高寒草地燕麦根际解植酸磷促生菌鉴定及其优势菌假单胞菌属菌株功能特性

doi:10.13560/j.cnki.biotech.bull.1985.2022-0686

摘要/Abstract

摘要：

旨为从高寒草地燕麦根际定向筛选解植酸磷微生物资源，筛选促生潜力菌株，分析植酸酶编码基因。采用国际植物研究所磷酸盐生长培养基（NBRIP）分离及筛选菌株，16S rRNA基因鉴定其分类地位，并测定菌株植酸酶活性及促生特性，结合简并PCR和高效热不对称交错PCR（hiTAIL-PCR）扩增植酸酶基因完整序列，并进行生物信息学分析及异源表达。共获得107株菌株，其中51株能在NBRIP培养基上形成清晰溶磷圈，鉴定为2门10科11属，以假单胞菌（Pseudomonas）为优势菌。14株不同种假单胞菌均检测出植酸酶活性，具有溶解有机/无机磷、分泌IAA（3-indoleacetic acid）、固氮及拮抗植物病原菌的促生活性。获得了3株菌株的植酸酶（PHY65、PHY101和PHY131）序列，预测为β-螺旋植酸酶（β-propeller phytases，BPPhy）家族蛋白，其中重组PHY65的比活性为28.2 U/mg。研究结果可为解磷生物菌剂的研发与利用提供优良菌株资源，为植酸酶的生产应用提供理论基础。

关键词: 燕麦, 植酸磷, 根际促生细菌, 16S rRNA基因鉴定, 植酸酶基因

Abstract:

This work aims to directly screen the microbial resources dissolving phytate phosphorus in Avena sativa rhizosphere from alpine grassland and plant growth promoting bacteria（PGPB）and to analyze the genes encodingphytase. The strains were isolated and screened using national botanical research institute's phosphate growth medium（NBRIP）and the 16S rRNA gene sequencing were used to identify the strains. The dominant strains were used to determine phytase activity and growth-promoting characteristics. The complete sequence of phytase was amplified by degenerate PCR and high-efficiency thermal asymmetric interleaved PCR（hiTAIL-PCR）, and bioinformatics analysis and heterologous expression of new phytase. A total of 107 strains were obtained, of which 51 strains formed a clear phosphate solubilizing circle on NBRIP medium, and the strains belonged to two phyla, 10 families and 11 genera, which Pseudomonas（52.94%）were the predominant bacteria. Fourteen Pseudomonas sp. were detected to have phytase activity, and to have the growth-promoting ability of dissolving organic/inorganic phosphorus, secreting 3-indoleacetic acid（IAA）, fixing nitrogen and antagonizing phytopathogenic fungi. Three BPPhy（PHY65, PHY101 and PHY131）sequences of strains were cloned from fourteen Pseudomonas sp., which were predicted to be β-propeller phytases（BPPhy）family proteins, and the specific activity of recombinant PHY65 was 28.2 U/mg. The results may provide excellent strain resources for the development and utilization of phosphate solubilizing agents, and provide a theoretical basis for the production and application of phytase.

Key words: Avena sativa, phytate phosphorus, plant growth promoting bacteria, 16S rRNA identification, phytase gene

李琦, 杨晓蕾, 李晓林, 申友磊, 李建宏, 姚拓. 高寒草地燕麦根际解植酸磷促生菌鉴定及其优势菌假单胞菌属菌株功能特性[J]. 生物技术通报, 2023, 39(3): 243-253.

LI Qi, YANG Xiao-lei, LI Xiao-lin, SHEN You-lei, LI Jian-hong, YAO Tuo. Identification of Phytate Phosphorus-solubilizing PGPB in Avena sativa Rhizosphere from Alpine Grassland and Functional Characteristics of Dominant Genus Pseudomonas sp.[J]. Biotechnology Bulletin, 2023, 39(3): 243-253.

