景德镇高岭土中解磷菌的分离及全基因组测序分析

Isolation and whole genome sequencing analysis of phosphatesolubilizing bacteria from Jingdezhen Kaolin

【目的】从景德镇高岭土中分离解磷菌并进行全基因组测序分析,以期为解磷微生物的挖掘及探究其解磷性状和解磷机制提供研究基础。【方法】对景德镇高岭土中的解磷菌进行分离,采用溶磷指数对菌株分解无机磷和有机磷能力进行初步评估,采用钼锑抗比色法对菌株的解磷能力进行定量分析,采用碱性磷酸酶(AKP/ALP)检测试剂盒对菌株碱性磷酸酶活性进行测定,采用16S rDNA对菌株进行鉴定,选取解磷能力较强的菌株进行全基因测序,并对其解磷相关基因进行分析。【结果】从景德镇高岭土中分离到5株既能分解有机磷又能分解无机磷的解磷细菌,分别命名为GP1、GP2、GP3、GP4和GP5。5株菌株均能产生碱性磷酸酶,其中,GP1分解无机磷能力最强,其发酵液中可溶性磷含量最高(61.77 mg/L);GP4分解有机磷能力最强,其发酵液中可溶性磷含量最高(30.22 mg/L)。16S rDNA测序结果显示,GP1和GP3为伯克霍尔德氏菌属(Burkholderia),GP2为副伯克霍尔德属(Paraburkholderia),GP4和GP5为藤黄色杆菌属(Luteibacter)。管家基因鉴定结果表明GP1为吡咯菌素伯克霍尔德氏菌(Burkholderia pyrrocinia)。基因注释结果显示,菌株GP1含有8个无机磷分解相关基因(pqqA、pqqB、pqqC、pqqD、pqqE、gdh、gltA、ppa)、4个有机磷分解基因(phoA、phnA、phnW、phnY)、8个磷化合物转运基因(ugpB、ugpA、ugpE、ugpC、pstB、pstA、pstC、pstS)及2个磷缺乏响应双组分调节基因(phoR、phoB)。【结论】景德镇高岭土中存在解磷细菌,从中分离的解磷细菌具有良好的分解有机磷和无机磷能力,是潜在的良好微生物肥料开发资源。GP1分解Ca3(PO4)2的机制可能是分泌葡萄糖酸、柠檬酸及无机焦磷酸酶,分解有机磷的机制可能是通过分泌碱性磷酸酶及C-P键裂解酶。GP1还具有适应低磷环境的潜力。

【Objective】To isolate phosphate-solubilizing bacteria from Jingdezhen Kaolin and conducted whole genome sequencing analysis,in order to provide a research basis for the exploration of phosphorus-solubilizing microorganisms and their phosphorus-solubilizing characteristics and mechanisms.【Method】Phosphate solubilizing bacteria were isolated from Jingdezhen Kaolin,and the ability of the strains to decompose inorganic and organic phosphorus were initially evaluated using soluble phosphorus index.The phosphorus solubilization ability of the strains was quantitatively analyzed using Mo-Sb colorimetric method and the alkaline phosphatase activity of the strains were measured using alkaline phosphatase(AKP/ALP)reagent kit.The strains were identified using 16S rDNA,and the strains with the strongest phosphorus solubilization ability were selected for whole genome sequencing,and their phosphorus solubilization-related genes were analyzed.【Result】Five strains of phosphate-solubilizing bacteria,named GP1,GP2,GP3,GP4 and GP5,were isolated from Jingdezhen Kaolin,which could dissolve both organic and inorganic phosphorus.All five strains could produce alkaline phosphatase.Among them,GP1 had the strongest ability to dissolve inorganic phosphorus and its fermentation broth contained the highest content of soluble phosphorus(61.77 mg/L).GP4 had the strongest ability to decompose organic phosphorus,with the highest content of soluble phosphorus in its fermentation broth(30.22 mg/L).The sequencing results of 16S rDNA showed that GP1 and GP3 belonged to the genus Burkholderia sp.,GP2 belonged to the genus Paraburkholderia sp.,and GP4 and GP5 belonged to the Luteiobacter sp.The results of the housekeeping gene identification indicated that GP1 was Burkholderia pyrrocinia.The results of gene annotation showed that strain GP1 contained eight inorganic phosphorus solubilization related genes(pqqA,pqqB,pqqC,pqqD,pqqE,gdh,gltA,ppa),four organic phosphorus solubilization genes(phoA,phnA,phnW,phnY),eight phosphorus compound transport genes(ugpB,ugpA,ugpE,ugpC,pstB,pstA,pstC,pstS),and two phosphorus deficiency responsive dual component regulatory genes(phoR,phoB).【Conclusion】Phosphate-solubilizing bacteria exist in Jingdezhen Kaolin,and the isolated bacteria from it have good ability to decompose organic and inorganic phosphorus.,which is a good potential resource for the development of microbial fertilizer.The mechanism by which GP1 solubilizes Ca3(PO4)2 may be by secretion gluconic acid,citric acid,and inorganic pyrophosphatase.The mechanism by which GP1 decomposes organic phosphorus may be through the secretion of alkaline phosphatase and C-P bond lyases.GP1 also has the potential to adapt to low phosphorus environments.