Establishment of Agrobacterium tumefaciens-Mediated Transformation of Cladonia macilenta, a Model Lichen-Forming Fungus
Abstract
:1. Introduction
2. Materials and Methods
2.1. Strains and Growth Conditions
2.2. Sensitivity of the C. macilenta Mycobiont to Hygromycin B
2.3. Optimization of ATMT
2.4. Statistical Analysis
2.5. Genomic DNA Extraction
2.6. Real-Time PCR Conditions for T-DNA Copy Number Variation Analysis
2.7. Relative Quantification of T-DNA Copy Number
2.8. TAIL-PCR and Sequencing
2.9. Microscopy
2.10. Data Availability
3. Results
3.1. The Mycobiont of C. macilienta
3.2. Optimization of ATMT Method for C. macilienta
3.3. T-DNA Copy Number Variation of Transformants
3.4. Identification of T-DNA Insertion Sites
3.5. Phenotyping of C. macilenta Transformants
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Parameter | d.f. | F Statistics | p-Value | GES 1 |
---|---|---|---|---|
Biomass input (i) | 1 | 0.980 | 0.327 | 0.018 |
Co-cultivation period (ii) | 2 | 5.522 | 0.007 * | 0.170 |
Incubation temperature (iii) | 2 | 0.671 | 0.515 | 0.024 |
(i):(ii) | 2 | 1.069 | 0.351 | 0.038 |
(i):(iii) | 2 | 0.131 | 0.878 | 0.005 |
(ii):(iii) | 4 | 0.297 | 0.879 | 0.022 |
(i):(ii):(iii) | 4 | 0.551 | 0.699 | 0.039 |
Strain | Flanking Sequence of T-DNA 1 | Length 2 (Identity) | Insertion Site 3 | Gene ID | Pfam Domain (E-Value) |
---|---|---|---|---|---|
CmT-2 | ATGATCATAGAAAGGATGCC | 134 (100) | 17:318521 | Cma_08524 | no domain found |
CmT-5 | CAGGACGTCGATTGTAGCAC | 149 (99) | 5:324280 | intergenic | - |
CmT-7 | CTGGAGGAGAATCAGGAGGT | 237 (100) | 2:2003077 | intergenic | - |
CmT-9 | CGGCCGGGGAAAACCGTTCG | 133 (100) | 15:985193 | intergenic | - |
CmT-41 | TCCGCTTTTTGGCAGGCTGC | 142 (99) | 21:390480 | intergenic | - |
CmT-45 | AAGGAAACCTTACGCTAGTG | 130 (100) | 0:1782331 | intergenic | - |
CmT-79 | TGGAGGGATGATCTCTATGG | 105 (100) | 16:437828 | intergenic | - |
CmT-81 | ATTTTTTGTCCTGTCTGAGG | 61 (100) | 2:1031916 | Cma_01716 | PF00364 (7.9 × 10−16) |
CmT-82 | TAGGAAATCAATGGGTTAAG | 527 (100) | 4:843652 | Cma_02877 | PF01812 (1.5 × 10−31) |
CmT-83 | GTTCACCCCTCTTCGCAATA | 642 (100) | 20:650595 | Cma_09655 | PF12796 (3.8 × 10−23) |
CmT-85 | TAGCGGATGCTTTAGGCGAA | 88 (100) | 14:378660 | intergenic | - |
CmT-86 | CCTAGAGTAAGGTAGGTATG | 126 (100) | 20:271306 | intergenic | - |
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Liu, R.; Kim, W.; Paguirigan, J.A.; Jeong, M.-H.; Hur, J.-S. Establishment of Agrobacterium tumefaciens-Mediated Transformation of Cladonia macilenta, a Model Lichen-Forming Fungus. J. Fungi 2021, 7, 252. https://doi.org/10.3390/jof7040252
Liu R, Kim W, Paguirigan JA, Jeong M-H, Hur J-S. Establishment of Agrobacterium tumefaciens-Mediated Transformation of Cladonia macilenta, a Model Lichen-Forming Fungus. Journal of Fungi. 2021; 7(4):252. https://doi.org/10.3390/jof7040252
Chicago/Turabian StyleLiu, Rundong, Wonyong Kim, Jaycee Augusto Paguirigan, Min-Hye Jeong, and Jae-Seoun Hur. 2021. "Establishment of Agrobacterium tumefaciens-Mediated Transformation of Cladonia macilenta, a Model Lichen-Forming Fungus" Journal of Fungi 7, no. 4: 252. https://doi.org/10.3390/jof7040252