suhB基因对绿针假单胞菌HT66生防能力的影响

Effect of suhB gene on biocontrol ability of Pseudomonas chlororaphis HT66

摘要:

【背景】绿针假单胞菌(Pseudomonas chlororaphis) HT66是一株兼具生防安全性和吩嗪-1-甲酰胺(Phenazine-1-Carboxamide，PCN)高产的植物根际促生菌，在生物防治、生态农业及可持续发展农业领域具有广阔的应用前景。非编码RNA (ncRNA) SuhB参与了细胞中多个过程的代谢调控。【目的】探究suhB基因对绿针假单胞菌HT66生防能力的影响。【方法】以同源重组的方法无痕敲除suhB基因构建突变菌株HT66ΔsuhB，利用质粒回补suhB基因构建突变菌株HT66ΔsuhB-pBBR-suhB，研究suhB基因对菌株生长状态、生物膜形成、群集运动及PCN合成的影响。【结果】缺失suhB基因后，菌株HT66生长缓慢，平台期滞后12 h，而且生物量减少为野生型的61.6%；在KMB培养基中单位细胞PCN产量最高达109.5 mg/g，为野生株的2.1倍；生物膜形成量明显增加，为野生型的1.8倍；在运动性检测平板上，野生株的运动半径为21 mm，而suhB突变株的运动半径缩减至9.7 mm，群集运动能力明显下降。suhB基因回补突变株上述生物学功能同野生株相似。在突变株HT66ΔsuhB中，pME6015-phzI-lacZ融合质粒表达的LacZ酶活与野生型差异不显著；pME6015-phzR-lacZ融合质粒表达的LacZ酶活显著上升，为野生型的3.1倍；pME6522-phzAp-lacZ融合质粒表达的LacZ酶活为野生型的1.8倍。【结论】绿针假单胞菌HT66中suhB基因参与了菌株生长、生物膜形成、群集运动及PCN合成等多个过程的调控。本研究为该菌株的代谢改造与生防应用提供了理论基础。

Abstract:

[Background] Pseudomonas chlororaphis HT66 is a plant growth-promoting rhizobacterium (PGPR) with biocontrol safety and high yield of phenazine-1-carboxamide (PCN). It has broad application prospects in biological control, ecological agriculture and sustainable agriculture. SuhB, a small non-coding RNA, involved in the metabolic regulation of multiple processes in cells. [Objective] This study explored the effect of suhB gene on the biocontrol ability of in P. chlororaphis HT66. [Methods] we constructed the suhB deletion mutant HT66ΔsuhB by homologous recombination method and the suhB complemented mutant HT66ΔsuhB-pBBR-suhB by plasmid. They were used to explore the effect of suhB gene on strain growth, biofilm formation, swarming motility and PCN synthesis. [Results] The mutant HT66ΔsuhB grew slowly, the plateau period delayed by 12 h and its biomass decreased to 61.6% of the wild type. The maximum yield of mutant strain in KMB medium can reach 109.5 mg/g (per DCW), which was 2.1 times that of wild type. The biofilm formation increased significantly, which was 1.8 times that of the wild type. However, the swarming motility of HT66ΔsuhB was defective. On the swarm plate, the movement radius of the wild strain was 21 mm, while the movement radius of the mutant strain was 9.7 mm. The suhB gene complemented mutant is similar to wild type in the above aspects. Compared with the wild type, there is no difference on the expression of phzI at the overall level in mutant HT66ΔsuhB. On the contrary, the expression of phzR increased significantly, which is 3.1 times that of the wild type. In addition, the expression of phzAp at the transcriptional level is 1.8 times that of the wild type. [Conclusion] The regulation of suhB gene in P. chlororaphis HT66 participates in the growth, biofilm formation, swarming motility and PCN synthesis. This research provides a theoretical basis for the metabolic transformation and biocontrol applications of P. chlororaphis HT66.