetric interlaced PCR (TAIL-PCR) was used to amplify the upstream unknown sequence adjoining tuf gene in jujube witches'-broom phytoplasma. Recombinant expression system was successfully constructed with promoter-probe vector pSUPV4 and tuf gene upstream sequences from paulownia and jujube witches'-broom phytoplasma. Genetic variation and promoter activity of tuf gene upstream regulatory sequences from 16SrI group phytoplasma strains including paulownia witches'-broom, chinaberry witches'-broom, lettuce yellows, mulberry dwarf and periwinkle virescence phytoplasma strains and 16SrV group strains including jujube witches'-broom, cherry lethal yellow, Bischofia polycarpa witches'-broom phytoplasma strains were analyzed and identified. [Results] The sequence length of intergenic region between tuf and its upstream gene, fusA, in 16SrI group strains was 129 to 130 bp, with putative complete promoter conservative structure. 130 bp upstream sequence of tuf gene in paulownia witches'-broom phytoplasma had promoter activity. There were four variation types in the intergenic region sequences from five kinds of 16SrI group phytoplasmas including 35 strains; the sequence length of intergenic region between fusA and tuf in 16SrV group strains was 53 to 54 bp, with predicted incomplete promoter structure. No promoter activity was found in 144 bp and 346 bp upstream sequence of tuf gene in jujube witches'-broom phytoplasma. There were two variation types in the intergenic region sequences from three kinds of 16SrV group phytoplasmas including 20 strains. The fusA-tuf intergenic region sequences were comparatively conservative, and the phytoplasma strains belonging to different groups were distinguished clearly by the phylogenetic tree constructed based on the sequences. [Conclusion] The methods and results of the study would lay a beneficial foundation in further investigating phytoplasma gene expression and regulation, revealing phytoplasma regularity of growth and propagation as well as its pathogenicity mechanism.