泡桐丛枝和枣疯病植原体tuf基因上游序列结构、功能和遗传变异比较分析

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

【目的】探究泡桐丛枝和枣疯病植原体tuf基因上游序列结构、功能差异及其遗传多样性。【方法】利用热不对称交错式PCR(TAIL-PCR)扩增枣疯病植原体tuf基因上游未知序列,利用启动子探针载体pSUPV4构建了泡桐丛枝和枣疯病植原体tuf基因上游序列的大肠杆菌异源表达体系,分析泡桐丛枝、苦楝丛枝、莴苣黄化、桑萎缩、长春花绿变等16SrI组和枣疯病、樱桃致死黄化、重阳木丛枝等16SrV组株系tuf基因上游调控序列的遗传变异特征和启动子活性。【结果】泡桐丛枝等16SrI组植原体株系tuf基因和其上游fus A基因之间的间区序列长129-130 bp,预测有完整的启动子保守结构。泡桐丛枝植原体tuf基因上游130 bp片段具有启动子活性,此间区序列在5种35株16SrI组株系中存在4种变异类型;枣疯病植原体等16SrV组株系fusA和tuf基因间区长53-54 bp,未预测到完整启动子结构。枣疯病植原体tuf基因上游144 bp和346 bp片段均未检测到启动子活性,fus A和tuf基因间区序列在3种20株16SrV组株系中存在2种变异类型。fus A-tuf基因间区序列相对保守,基于此序列构建的进化树可清晰区分不同组别的植原体株系。【结论】研究方法和结果为深入研究植原体基因表达与调控、揭示植原体生长繁殖规律及其致病机理等奠定了良好的基础。

[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 tufgene 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 tufgene 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 tufin 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.