Biotechnology Bulletin ›› 2023, Vol. 39 ›› Issue (3): 243-253.doi: 10.13560/j.cnki.biotech.bull.1985.2022-0686
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LI Qi1(), YANG Xiao-lei1, LI Xiao-lin2, SHEN You-lei1, LI Jian-hong1, YAO Tuo1()
Received:
2022-06-04
Online:
2023-03-26
Published:
2023-04-10
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.
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 |
Table 1 Primers used in hiTAIL-PCR
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 |
Fig. 1 Phylogenetic tree of culturable bacteria from rhizosphere of Avena sativa and their closest relatives based on 16S rRNA gene sequences The effective sequence length was 1 100 bp, the bootstrap values were 1 000 replications in the Neighbour-Joining tree, and the serial number in parentheses denotes the GenBank accession number of the strains
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 | |
---|---|---|---|---|---|
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 | ﹣ | ﹣ |
Table 2 Characteristics of different species of Pseudomonas sp.
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 | |
---|---|---|---|---|---|
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 | |||
---|---|---|---|---|---|---|---|---|
pH | 溶磷量 P-solubilization/ (μg·mL-1) | pH | 溶磷量 P-solubilization/ (μg·mL-1) | 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 | ﹢ | ﹣ |
Table 3 Plant growth promoting characteristics of strains
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 | |||
---|---|---|---|---|---|---|---|---|
pH | 溶磷量 P-solubilization/ (μg·mL-1) | pH | 溶磷量 P-solubilization/ (μg·mL-1) | 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 | ﹢ | ﹣ |
基因编号 Gene code | 酶家族 Enzyme family | 相对分子质量 Molecular weight/kD | 理论等电点 Theoretical pI | 最相似菌株(UniProt ID) Most similar strain | 相似度 Similarity/% | GenBank登录号 GenBank accession No. |
---|---|---|---|---|---|---|
PHY65 | 3-phytase(EC 3.1.3.8) | 69.6 | 4.88 | Pseudomonas mucidolens (A0A1H2MGK9) | 78.3 | OM935858 |
PHY101 | 3-phytase(EC 3.1.3.8) | 70.1 | 4.86 | Pseudomonas fluorescens (Q4KAB7) | 79.4 | OM935859 |
PHY131 | 3-phytase(EC 3.1.3.8) | 69.8 | 4.84 | Pseudomonas fluorescens (Q4KAB7) | 78.1 | OM935857 |
Table 4 Basic physicochemical properties of amino acid sequences of phytase
基因编号 Gene code | 酶家族 Enzyme family | 相对分子质量 Molecular weight/kD | 理论等电点 Theoretical pI | 最相似菌株(UniProt ID) Most similar strain | 相似度 Similarity/% | GenBank登录号 GenBank accession No. |
---|---|---|---|---|---|---|
PHY65 | 3-phytase(EC 3.1.3.8) | 69.6 | 4.88 | Pseudomonas mucidolens (A0A1H2MGK9) | 78.3 | OM935858 |
PHY101 | 3-phytase(EC 3.1.3.8) | 70.1 | 4.86 | Pseudomonas fluorescens (Q4KAB7) | 79.4 | OM935859 |
PHY131 | 3-phytase(EC 3.1.3.8) | 69.8 | 4.84 | Pseudomonas fluorescens (Q4KAB7) | 78.1 | OM935857 |
Fig. 3 Amino acid multiple sequence alignment of PHY65, PHY101 and PHY131 The shaded part represents the degree of similarity of the sequences, black indicates that the sequences have completely identical and gray indicates that the sequences have highly similar. The sequences used in the figure are all from GenBank, and the access numbers from top to bottom are: P. baetica PHY65(OM935858); P. mandelii PHY101(OM935859); P. reinekei PHY131(OM935857); P. mucidolens(WP084377901.1); P. fluorescens(WP014338283.1); P. alcaligenes(WP021702177.1); P. mangrovi(WP108107190.1)
Fig. 4 SDS-PAGE analysis of protein expression M: Protein marker. Lane 1: The strain secretes supernatant. Lane 2: Effluent solution through column. Lane 3-6: Elution solution through column
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