【目的】功能性微生物可以协助土壤和生态系统重建。通过插入外源质粒分析不同菌株在自然界中的繁殖能力、生存能力等,研究硅酸盐岩高效溶蚀细菌野生型假单胞菌Pseudomonas protegens菌株NLX-4的抗性质粒标记方法,并探究该改良菌株受转入基因的影响,为岩土改良功能性菌株的进一步应用开发奠定基础。【方法】以筛选出的硅酸盐岩溶蚀细菌NLX-4为试验菌株,采用电转化方式转入质粒,获得具有氨苄青霉素抗性的新型突变株;通过稀释涂布法分10⁴、10⁵、10⁶共3个梯度对其氨苄青霉素抗性能力进行检验,通过平板生长法和生长曲线测定法,以2 h为间隔,测定两菌株OD₆₀₀值,对比突变株与原菌株的生长能力,通过盆栽试验测定植物根围土壤机械粒径的变化,并参考富集率和平均质量直径指标分析菌株溶蚀能力差异。【结果】相较于原菌株NLX-4,突变株NLX-41(OD₆₀₀=1)成功获得氨苄青霉素抗性基因,能够以不同浓度梯度在氨苄青霉素抗性平板上长出菌落。在生长速度上,突变株NLX-41与原菌株均在36 h时菌浓度达到峰值,吸光度稳定在2.8左右;溶蚀能力上NLX-41和NLX-4处理组的粉粒、极细砂、细砂含量相较于对照组均有显著性差异(P<0.01)。突变株平均质量直径为原菌株的96.46%,原菌株和突变株处理下中砂富集率分别降低19%、24%,而细砂富集率分别提高20%、25%。【结论】通过电转化获得1株突变株NLX-41,其具有氨苄青霉素抗性的同时仍能保持高效的生长能力、溶蚀能力。采用本方法提高了该菌株的筛选效率,便于该功能性微生物的快速鉴定。同时,掌握了该功能性菌株的遗传转化方法,便于在未来不同情境下转入目的基因,提升该菌株应用潜力。

【Objective】Functional microorganisms play crucial roles in facilitating soil restoration and ecosystem reconstruction. This study aims to establish a method for labeling the highly efficient silicate-dissolving bacterial strain Pseudomonas protegens NLX-4 with an antibiotic resistance plasmid by introducing exogenous plasmids to evaluate the reproductive capacity, survival ability, and other characteristics of different strains in natural environments. The research further investigates the effects of genetic modification on strain performance, providing a foundation for the broader application of functional strains in geotechnical improvement.【Method】The silicate-dissolving bacterial strain P. protegens NLX-4 was used as the experimental strain.A plasmid was introduced via electroporation to generate a novel mutant strain with ampicillin resistance.The ampicillin resistance was tested using the dilution coating method at three gradients(10⁴,10⁵,10⁶).Growth capabilities were compared between the mutant and original strains through plate growth assays and growth curve measurements (OD₆₀₀ values recorded at 2-hour intervals).Soil mechanical particle size changes in plant rhizospheres were analyzed via pot experiments,with dissolution capacity differences evaluated using enrichment rates and average mass diameter indices.【Result】Compared to the original strain P. protegens NLX-4,the mutant strain P. protegens NLX-41(OD₆₀₀=1) successfully acquire the ampicillin resistance gene and form colonies on ampicillin resistance plates at all concentration gradients.In terms of growth rate,both the mutant strain NLX-41 and the original strain reach the peak bacterial concentration at 36 h,with the absorbance stabilizing at approximately 2.8.In terms of dissolution ability,the contents of silt,very fine sand,and fine sand in the NLX-41 group and NLX-4 group show significant differences compared with the control group(P<0.01).The average weight diameter of the mutant strain is 96.46% of that of the original strain.Under the treatment of the original strain and the mutant strain,the enrichment rates of medium sand decrease by 19% and 24%,respectively,while the enrichment rates of fine sand increase by 20% and 25% respectively.【Conclusion】A mutant strain,NLX-41, is obtained via electroporation,retaining high-efficiency growth and dissolution capabilities while exhibiting ampicillin resistance.This method enhances the screening efficiency of the strain,facilitating rapid identification of functional microorganisms.Additionally,the established genetic transformation protocol enables future insertion of target genes under diverse scenarios,expanding its application potential.