一株玉米根际促生菌Leclercia adecarboxylata LN01促生效果研究及其基因组分析

doi:10.13560/j.cnki.biotech.bull.1985.2024-0487

摘要/Abstract

摘要：

【目的】有益植物根际微生物通过加强土壤与农作物间互作以促进农作物生长。菌株LN01是一株玉米根际促生菌，探究菌株LN01对玉米植株生长的促进机制。【方法】采用16S rRNA和全基因组学测序，明确菌株LN01分类学地位；其次，通过盆栽实验验证菌株LN01对玉米的促生效果，通过全基因组数据挖掘与促生特性相关基因信息并借助碳氮分析仪、钼锑抗比色法、火焰分光光度计、Salkowski比色法、Chrome Azurol Sulfonate（CAS）蓝色定性检测平板研究菌株LN01固氮、溶磷、解钾、产吲哚乙酸（IAA）和铁载体能力。【结果】菌株LN01促进玉米植株生物量累积并有效提高土壤营养成分。经鉴定菌株LN01为Leclercia adecarboxylata，菌株LN01基因组中存在参与铁获取（fhuBCDEF、afuABC、efeOBU和fepCDG）、磷酸盐溶解（pstABCS、phoABER、phnACDEFGHIJKLP和ugpABCE）、生物固氮（nirBCD、nasA、glnA、gltBD和nrtABC）、解钾（kdpABC、kefBCFG、trkAH和kup）的基因簇和IAA生物合成基因簇（trpABSCFRGD）。通过antiSMASH分析，在整个基因组中发现了促进植物生长的4种次级代谢物的生物基因合成簇，包括非核糖体肽合成酶（NRPS）、萜烯、硫肽和芳基多烯。菌株LN01固氮量、磷酸盐溶解活性、解钾能力、IAA产量分别为15.14、58.33、15.6和 42.2 mg/L，并具有产生铁载体能力。【结论】菌株LN01具有固氮溶磷、解钾作用、铁载体生产及IAA分泌相关基因，帮助玉米植株固定营养成分，促进玉米植株发育。

关键词: Leclercia adecarboxylata LN01, 根际促生菌, 全基因组测序, 促生效果

Abstract:

【Objective】Beneficial plant inter-root microorganisms can enhance soil-crop interactions and promote crop growth. The aim of this paper is to investigate the growth promotion mechanism of strain LN01, a maize PGPR (Plant growth-promoting rhizobacteria) strain, on maize plants.【Method】Firstly, 16S rRNA and whole genomics sequencing were used to clarify the taxonomic status of strain LN01. Secondly, a pot experiment was conducted to validate the growth-promoting effects of strain LN01 on maize. Whole-genome data mining was performed to identify genes related to plant growth promotion, and carbon-nitrogen analyzers, molybdenum-antimony colorimetric methods, flame photometers, Salkowski colorimetric methods, and Chrome Azurol Sulfonate（CAS）blue qualitative detection plates were used to study the nitrogen fixation, phosphate solubilization, potassium solubilization, indole-3-acetic acid（IAA）production, and siderophore capabilities of strain LN01.【Result】The strain LN01 promoted biomass accumulation in maize plants and effectively increased soil nutrient content. Strain LN01 was identified as Leclercia adecarboxylata. There were gene clusters in strain LN01's genome for these, including iron acquisition（fhuBCDEF, afuABC, efeOBU, and fepCDG）, phosphate solubilization（pstABCS, phoABER, phnACDEFGHIJKLP, and ugpABCE）, biological nitrogen fixation（nirBCD, nasA, glnA, gltBD, and nrtABC）, potassium solubilization（kdpABC, kefBCFG, trkAH, and kup）and IAA biosynthesis（trpABSCFRGD）. The biogenic gene synthesis clusters for four secondary metabolites that promoted plant growth, including non-ribosomal peptide synthases（NRPS）, terpenes, thiopeptides, and aryl polyenes, were identified throughout the genome by antiSMASH analysis. Strain LN01 had the capacity of nitrogen fixation, phosphate solubilizing activity, potassium solubilizing capacity, and IAA production by 15.14, 58.33, 15.6 and 42.2 mg/L, respectively, as well as iron-carrier production capacity.【Conclusion】Strain LN01 possesses genes related to nitrogen-fixing and phosphorus-solubilizing, potassium solubilization, iron carrier production and IAA secretion, which helps maize plants fix nutrients and promote maize plant development.

Key words: Leclercia adecarboxylata LN01, rhizosphere growth-promoting bacteria, whole-genome sequencing, growth-promoting effect

张婷, 万雨欣, 徐伟慧, 王志刚, 陈文晶, 胡云龙. 一株玉米根际促生菌Leclercia adecarboxylata LN01促生效果研究及其基因组分析[J]. 生物技术通报, 2025, 41(1): 263-275.

ZHANG Ting, WAN Yu-xin, XU Wei-hui, WANG Zhi-gang, CHEN Wen-jing, HU Yun-long. Growth-promoting Effects of a Rhizosphere Growth-promoting Bacterium Leclercia adecarboxylata LN01 in Maize Plants and Its Whole-genome Analysis[J]. Biotechnology Bulletin, 2025, 41(1): 263-275.

