Cloning, Heterologous Expression, and Antifungal Activity Evaluation of a Novel Truncated TasA Protein from Bacillus amyloliquefaciens BS-3
Abstract
1. Introduction
2. Result
2.1. Sequence and Structure Analysis
2.2. Cloning, Heterologous Expression, and Purification of TasA Gene
2.3. Detection of Antifungal Activity of Recombinant Protein TasA on Spores of Colletotrichum Acutatum
2.4. Inhibitory Effect of Recombinant Protein TasA on Oidium Heveae Steinmann
2.5. Inhibitory Effect of Recombinant Protein TasA on Plant Pathogenic Fungi
3. Discussion
4. Materials and Methods
4.1. Microbial Strainsand Chemical Reagent
4.2. Sequence Analysis
4.3. Gene Cloning
4.4. Construction of Prokaryotic Expression Vector
4.5. Expression and Purification of Protein
4.6. Inhibitory Effect of Recombinant Protein TasA on the Spores of Colletotrichum Acutatumfrom Rubber Trees
4.7. Inhibitory Effect of Recombinant Protein TasA on the Spores of Oidium Heveae Steinmann from Rubber Trees
4.8. Determination of Inhibitory Effect of Recombinant Protein TasA on Plant Pathogenic Fungi
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Conflicts of Interest
References
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Number | GenBank | Query Coverage (%) | Similarity (%) | |
---|---|---|---|---|
B. amyloliquefaciens | 205 | CP054415.1 | 100 | 99.18 |
H | CP041693.1 | 100 | 99.18 | |
HK1 | CP018902.1 | 100 | 99.18 | |
SRCM101267 | CP021505.1 | 100 | 99.18 | |
SRCM123386 | CP128501.1 | 100 | 99.18 | |
SRCM124317 | CP116011.1 | 100 | 99.18 | |
B. subtilis | ATCC 13952 | CP009748.1 | 100 | 99.18 |
Sequence | Positions in Master Proteins | Theo. MH + Da | Charge: Sequest HT | m/z Da: Sequest HT |
---|---|---|---|---|
VHHHHHHMNDVK | 5–16 | 1527.71346 | 3 | 509.91019 |
EQSANVNLSNLKPGDKLTK | 33–51 | 2056.10331 | 3 | 686.04144 |
AASLKDLYLMSAK | 115–127 | 1426.76103 | 3 | 476.25912 |
EVLMALNFTDFKGAK | 65–79 | 1683.87745 | 2 | 842.44513 |
VHHHHHHMNDVKSTDATFASGTLDLSAK | 5–32 | 3093.47077 | 4 | 774.12451 |
EGGNGYPKNIILK | 102–114 | 1402.76889 | 2 | 701.88715 |
NNGSLAIKEVLMALNFTDFK | 57–76 | 2225.16347 | 3 | 742.38464 |
QDKAAAEAISILIR | 128–141 | 1498.85877 | 2 | 749.93365 |
NIILKAASLK | 110–119 | 1070.69321 | 2 | 535.85052 |
GNESAEDFLSQFEITVLTVGK | 81–101 | 2284.13433 | 2 | 1142.57556 |
QDKAAAEAISILIRSS | 128–143 | 1672.92282 | 3 | 558.31409 |
EQSANVNLSNLKPGDKLTKDFEFR | 33–56 | 2750.41078 | 4 | 688.35828 |
STDATFASGTLDLSAKEQSANVNLSNLKPGDKLTK | 17–51 | 3621.86062 | 4 | 906.22137 |
STDATFASGTLDLSAKEQSANVNLSNLKPGDK | 17–48 | 3279.63392 | 4 | 820.66321 |
MNHKVHHHHHHMNDVKSTDATFASGTLDLSAK | 1–32 | 3645.71863 | 5 | 729.94092 |
DFEFRNNGSLAIK | 52–64 | 1510.76487 | 2 | 755.88629 |
NIILKAASLKDLYLMSAK | 110–127 | 1992.1562 | 3 | 664.72455 |
DLYLMSAKQDK | 120–130 | 1327.65623 | 2 | 664.33289 |
EQSANVNLSNLKPGDK | 33–48 | 1713.8766 | 2 | 857.44183 |
VHHHHHHMNDVK | 5–16 | 1543.70837 | 4 | 386.68253 |
MNHKVHHHHHHMNDVK | 1–16 | 2095.95622 | 3 | 699.32373 |