图/表 8

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引物名称Primer name	引物序列Primer sequence（5'-3'）	用途Usage
B6-5 LB-0a	ATTCACCCTGAGCAGCCCTCTA	GS6-5植酸酶基因两翼序列扩增
B6-5 LB-1a	ACGATGGACTCCAGTCCGGCCCGCGTACTTGGCACCAACAAGAAGCA
B6-5 LB-2a	TCGGGCGCCTCAACAACGTCGATA
B6-5 RB-0a	GACCGCCAGTTCCTGCAACAA
B6-5 RB-1a	ACGATGGACTCCAGTCCGGCCAGGCCCTGCTTCTTGTTGGTGCCA
B6-5 RB-2a	ATCCACACCGCTGGATCATCGGCT
B10-1 LB-0a	GGATTCACCCGCAGCAACCGT	GS10-1植酸酶基因两翼序列扩增
B10-1 LB-1a	ACGATGGACTCCAGTCCGGCCAAGCAGGGACTGATGGCGTACGACC
B10-1 LB-2a	CCGGTGGGGCGCCTGAACAATGTC
B10-1 RB-0a	GCGCAAGTCGACATTGTTCAG
B10-1 RB-1a	ACGATGGACTCCAGTCCGGCCTGCCTTGCAGGTCGTACGCCATCAG
B10-1 RB-2a	GTGCCCAGTACACGGCTCAGCGAC
B13-1 LB-0a	CCGCCGTATGGATTCACCCGCAGCA	GS13-1植酸酶基因两翼序列扩增
B13-1 LB-1a	ACGATGGACTCCAGTCCGGCCCGCTGAGCCGTGTACTGGGCACCAA
B13-1 LB-2a	ACTGCTACAGGAGTTGCCGGTGGGG
B13-1 RB-0a	CGCCCCACCGGCAACTCCTGTAGCA
B13-1 RB-1a	ACGATGGACTCCAGTCCGGCCTTGCAGGTCGTACGCCATCAGTCCC
B13-1 RB-2a	GCGACGGTTGCTGCGGGTGAATCCA

引物名称Primer name	引物序列Primer sequence（5'-3'）	用途Usage
B6-5 LB-0a	ATTCACCCTGAGCAGCCCTCTA	GS6-5植酸酶基因两翼序列扩增
B6-5 LB-1a	ACGATGGACTCCAGTCCGGCCCGCGTACTTGGCACCAACAAGAAGCA
B6-5 LB-2a	TCGGGCGCCTCAACAACGTCGATA
B6-5 RB-0a	GACCGCCAGTTCCTGCAACAA
B6-5 RB-1a	ACGATGGACTCCAGTCCGGCCAGGCCCTGCTTCTTGTTGGTGCCA
B6-5 RB-2a	ATCCACACCGCTGGATCATCGGCT
B10-1 LB-0a	GGATTCACCCGCAGCAACCGT	GS10-1植酸酶基因两翼序列扩增
B10-1 LB-1a	ACGATGGACTCCAGTCCGGCCAAGCAGGGACTGATGGCGTACGACC
B10-1 LB-2a	CCGGTGGGGCGCCTGAACAATGTC
B10-1 RB-0a	GCGCAAGTCGACATTGTTCAG
B10-1 RB-1a	ACGATGGACTCCAGTCCGGCCTGCCTTGCAGGTCGTACGCCATCAG
B10-1 RB-2a	GTGCCCAGTACACGGCTCAGCGAC
B13-1 LB-0a	CCGCCGTATGGATTCACCCGCAGCA	GS13-1植酸酶基因两翼序列扩增
B13-1 LB-1a	ACGATGGACTCCAGTCCGGCCCGCTGAGCCGTGTACTGGGCACCAA
B13-1 LB-2a	ACTGCTACAGGAGTTGCCGGTGGGG
B13-1 RB-0a	CGCCCCACCGGCAACTCCTGTAGCA
B13-1 RB-1a	ACGATGGACTCCAGTCCGGCCTTGCAGGTCGTACGCCATCAGTCCC
B13-1 RB-2a	GCGACGGTTGCTGCGGGTGAATCCA

菌株编号Strain code	相似序列 Closest sequence	相似度 Similarity/%	植酸酶活性 Phytase activity/（U·mL^-1）	植酸酶基因扩增 Phytase gene presence
菌株编号Strain code	相似序列 Closest sequence	相似度 Similarity/%	植酸酶活性 Phytase activity/（U·mL^-1）	BPPhy	HAPhy
GB5-5	Pseudomonas canadensis 2-92	100.00	0.103±0.007f	﹣	﹣
GB5-6	Pseudomonas simiae OLiT	100.00	0.119±0.005ef	﹣	﹣
GB13-2	Pseudomonas umsongensis DSM 16611	99.78	0.149±0.021bc	﹣	﹣
GS1-5	Pseudomonas brassicacearum subsp. neoaurantiaca CIP 109457	99.85	0.129±0.007cde	﹣	﹣
GS2-2	Pseudomonas graminis DSM 11363	99.78	0.124±0.006def	﹣	﹣
GS3-1	Pseudomonas neuropathica P155	99.84	0.189±0.025a	﹣	﹣
GS3-2	Pseudomonas izuensis IzPS43	99.78	0.146±0.012bcd	﹣	﹣
GS5-5	Pseudomonas helmanticensis OHA11	99.93	0.129±0.003cde	﹣	﹣
GS6-5	Pseudomonas baeticaa390	100.00	0.161±0.012b	﹢	﹣
GS10-1	Pseudomonas mandelii NBRC 103147	99.71	0.139±0.011bcde	﹢	﹣
GS10-3	Pseudomonas edaphica RD25	99.85	0.137±0.016cde	﹣	﹣
GS10-4	Pseudomonas alloputida VKh7	99.63	0.123±0.006def	﹣	﹣
GS13-1	Pseudomonas reinekei MT1T	99.78	0.141±0.005bcde	﹢	﹣
GS13-2	Pseudomonas donghuensis HYS Scaffold49	100.00	0.129±0.004cde	﹣	﹣