图/表 12

表1 供试培养基

Table 1 Test medium

培养基Medium	组分Composition/（g·L^-1）
NBRIP 培养基^[12] National Botanical Research Institute's phosphate medium	葡萄糖10.0，Ca₃ (PO₄)₂ 5.0，MgCl₂ ·6H₂O 0.2， (NH₄)₂SO₄ 0.1，琼脂20.0，液体培养基不加琼脂
亚历山大罗夫培养基^[13] Alexandrov medium	蔗糖5.0，Na₂HPO₄ 2.0，MgSO₄ 0.5，CaCO₃ 0.1，FeCl₃ 0.005，钾长石1.0，溴百里酚蓝（100 mg/L）20 mL，琼脂20.0，pH 7.0
解钾液体发酵培养基^[14] Potassium bacteria medium	蔗糖10.0，Na₂HPO₄ 1.0，MgSO₄ 1.0，CaCO₃ 0.1，FeCl₃ 0.005，钾长石10.0，(NH₄)₂SO₄ 0.5
阿须贝无氮培养基^[15] Ashby’S medium	KH₂PO₄0.2，MgSO₄0.2，NaCl 0.2，CaCO₃ 5.0，甘露醇10.0，CaSO₄ 0.1，琼脂20.0，pH 7.0，液体培养基不加琼脂
牛肉膏蛋白胨培养基^[16] Nutrient broth medium	蛋白胨10.0，NaCl 5.0，牛肉膏3.0，琼脂20.0，pH 7.0，液体培养基不加琼脂
CAS培养基Chrome azurol sulfonate medium	CAS培养基10.87，琼脂20.0，pH 7.0

图1 基于16S rRNA和31个看家基因构建系统发育树

Fig. 1 Phylogenetic tree construction based on 16S rRNA or 31 housekeeping genes

图2 菌株LN01对玉米幼苗的促生效应及综合能力评价 A: 不同处理下的14日龄玉米幼苗；B: 生物量；C: 株高；D: 茎粗；E: 土壤全氮；F: 土壤有效磷；G: 土壤有效钾

Fig. 2 Evaluation of the growth-promoting effect and comprehensive ability of strain LN01 on maize seedlings A: 14-day-old maize seedlings under different treatments; B: biomass; C: plant height; D: thick stem; E: soil total nitrogen; F: soil available phosphorus; G: soil available potassium. The different lowercase letters indicate significant differences among treatments（P<0.05）. The same below

表2 菌株LN01的基因组信息

Table 2 Genome characteristics of strain LN01

菌株LN01特征 Characteristics of LN01	数值 Value
Genome size/bp	5 379 004
GC content/%	55.1
Chromosome	1
Plasmid	6
tRNA	87
rRNA（5S, 16S, 23S）	25
Protein-coding genes（CDS）	5 004
Genomic islands	14
Secondary metabolite gene clusters	4
Genes assigned to NR	4 873
Genes assigned to COGs	4 085
Genes assigned to GO	2 793
Genes assigned to KEGG	3 285
Genes assigned to Pfam	4 400
Genes assigned to Swiss-Prot	4 219

图3 菌株LN01 基因组图谱及COG功能注释注释 A: 基因组图谱；B: 基因组COG 功能注释

Fig. 3 Genomic mapping and COG functional annotation of strain LN01 A: Genomic mapping; B: genomic COG functional annotation

图4 菌株LN01基因组功能注释及通路注释 A: 基因组GO 功能注释；B: KEGG 通路注释

Fig. 4 Genome functional and pathway annotation of strain LN01 A: Genomic GO functional annotation; B: KEGG pathway annotation

表3 预测LN01基因组中与PGP相关的基因

Table 3 Predicted genes associated with PGP in LN01 genome

促生特性 PGP traits	基因 Gene
IAA	trpA trpB trpS trpCF trpR trpGD ipdC amiE patB
Potassium solubilization and uptake	kdpA kdpB kdpC kefB kefC kefF kefG trkA trkH kup
Siderophore	tonB exbB fhuB fhuC fhuD fhuE fhuF efeO efeB efeU fepD fepG fepC ftsB afuB afuC afuA
Nitrogen generation	glnG nirB nirC nirD nasA nrtA nrtB nrtC nrtB nasA nirA glnA gltB gltD
Phosphate solubilization and uptake	pstS pstC pstA pstB phnA phnC phnD phnE phnF phnG phnH phnI phnJ phnK phnL phnN phnP phoA phoB phoE phoR ugpA ugpB ugpC ugpE

图5 菌株LN01 基因组中具有PGP作用的基因

Fig. 5 Genes with PGP action in the genome of strain LN01

图6 LN01基因组关键生物合成基因簇（BGCs）图谱

Fig. 6 Mapping of key biosynthetic gene clusters（BGCs）in the LN01 genome

图7 菌株LN01的促生功能定量及定性测定 A: 产铁载体及固氮能力定性结果；B: 解磷及解钾定性结果；C: 菌株发酵液中总氮含量；D: 可溶性钾含量。* 表示与对照组相比差异显著（P<0.05），**P<0.01，***P<0.001，****P<0.000 1

Fig. 7 Quantitative and qualitative determination of the growth-promoting function of strain LN01 A: Qualitative results of iron production carrier and nitrogen fixation capacity.B: Qualitative results of phosphorus and potassium solubilization. C: Total nitrogen content in fermentation broth of the strains. D: Soluble potassium content. * denotes significant difference（P<0.05）compared with the control, ** P<0.01, *** P<0.001, and ****denotes P<0.000 1

表4 菌株分泌IAA 及溶磷能力定性检测结果和定量检测数据

Table 4 Qualitative and quantitative detection of IAA secretion and phosphorus dissolution

菌株 Strain	定性结果 Qualitative result	吸光值 Optical density	产率 Productive rate/（mg·L^-1）
LN01	IAA（+）	OD_530nm =0.061 3	42.200
LN01	溶磷（+）	OD_700nm =1.762 0	58.329

图8 菌株LN01的促生能力及促生效应相关分析

Fig. 8 Correlation analysis between growth-promoting ability and growth-promoting effect of strain LN01

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