Identification of Phytate Phosphorus-solubilizing PGPB in Avena sativa Rhizosphere from Alpine Grassland and Functional Characteristics of Dominant Genus Pseudomonas sp.

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

Abstract

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

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.

Figures/Tables 8

References 31

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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	﹢	﹣