菌株编号Strain code	相似序列 Closest sequence	相似度 Similarity/%	植酸酶活性 Phytase activity/（U·mL^-1）	植酸酶基因扩增 Phytase gene presence
菌株编号Strain code	相似序列 Closest sequence	相似度 Similarity/%	植酸酶活性 Phytase activity/（U·mL^-1）	BPPhy	HAPhy
GB5-5	Pseudomonas canadensis 2-92	100.00	0.103±0.007f	﹣	﹣
GB5-6	Pseudomonas simiae OLiT	100.00	0.119±0.005ef	﹣	﹣
GB13-2	Pseudomonas umsongensis DSM 16611	99.78	0.149±0.021bc	﹣	﹣
GS1-5	Pseudomonas brassicacearum subsp. neoaurantiaca CIP 109457	99.85	0.129±0.007cde	﹣	﹣
GS2-2	Pseudomonas graminis DSM 11363	99.78	0.124±0.006def	﹣	﹣
GS3-1	Pseudomonas neuropathica P155	99.84	0.189±0.025a	﹣	﹣
GS3-2	Pseudomonas izuensis IzPS43	99.78	0.146±0.012bcd	﹣	﹣
GS5-5	Pseudomonas helmanticensis OHA11	99.93	0.129±0.003cde	﹣	﹣
GS6-5	Pseudomonas baeticaa390	100.00	0.161±0.012b	﹢	﹣
GS10-1	Pseudomonas mandelii NBRC 103147	99.71	0.139±0.011bcde	﹢	﹣
GS10-3	Pseudomonas edaphica RD25	99.85	0.137±0.016cde	﹣	﹣
GS10-4	Pseudomonas alloputida VKh7	99.63	0.123±0.006def	﹣	﹣
GS13-1	Pseudomonas reinekei MT1T	99.78	0.141±0.005bcde	﹢	﹣
GS13-2	Pseudomonas donghuensis HYS Scaffold49	100.00	0.129±0.004cde	﹣	﹣

菌株编号Strain code	有机磷 Organic phosphorus		无机磷 Inorganic phosphorus		IAA分泌量 IAA production/ （μg·mL^-1）	固氮酶活性 Nitrogenase activity/[nmol（C₂H₄）/（h·mL）]	拮抗病原菌活性 Antifungal activity
菌株编号Strain code	pH	溶磷量 P-solubilization/ （μg·mL^-1）	pH	溶磷量 P-solubilization/ （μg·mL^-1）	IAA分泌量 IAA production/ （μg·mL^-1）	固氮酶活性 Nitrogenase activity/[nmol（C₂H₄）/（h·mL）]	立枯丝核菌Rhizoctonia solani	尖刀镰孢菌Fusarium oxysporum
GB5-5	4.28	460.73±40.36e	5.17	412.29±39.70b	0.24±0.05f	51.29±2.56g	﹢	﹣
GB5-6	3.77	547.99±9.02a	5.31	338.48±6.28c	﹣	48.37±3.56g	﹣	﹣
GB13-2	3.65	535.06±11.82ab	5.98	370.34±4.11bc	0.41±0.11d	68.44±1.90f	﹣	﹣
GS1-5	3.43	466.39±6.40e	5.15	292.57±17.41d	0.54±0.09d	89.57±8.06abc	﹣	﹣
GS2-2	3.53	496.05±2.30cd	5.26	418.78±68.58bc	0.97±0.17c	80.98±3.34cd	﹢	﹣
GS3-1	3.68	524.44±1.63ab	5.96	264.82±1.99d	﹣	85.50±4.47bcd	﹢	﹣
GS3-2	3.23	522.83±4.20abc	5.43	419.14±7.41b	﹣	69.51±6.06ef	﹣	﹣
GS5-5	3.80	474.35±14.09de	6.02	372.63±14.73bc	0.81±0.08c	92.40±3.53ab	﹣	﹣
GS6-5	3.31	510.25±19.59bc	5.09	468.66±60.61a	﹣	84.90±3.79bcd	﹣	﹢
GS10-1	2.97	542.33±9.51a	5.07	391.02±11.87b	1.78±0.05a	90.39±5.97abc	﹢	﹢
GS10-3	3.56	425.29±13.31f	5.19	397.51±17.99b	﹣	82.66±1.85bcd	﹢	﹢
GS10-4	3.52	465.23±12.00e	5.48	380.92±5.55bc	0.36±0.06e	80.92±5.55cd	﹢	﹣
GS13-1	3.65	478.05±24.76de	5.35	396.06±7.41b	﹣	77.94±4.89de	﹣	﹣
GS13-2	3.88	480.01±5.74de	5.28	421.66±9.98b	1.27±0.52 bc	97.47±10.12a	﹢	﹣