The Effects of swnN Gene Function of Endophytic Fungus Alternaria oxytropis OW 7.8 on Its Swainsonine Biosynthesis
Abstract
:1. Introduction
2. Results
2.1. Cloning and Bioinformatics Analysis of the swnN Gene in A. oxytropis OW 7.8
2.2. Sensitivity Screening of A. oxytropis OW 7.8 to Hyg B
2.3. Identification for Transformants of ΔswnN Colonies
2.4. Morphology of Colonies and Mycelia
2.5. Screening of ΔswnN for Glufosinate Sensitivity
2.6. Screening and Identification of ΔswnN/swnN
2.7. SW Levels in A. oxytropis OW 7.8, ΔswnN, and ΔswnN/swnN Mycelia
2.8. Transcriptome Sequencing Analysis of A. oxytropis OW 7.8 and ΔswnN
2.9. Metabolomic Analysis of A. oxytropis OW 7.8 and ΔswnN
2.10. Expression of sac, P5CR, and SWN Cluster Genes in A. oxytropis OW 7.8 and ΔswnN
2.11. SW Synthesis Pathway in A. oxytropis OW 7.8
3. Discussion
4. Materials and Methods
4.1. Strain
4.2. Extraction of Genomic DNA and Identification of swnN Gene from A. oxytropis OW 7.8
4.3. Total RNA Extraction and swnN cDNA Cloning of A. oxytropis OW 7.8
4.4. Construction of Vectors
4.4.1. Construction of the swnN Gene Knockout Vector
4.4.2. Construction of the swnN Gene Complementation Vector
4.5. Sensitivity Test of A. oxytropis OW 7.8 to Hygromycin B
4.6. Preparation and Transformation of A. oxytropis OW 7.8 Protoplasts
4.7. Screening and Identification of Gene Knockout Transformants
4.8. Sensitivity Test of ΔswnN to Glufosinate
4.9. Preparation and Transformation of ΔswnN Protoplasts
4.10. Screening and Identification of Gene Complementation Transformants
4.11. Colony and Mycelia Morphology
4.12. Extraction and Detection of SW in Mycelia of A. oxytropis OW 7.8, ΔswnN, and ΔswnN/swnN
4.13. Transcriptome Analysis of A. oxytropis OW 7.8 and ΔswnN
4.14. Metabolome Detection and Analysis of A. oxytropis OW 7.8 and ΔswnN
4.15. RT-qPCR Detection of Genes Closely Related to SW Synthesis in A. oxytropis OW 7.8 and ΔswnN
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Gene | Encoding Product | Function Prediction |
---|---|---|
swnA | Aminotransferase | Catalyzing the synthesis of Pyrroline-6-carboxylate (P6C) from L-Lysine |
swnR | Dehydrogenase or reductase | Catalyzing the synthesis of L-PA from P6C |
swnK | Multifunctional protein | Catalyzing the synthesis of 1-Oxoindolizidine (or 1-Hydroxyindolizine) from L-PA |
swnN | Dehydrogenase or reductase | Catalyzing the synthesis of 1-Hydroxyindolizine from 1-Oxoindolizidine |
swnH1 | Fe (II)/α-Ketoglutarate-dependent dioxygenase | Catalyzing the synthesis of SW from 1,2-Dihydroxyindolizine |
swnH2 | Fe (II)/α-Ketoglutarate-dependent dioxygenase | Catalyzing the synthesis of 1,2-Dihydroxyindolizine form 1-Hydroxyindolizine |
swnT | Transmembrane transporter | Transport of SW |
Screening Mode | Total of Metabolites | Total of DEMs | Up-Regulated | Down-Regulated |
---|---|---|---|---|
Positive | 801 | 462 | 258 | 204 |
Negative | 472 | 271 | 172 | 99 |
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Liu, C.; Ding, N.; Lu, P.; Yuan, B.; Li, Y.; Jiang, K. The Effects of swnN Gene Function of Endophytic Fungus Alternaria oxytropis OW 7.8 on Its Swainsonine Biosynthesis. Int. J. Mol. Sci. 2024, 25, 10310. https://doi.org/10.3390/ijms251910310
Liu C, Ding N, Lu P, Yuan B, Li Y, Jiang K. The Effects of swnN Gene Function of Endophytic Fungus Alternaria oxytropis OW 7.8 on Its Swainsonine Biosynthesis. International Journal of Molecular Sciences. 2024; 25(19):10310. https://doi.org/10.3390/ijms251910310
Chicago/Turabian StyleLiu, Chang, Ning Ding, Ping Lu, Bo Yuan, Yuling Li, and Kai Jiang. 2024. "The Effects of swnN Gene Function of Endophytic Fungus Alternaria oxytropis OW 7.8 on Its Swainsonine Biosynthesis" International Journal of Molecular Sciences 25, no. 19: 10310. https://doi.org/10.3390/ijms251910310