解磷菌PSB-R全基因组测序鉴定及其解磷特性分析

doi:10.13560/j.cnki.biotech.bull.1985.2022-0448

摘要/Abstract

摘要：

采用生理生化指标结合分子生物学技术对解磷菌PSB-R进行分类学鉴定，确定为粘质沙雷氏菌（Serratia marcescens），通过二代测序平台Illumina NovaSeq PE150对PSB-R进行全基因组测序，分析预测了与解磷能力相关基因及其他植物促生基因组成情况。通过响应面优化试验检测了PSB-R最大解磷能力为805.199 mg/L，连续培养10代后解磷能力稳定且对多种难溶性磷酸盐均具有溶解能力。本研究为解磷菌解磷机制的进一步研究提供了基因组数据基础，同时证实PSB-R具有应用于菌肥的潜力，为后续解磷菌肥的研制提供了研究基础。

关键词: 解磷细菌, 粘质沙雷氏菌, 响应面优化, 全基因组测序

Abstract:

The phosphate-solubilizing bacterium PSB-R was identified as Serratia marcescens by physiological and biochemical indicators combined with molecular biology technology. Second-generation sequencing platform Illumina NovaSeq PE150 was used to have the whole genome of PSB-R sequenced, and to analyze and predict the composition of genes related to phosphate-solubilizing ability and other plant growth-promoting genes. The response surface optimization test showed that the maximum phosphorus dissolving capacity of PSB-R was 805.199 mg/L. After 10 generations of continuous culture, the phosphorus dissolving capacity was stable and had the ability to dissolve a variety of insoluble phosphates. This study provides a genomic data basis for the further study of the mechanism of phosphate solubilization by phosphate solubilizing bacteria, and confirms that there is a potential for PSB-R to be applied to bacterial fertilizer, which provides a research basis for the subsequent development of phosphate-solubilizing bacterial fertilizer.

Key words: phosphate-solubilizing bacteria, Serratia marcescens, response surface optimization, whole genome sequencing

王帅, 吕鸿睿, 张昊, 吴占文, 肖翠红, 孙冬梅. 解磷菌PSB-R全基因组测序鉴定及其解磷特性分析[J]. 生物技术通报, 2023, 39(1): 274-283.

WANG Shuai, LV Hong-rui, ZHANG Hao, WU Zhan-wen, XIAO Cui-hong, SUN Dong-mei. Whole-Genome Sequencing Identification of Phosphate-solubilizing Bacteria PSB-R and Analysis of Its Phosphate-solubilizing Properties[J]. Biotechnology Bulletin, 2023, 39(1): 274-283.

