CbrB在防御假单胞菌Pf-5生物防治中的作用

Role of CbrB in the biocontrol strain Pseudomonas protegens Pf-5

摘要:

【目的】从基因水平探究碳分解代谢阻遏调控系统中CbrB对防御假单胞菌(Pseudomonas protegens) Pf-5生物防治能力的影响。【方法】通过基于pJQ200SK的二次同源重组法构建cbrB编码区无痕缺失突变菌株Pf5274，采用基于pBBR1Am的质粒回补法构建cbrB互补菌株及其对照菌株，通过测定菌株OD₆₀₀、结晶紫染色法、琼脂平板法、平板对峙法和pltL'-'lacZ融合报告菌株，分析CbrB对防御假单胞菌Pf-5生长、生物膜形成、运动性、抗真菌活性和藤黄绿脓菌素合成的影响。【结果】CbrB在天然培养基LB中显著抑制Pf-5的生长，而在基础培养基M9-葡萄糖中则显著促进Pf-5的生长；CbrB显著促进Pf-5的运动性，但是却抑制其生物膜形成、抗真菌活性及藤黄绿脓菌素合成。【结论】CbrB在调控防御假单胞菌Pf-5生物防治方面具有一定的作用，具体表现在菌株生长、生物膜形成、运动性、抗真菌活性及藤黄绿脓菌素合成等方面。本研究为利用基因工程技术提高生防菌株生物防治能力提供理论依据，并为深度发掘藤黄绿脓菌素生物合成奠定基础。

Abstract:

[Objective] To explore the effects of CbrB from the carbon catabolite repression system on the biocontrol performance of Pseudomonas protegens Pf-5. [Methods] The mutant Pf5274 with the markerless deletion of the coding region of cbrB was constructed by a double-crossover recombination event based on pJQ200SK. Moreover, the complementary strain of cbrB and the control strains were constructed by the plasmid complementation method based on pBBR1Am. Lastly, the effects of CbrB on the growth, biofilm formation, motility, antifungal activity, and pyoluteorin synthesis of Pf-5 were analyzed by the measurement of OD₆₀₀, crystal violet staining, agar plate culture, plate confrontation method, and pltL'-'lacZ fusion report strains, respectively. [Results] CbrB greatly slowed the growth of Pf-5 in the natural medium LB, but greatly sped up the growth of Pf-5 in the basic medium M9-glucose. In addition, CbrB significantly promoted the motility but inhibited the biofilm formation, antifungal activity, and pyoluteorin synthesis of Pf-5. [Conclusion] CbrB plays a role in regulating the growth, biofilm formation, motility, antifungal activity, and pyoluteorin synthesis of P. protegens Pf-5, thus regulating the biocontrol performance of this strain. This study provides a theoretical basis for the biocontrol capabilities of strains by genetic engineering and lays a foundation for probing into the biosynthesis of pyoluteorin.