不同致病力青枯雷尔氏菌的甲基化敏感扩增多态分析

Methylation sensitive amplification polymorphism of Ralstonia solanacearum strains with different pathogenicity

摘要:

【目的】 探究不同致病力青枯雷尔氏菌连续传代过程中致病力、DNA甲基化水平与模式的变化。【方法】 将不同致病力青枯雷尔氏菌连续传代培养50次，通过弱化指数(attenuated index, AI)和接种番茄盆栽苗，分析其F₁和F₅₀菌株致病力变化；采用甲基化敏感扩增多态(methylation sensitive amplification polymorphism, MSAP)技术分析不同致病力和传代数青枯雷尔氏菌DNA甲基化水平变化；利用荧光定量PCR (real-time fluorescent quantitative PCR, qRT-PCR)技术分析DNA甲基化和去甲基化相关酶基因的表达量变化。【结果】 经连续传代50次后，强致病力菌株FJAT15304和过渡型菌株FJAT445均变为无致病力菌株，而无致病力菌株FJAT15249仍然保持其无致病力特性。MSAP分析显示，与F₁菌株相比，强致病力菌株传代50次的FJAT15304.F₅₀和致病力衰退型菌株传代50次的FJAT445.F₅₀的总甲基化率分别增加7.82%和38.22%；无致病力菌株FJAT15249的F₁和F₅₀的总甲基化率均为33.33%；强致病力和过渡型菌株的主要甲基化模式为全甲基化，其全甲基化率高于半甲基化率，而所有无致病力菌株主要甲基化模式为半甲基化。qRT-PCR分析表明，强致病力和过渡型菌株连续传代致病力衰退菌株的DNA甲基化酶相关基因dam、dcm和ftsZ表达量显著增加，而去甲基化酶相关基因alkB表达量显著降低，推测DNA甲基化水平变化在致病力衰退过程起重要作用。【结论】 青枯雷尔氏菌连续传代出现致病力衰退现象，这种致病力衰退可能与DNA甲基化水平有关。本研究为利用无致病力菌株防治青枯病害提供依据。

关键词:青枯雷尔氏菌;连续传代培养;DNA甲基化;甲基化敏感扩增多态性

Abstract:

[Objective] To investigate the changes of pathogenicity and DNA methylation levels and patterns of Ralstonia solanacearum strains with different pathogenicity during consecutive subculture. [Methods] R. solanacearum strains with different pathogenicity were consecutively subcultured for 50 passages. The pathogenicity of different strains was determined by the attenuated index (AI) method and the pot experiments. Methylation sensitive amplification polymorphism (MSAP) analysis was performed to profile the DNA methylation levels of different strains. Moreover, the relative expression levels of genes related to methylases and demethylases were determined by real-time fluorescent quantitative PCR (qRT-PCR). [Results] After 50 passages, both of the virulent strain FJAT15304 and the intermediate strain FJAT445 evolved into avirulent strains, while the avirulent strain FJAT15249 remained to be avirulent. Compared with F₁ strains, FJAT15304.F₅₀ and FJAT445.F₅₀ showed the total methylation rates increasing by 7.82% and 38.22%, respectively. However, both of FJAT15249.F₁ and FJAT15249.F₅₀ had the total methylation rate of 33.33%. Full methylation was the main pattern in the virulent and intermediate strains, while hemi-methylation was the main pattern in all the avirulent strains. Compared with F₁ strains, strains FJAT15304.F₅₀ and FJAT445.F₅₀ showed up-regulated expression of three methylase-related genes dam, dcm, and ftsZ and down-regulated expression of demethylase-related gene alkB, which suggested that the change of DNA methylation might play a key role in the debilitation of pathogenicity. [Conclusion] The pathogenicity of R. solanacearum attenuates during the consecutive subculture, which might be related to the level of DNA methylation. The findings provide a scientific basis for the application of avirulent strains in the biocontrol of bacterial wilt.

Key words:Ralstonia solanacearum;consecutive subculture;DNA methylation;methylation sensitive amplification polymorphism