图/表 9

参考文献 27

[1]	王雪郦, 张芮瑞, 周少奇, 等. 不良环境解磷微生物研究进展[J]. 河南农业科学, 2020, 49(7): 8-17.
	Wang XL, Zhang RR, Zhou SQ, et al. Research progress of phosphate-solubilizing microorganisms in bad environments[J]. J Henan Agric Sci, 2020, 49(7): 8-17.
[2]	吉蓉. 土壤解磷微生物及其解磷机制综述[J]. 甘肃农业科技, 2013(8): 42-45.
	Ji R. Research summary on phosphate dissolution of phosphate solubilizing microorganisms[J]. Gansu Agric Sci Technol, 2013(8): 42-45.
[3]	Schindler DW, Hecky RE, Findlay DL, et al. Eutrophication of lakes cannot be controlled by reducing nitrogen input: results of a 37-year whole-ecosystem experiment[J]. Proc Natl Acad Sci USA, 2008, 105(32): 11254-11258. doi: 10.1073/pnas.0805108105 pmid: 18667696
[4]	Boubekri K, Soumare A, Mardad I, et al. The screening of potassium- and phosphate-solubilizing actinobacteria and the assessment of their ability to promote wheat growth parameters[J]. Microorganisms, 2021, 9(3): 470. doi: 10.3390/microorganisms9030470 URL
[5]	Whitelaw MA. Growth promotion of plants inoculated with phosphate-solubilizing fungi[J]. Adv Agron, 1999, 69: 99-151.
[6]	Alori ET, Glick BR, Babalola OO. Microbial phosphorus solubilization and its potential for use in sustainable agriculture[J]. Front Microbiol, 2017, 8: 971. doi: 10.3389/fmicb.2017.00971 pmid: 28626450
[7]	Xie JG, Yan ZQ, Wang GF, et al. A bacterium isolated from soil in a Karst rocky desertification region has efficient phosphate-solubilizing and plant growth-promoting ability[J]. Front Microbiol, 2021, 11: 625450. doi: 10.3389/fmicb.2020.625450 URL
[8]	Reyes I, Bernier L, Antoun H. Rock phosphate solubilization and colonization of maize rhizosphere by wild and genetically modified strains of Penicillium rugulosum[J]. Microb Ecol, 2002, 44(1): 39-48. pmid: 12019460
[9]	池景良, 郝敏, 王志学, 等. 解磷微生物研究及应用进展[J]. 微生物学杂志, 2021, 41(1): 1-7.
	Chi JL, Hao M, Wang ZX, et al. Advances in research and application of phosphorus-solubilizing microorganism[J]. J Microbiol, 2021, 41(1): 1-7.
[10]	王俊娟, 阎爱华, 王薇, 等. 铁尾矿区油松根际溶磷泛菌D2的筛选鉴定及溶磷特性[J]. 应用生态学报, 2016, 27(11): 3705-3711. doi: 10.13287/j.1001-9332.201611.003
	Wang JJ, Yan AH, Wang W, et al. Screening, identification and phosphate-solubilizing characteristics of phosphate-solubilizing bacteria strain D2(Pantoea sp.)in rhizosphere of Pinus tabuliformis in iron tailings yard[J]. Chin J Appl Ecol, 2016, 27(11): 3705-3711.
[11]	李海云, 姚拓, 张榕, 等. 红三叶根际溶磷菌株分泌有机酸与溶磷能力的相关性研究[J]. 草业学报, 2018, 27(12): 113-121.
	Li HY, Yao T, Zhang R, et al. Relationship between organic acids secreted from rhizosphere phosphate-solubilizing bacteria in Trifolium pratense and phosphate-solubilizing ability[J]. Acta Prataculturae Sin, 2018, 27(12): 113-121.
[12]	Zhu FL, Qu LY, Hong XG, et al. Isolation and characterization of a phosphate-solubilizing halophilic bacterium Kushneria sp. YCWA18 from daqiao saltern on the coast of Yellow Sea of China[J]. Evid Based Complementary Altern Med, 2011, 2011: 615032.
[13]	Ochoa-Loza FJ, Artiola JF, Maier RM. Stability constants for the complexation of various metals with a rhamnolipid biosurfactant[J]. J Environ Qual, 2001, 30(2): 479-485. pmid: 11285908
[14]	Ludueña LM, Anzuay MS, Angelini JG, et al. Strain Serratia sp. S119: a potential biofertilizer for peanut and maize and a model bacterium to study phosphate solubilization mechanisms[J]. Appl Soil Ecol, 2018, 126: 107-112. doi: 10.1016/j.apsoil.2017.12.024 URL
