罗非鱼优势耐冰温假单胞菌的分离鉴定及耐冰温基因

Isolation and Identification of Dominant Pseudomonas spp.from Tilapia under Super-chilled Storage and Ice-tolerant Genes of Pseudomonas Strains

【目的】分析罗非鱼(Oreochromis mossambicus)中优势耐冰温假单胞菌(Pseudomonas)特有的基因特征和耐冰温基因。【方法】采用全长16S rDNA测序找出冰温贮藏罗非鱼中的优势假单胞菌,进行分离、鉴定;通过Illumina平台对其进行基因组从头测序,并通过基因家族分析确定耐冰温假单胞菌的核心基因家族,再与不耐冰温的铜绿假单胞菌(Pseudomonas aeruginosa PAO1)的基因组比较,筛选出耐冰温假单胞菌特有的核心基因家族;结合文献报道的假单胞菌的寒冷适应基因筛查出耐冰温假单胞菌潜在的耐冰温基因。【结果与结论】发现并分离出冰温贮藏罗非鱼中丰度最高的5株腐败菌,经鉴定均为布氏假单胞菌(Pseudomonas bubulae)和莓实假单胞菌(Pseudomonas fragi)。这5株假单胞菌的核心基因家族有3 692个,其中861个在不耐冰温的铜绿假单胞菌的基因组中不存在,此861个特有的核心基因家族在群体感应、类固醇的降解、某些氨基酸和肽的代谢、脂肪酸降解、抗坏血酸和醛糖酸代谢、柠檬烯和蒎烯降解、丁酸代谢、苯乙烯降解和香叶醇降解等途径中显著富集。进一步筛查发现上述5株耐冰温假单胞菌带有不耐冰温的铜绿假单胞菌没有的des、cbpM、ousA、treP、treR、trxC和grX等基因。

【Objective】The aim of this study is to analyze the unique genetic characteristics and icetolerant genes of dominant ice-tolerant Pseudomonas strains isolated from tilapia.【Methods】 The dominant Pseudomonas strains were first identified using full-length 16S rDNA sequencing from tilapia under super-chilled storage.These strains were then isolated,identified,and subjected to de novo genome sequencing using the Illumina platform.Gene family analysis was performed to determine the core gene families of these ice-tolerant Pseudomonas strains,which were subsequently compared to the genome of the ice-intolerant Pseudomonas aeruginosa PAO1 to identify the unique core gene families of these and then screened out the potential ice-tolerant genes in combination with the cold-adaptive genes of Pseudomonas reported in the literature.【Result and conclusion】 Five strains of spoilage bacteria with the highest abundance in tilapia under super-chilled storage were discovered and isolated,and they were identified as Pseudomonas bubulae and Pseudomonas fragi.These five Pseudomonas strains shared 3 692 core gene families,of which 861 were absent in the genome of ice-intolerant P.aeruginosa.These 861 unique core gene families were significantly enriched in pathways related to quorum sensing,steroid degradation,metabolism of certain amino acids and peptides,fatty acid degradation,ascorbate and aldarate metabolism,limonene and pinene degradation,butanoate metabolism,styrene degradation,geraniol degradation and others.Further screening revealed that these five ice-tolerant Pseudomonas strains possessed the potential ice-tolerant genes,such as des,cbpM,ousA,treP,treR,trxC,and grX,which were absent in the genome of ice-intolerant P.aeruginosa.