DLYLMSAK | 120–127 | 940.48083 | 1 | 940.48022 |
EGGNGYPK | 102–109 | 821.37881 | 2 | 411.19736 |
EVLMALNFTDFK | 65–76 | 1443.71883 | 2 | 722.36908 |
STDATFASGTLDLSAK | 17–32 | 1584.77516 | 2 | 792.8924 |
AASLKDLYLMSAKQDK | 115–130 | 1781.9466 | 4 | 446.24292 |
NNGSLAIK | 57–64 | 816.45739 | 2 | 408.73315 |
LTKDFEFR | 49–56 | 1055.55202 | 3 | 352.52206 |
AAAEAISILIR | 131–141 | 1127.67828 | 2 | 564.34387 |
DLYLMSAKQDK | 120–130 | 1311.66131 | 2 | 656.33484 |
KGNESAEDFLSQFEITVLTVGK | 80–101 | 2412.2293 | 3 | 804.75092 |
Concentrate/Time | 4.5 h | 6 h | 12 h |
---|---|---|---|
CK | 59.5% ± 0.36% | 35% ± 0.15% | 0% ± 0.00% |
30 μg/mL | 93.91% ± 1.35% ** | 91.95% ± 0.01% ** | 87.4% ± 0.42% ** |
60 μg/mL | 100% ± 0.00% ** | 100% ± 0.00% ** | 100% ± 0.00% ** |
Concentrate/Time | 6 h |
---|---|
CK | 38.75% ± 0.35% |
30 μg/mL | 88.38% ± 0.14% ** |
60 μg/mL | 100% ± 0.00% ** |
Pathogen | Concentrate (μg/mL) | Diameter (mm) | Inhibition Rate (%) |
---|---|---|---|
A. heveae | 150 | 31.29 ± 0.63 | 64.77 ± 1.34 ** |
100 | 19.14 ± 0.14 | 39.62 ± 0.87 * | |
50 | 12.62 ± 0.48 | 26.11 ± 1.26 * | |
C. acutatum | 150 | 48.63 ± 0.15 | 98.6 ± 1.09 ** |
100 | 23.07 ± 0.51 | 52.47 ± 1.19 * | |
50 | 18.17 ± 0.24 | 36.83 ± 1.87 * |
Primer | Restriction Enzyme | Sequences a |
---|---|---|
TasAS | F: 5′-ATGGGTATGAAAAAGAA-3′ | |
R: 5′-AGAACTTCGGATCAATATGCT′-3′ | ||
pET28a-TasA | EcoRΙ | F:5′-CAAATGGGTCGCGGATCCGAATTCGCATTTAATGATGTGAAGTCCAC-3′ |
NotI | R:5′-GTGGTGGTGCTCGAGTGCGGCCGCAGAACTTCGGATCAATATGCTGAT-3′ | |
pCZN1-TasA | NdeI | F:5′-ATGAATCACAAAGTGCATCATCATCATCATCATATGAACGATGTGAAAAGCA-3′ |
XbaI | R:5′-CTTTTAAGCAGAGATTACCTATCTAGATTAGCTGCTACGAATCA-3′ |
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Dai, L.-M.; He, L.-L.; Li, L.-L.; Liu, Y.-X.; Shi, Y.-P.; Su, H.-P.; Cai, Z.-Y. Cloning, Heterologous Expression, and Antifungal Activity Evaluation of a Novel Truncated TasA Protein from Bacillus amyloliquefaciens BS-3. Int. J. Mol. Sci. 2025, 26, 7529. https://doi.org/10.3390/ijms26157529
Dai L-M, He L-L, Li L-L, Liu Y-X, Shi Y-P, Su H-P, Cai Z-Y. Cloning, Heterologous Expression, and Antifungal Activity Evaluation of a Novel Truncated TasA Protein from Bacillus amyloliquefaciens BS-3. International Journal of Molecular Sciences. 2025; 26(15):7529. https://doi.org/10.3390/ijms26157529
Chicago/Turabian StyleDai, Li-Ming, Li-Li He, Lan-Lan Li, Yi-Xian Liu, Yu-Ping Shi, Hai-Peng Su, and Zhi-Ying Cai. 2025. "Cloning, Heterologous Expression, and Antifungal Activity Evaluation of a Novel Truncated TasA Protein from Bacillus amyloliquefaciens BS-3" International Journal of Molecular Sciences 26, no. 15: 7529. https://doi.org/10.3390/ijms26157529
APA StyleDai, L.-M., He, L.-L., Li, L.-L., Liu, Y.-X., Shi, Y.-P., Su, H.-P., & Cai, Z.-Y. (2025). Cloning, Heterologous Expression, and Antifungal Activity Evaluation of a Novel Truncated TasA Protein from Bacillus amyloliquefaciens BS-3. International Journal of Molecular Sciences, 26(15), 7529. https://doi.org/10.3390/ijms26157529