[15]	Ludueña LM, Anzuay MS, Angelini JG, et al. Role of bacterial pyrroloquinoline quinone in phosphate solubilizing ability and in plant growth promotion on strain Serratia sp. S119[J]. Symbiosis, 2017, 72(1): 31-43. doi: 10.1007/s13199-016-0434-7 URL
[16]	Chen ML, Ma YK, Wu S, et al. Genome warehouse: a public repository housing genome-scale data[J]. Genomics Proteomics Bioinformatics, 2021, 19(4): 584-589. doi: 10.1016/j.gpb.2021.04.001 URL
[17]	中华人民共和国农业部. NY/T 2421-2013植株全磷含量测定钼锑抗比色法[S]. 国家标准馆, 2013.
	Ministry of Agriculture and Rural Affairs of the People's Republic of China. NY/T 2421-2013 Determination of total phosphorus in plant. Vanadium molybdate blue colorimetric method[S]. National Library of Standards, 2013.
[18]	国家质量监督检验检疫总局, 中国国家标准化管理委员会. 尿素的测定方法第4部分:铁含量邻菲啰啉分光光度法: GB/T 2441.4—2010[S]. 北京: 中国标准出版社, 2011.
	General Administration of Quality Supervision, Inspection and Quarantine of the People's Republic of China, Standardization Administration of the People's Republic of China. Determination of urea—Part 4: iron content—1, 10-Phenanthroline spectrophotometric method: GB/T 2441.4—2010[S]. Beijing: Standards Press of China, 2011.
[19]	国家统计局. 第七次全国人口普查公报(第二号)[R]. 2021.
	National Bureau of Statistics. Communiqué of the Seventh National Population Census(No. 2)[R]. 2021.
[20]	唐岷宸, 李文静, 宋天顺, 等. 一株高效解磷菌的筛选及其解磷效果验证[J]. 生物技术通报, 2020, 36(6): 102-109. doi: 10.13560/j.cnki.biotech.bull.1985.2019-0969
	Tang MC, Li WJ, Song TS, et al. Screening of a highly efficient phosphate-solubilizing bacterium and validation of its phosphate-solubilizing effect[J]. Biotechnol Bull, 2020, 36(6): 102-109.
[21]	魏烈群. 荣成天鹅湖解磷菌的分离筛选及其对沉积物磷释放的影响[D]. 烟台: 烟台大学, 2021.
	Wei LQ. Isolation and screening of phosphate-solubilizing bacteria and the effect on phosphorus release from the sediments in Rongcheng Swan lake[D]. Yantai: Yantai University, 2021.
[22]	朱德旋, 杜春梅, 董锡文, 等. 一株寒地高效解无机磷细菌的分离鉴定及拮抗作用[J]. 微生物学报, 2020, 60(8): 1672-1682.
	Zhu DX, Du CM, Dong XW, et al. Identification and antagonism activity of an inorganic phosphorus-dissolving bacterial strain isolated from cold region[J]. Acta Microbiol Sin, 2020, 60(8): 1672-1682.
[23]	赵伟进, 王孝先, 杨洋, 等. 黑青稞根际促生菌筛选及其对种子萌发的影响[J]. 种子, 2018, 37(12): 1-5, 10.
	Zhao WJ, Wang XX, Yang Y, et al. Selection of rhizotrophic bacteria from rhizosphere and its effect on seed germination of black barley[J]. Seed, 2018, 37(12): 1-5, 10.
[24]	Zhao JJ, Wang S, Zhu XF, et al. Isolation and characterization of nodules endophytic bacteria Pseudomonas protegens Sneb1997 and Serratia plymuthica Sneb2001 for the biological control of root-knot nematode[J]. Appl Soil Ecol, 2021, 164: 103924. doi: 10.1016/j.apsoil.2021.103924 URL
[25]	陈佳兴, 秦琴, 邱树毅, 等. 磷尾矿土壤中解磷细菌的筛选及解磷能力的测定[J]. 生物技术通报, 2018, 34(6): 183-189. doi: 10.13560/j.cnki.biotech.bull.1985.2017-1122
	Chen JX, Qin Q, Qiu SY, et al. Isolation, identification of phosphate-solubilizing bacteria derived from phosphate tailing soil and their capacities[J]. Biotechnol Bull, 2018, 34(6): 183-189.
[26]	卢琴, 罗青平, 张腾飞, 等. 灰雁黏质沙雷菌的鉴定及其耐药性分析[J]. 动物医学进展, 2019, 40(2): 130-134.
	Lu Q, Luo QP, Zhang TF, et al. Identification and drug sensitivity test of Serratia marcescens in greylag goose[J]. Prog Vet Med, 2019, 40(2): 130-134.
[27]	乔志伟, 洪坚平, 谢英荷, 等. 石灰性土壤拉恩式溶磷细菌的筛选鉴定及溶磷特性[J]. 应用生态学报, 2013, 24(8): 2294-2300.
	Qiao ZW, Hong JP, Xie YH, et al. Screening, identification and phosphate-solubilizing characteristics of Rahnella sp. phosphate-solubilizing bacteria in calcareous soil[J]. Chin J Appl Ecol, 2013, 24(8): 2294-2300.

