摘要
绿针假单胞菌Pseudomonas chlororaphis YL-1是从大豆根围分离获得的,对多种病原细菌和病原真菌有较强抑制作用。为了探明绿针假单胞菌YL-1生防相关基因的功能,本研究以嗜铁素转录调控因子PvdS编码基因为对象,建立了一套基于负选择标记基因sacB的绿针假单胞菌无标记基因敲除技术,构建重组质粒p EX18-pvdS,通过改良的细菌接合转移技术将重组质粒导入野生型菌株YL-1中,利用同源重组技术获得缺失突变株ΔpvdS。生防相关性状研究结果表明,与野生型菌株YL-1相比,突变株ΔpvdS泳动能力和生长能力未发生改变,但是群集运动能力显著下降。同时突变株ΔpvdS合成嗜铁素的能力也显著下降,pvdS基因互补后突变株能恢复合成嗜铁素的功能。本文结果表明,已成功建立了适用于YL-1的基因定向敲除技术和功能基因互补体系,为深入研究YL-1的生防机制奠定了重要基础。
Strain YL-1 was isolated from soybean root tips and identified as Pseudomonas chlororaphis. This strain showed broad-spectrum antibacterial and antifungal activities against plant phytopathogens that are economically important in agriculture. In order to explore the function of its biocontrol-associated genes, an efficient genetic manipulation system was established in this study. The Bacillus subtilis sacB gene, whose product encodes levansucrase that is toxic to Gram-negative bacteria in the presence of sucrose, was considered as a counterselection marker in the system. We selected a well-characterized pvdS as a representative example to generate an in-frame deletion mutant, because the product of pvdS encodes a sigma factor that is known to control pyoverdine biosynthesis at transcription level. A sacB-containing suicide-vector, pEX18 was used, in which both the upstream and downstream homolog fragment of pvdS was cloned, creating a recombinant plasmid, pEX18-pvdS. The recombinant plasmid was transformed into wild type strain YL-1 via an optimized bacterial conjugal approach. Via a double-crossover homologous recombinant approach, an in-frame deletion mutant of pvdS, named as ΔpvdS was generated and validated. Compared to the wild type, this mutant exhibited equal swimming motility and growth capacity, but a significant decrease in swarming motility and pyoverdine production.The function of pyoverdine production in the ΔpvdS could be rescued by introducing a plasmid-borne pvdS into this mutant. Together, our studies established an effective system for both in-frame gene deletion and complementation in YL-1, which facilitates to uncover the biocontrol mechanisms of YL-1 in the future.
引文
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