项目Item	数据Data
Scaffold数量 Number of Scaffold	16
Contig数量 Number of Contig	16
（G+C）/%	59.75
基因组大小Genome size/bp	5 061 510
预测基因数量 Number of genes	4 742
基因总长度Total gene length/bp	4 393 551

项目Item	数据Data
Scaffold数量 Number of Scaffold	16
Contig数量 Number of Contig	16
（G+C）/%	59.75
基因组大小Genome size/bp	5 061 510
预测基因数量 Number of genes	4 742
基因总长度Total gene length/bp	4 393 551

基因号码Gene code	基因名称Gene name	产物名称 Product name	生物活性 Activity
PSB.R_GM000437	gcd	PQQ依赖型葡萄糖脱氢酶 Quinoprotein glucose dehydrogenase	葡萄糖酸合成 Gluconic acid synthesis
PSB.R GM001125	ppq B	PQQ合成蛋白B Cofactor PQQ biosynthesis protein B
PSB.R GM001126	ppq C	PQQ合成蛋白C Cofactor PQQ biosynthesis protein C
PSB.R GM001127	ppq D	PQQ合成蛋白D Cofactor PQQ biosynthesis protein D
PSB.R GM001128	ppq E	PQQ合成蛋白E Cofactor PQQ biosynthesis protein E
PSB.R GM001129	ppq F	PQQ合成蛋白F Cofactor PQQ biosynthesis protein F
PSB.R_GM000385	ent A	2,3-二氢-2,3-二羟基苯甲酸脱氢酶 2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase	铁载体产生 Siderophore production
PSB.R_GM003128	ent C	异分支酸合成酶C Isochorismate synthase C
PSB.R_GM003126	ent B	肠杆菌素合成亚基B Enterobactin synthase subunit B
PSB.R_GM003127	ent E	肠杆菌素合成亚基E Enterobactin synthase subunit E
PSB.R_GM003130	ent F	肠杆菌素合成亚基F Enterobactin synthase subunit F
PSB.R_GM003129	ent S	肠杆菌素（铁载体）外排蛋白 Enterobactin（siderophore）exporter
PSB.R_GM002907	exb B	生物聚合物转运蛋白B Biopolymer transport protein B
PSB.R_GM002910	exb D	生物聚合物转运蛋白D Biopolymer transport protein D
PSB.R_GM004736	bfr	细菌铁蛋白 Bacterioferritin
PSB.R_GM000070	ipd C	吲哚丙酮酸脱羧酶 Indolepyruvate decarboxylase	IAA产生 IAA production
PSB.R_GM000012	chi	几丁质酶 Chitinase	几丁质酶产生 Chitinase production
PSB.R_GM003955	ppx	外切聚磷酸酶 Exopolyphosphatase	磷酸盐降解酶 Phosphate degrading enzyme
PSB.R_GM003047	ppa	无机焦磷酸酶 Inorganic pyrophosphatase
PSB.R_GM001406	phn X	磷酸乙醛水解酶 Phosphonoacetaldehyde hydrolase
PSB.R_GM002025	pho A	碱性磷酸酶 Alkaline phosphatase

基因号码Gene code	基因名称Gene name	产物名称 Product name	生物活性 Activity
PSB.R_GM000437	gcd	PQQ依赖型葡萄糖脱氢酶 Quinoprotein glucose dehydrogenase	葡萄糖酸合成 Gluconic acid synthesis
PSB.R GM001125	ppq B	PQQ合成蛋白B Cofactor PQQ biosynthesis protein B
PSB.R GM001126	ppq C	PQQ合成蛋白C Cofactor PQQ biosynthesis protein C
PSB.R GM001127	ppq D	PQQ合成蛋白D Cofactor PQQ biosynthesis protein D
PSB.R GM001128	ppq E	PQQ合成蛋白E Cofactor PQQ biosynthesis protein E
PSB.R GM001129	ppq F	PQQ合成蛋白F Cofactor PQQ biosynthesis protein F
PSB.R_GM000385	ent A	2,3-二氢-2,3-二羟基苯甲酸脱氢酶 2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase	铁载体产生 Siderophore production
PSB.R_GM003128	ent C	异分支酸合成酶C Isochorismate synthase C
PSB.R_GM003126	ent B	肠杆菌素合成亚基B Enterobactin synthase subunit B
PSB.R_GM003127	ent E	肠杆菌素合成亚基E Enterobactin synthase subunit E
PSB.R_GM003130	ent F	肠杆菌素合成亚基F Enterobactin synthase subunit F
PSB.R_GM003129	ent S	肠杆菌素（铁载体）外排蛋白 Enterobactin（siderophore）exporter
PSB.R_GM002907	exb B	生物聚合物转运蛋白B Biopolymer transport protein B
PSB.R_GM002910	exb D	生物聚合物转运蛋白D Biopolymer transport protein D
PSB.R_GM004736	bfr	细菌铁蛋白 Bacterioferritin
PSB.R_GM000070	ipd C	吲哚丙酮酸脱羧酶 Indolepyruvate decarboxylase	IAA产生 IAA production
PSB.R_GM000012	chi	几丁质酶 Chitinase	几丁质酶产生 Chitinase production
PSB.R_GM003955	ppx	外切聚磷酸酶 Exopolyphosphatase	磷酸盐降解酶 Phosphate degrading enzyme
PSB.R_GM003047	ppa	无机焦磷酸酶 Inorganic pyrophosphatase
PSB.R_GM001406	phn X	磷酸乙醛水解酶 Phosphonoacetaldehyde hydrolase
PSB.R_GM002025	pho A	碱性磷酸酶 Alkaline phosphatase