Table 1.
Primers and probes used in the present study
| Primer/TaqMan probe | Sequencee | Target |
|---|---|---|
| CYP51AFa | 5′-ATCCCGGGATAAACTCTATACATCATAG-3′ (SmaI) | cyp51A gene of A. flavus |
| CYP51ARa | 5′-ATGCGGCCGCGACCTTACATGGAACGCCTT-3′ (NotI) | |
| CYP51BFa | 5′-ATGCGGCCGCGCCCGTGGGTTGCTGTCTTT-3′ (NotI) | cyp51B gene of A. flavus |
| CYP51BRa | 5′-ACGCATGCTTTTCGGTTCCTATCCACCC-3′ (SphI) | |
| CYP51CF1a | 5′-ATCCCGGGCACGTACACCAAACACTGCT-3′ (SmaI) | cyp51C gene of A. flavus |
| CYP51CR1a | 5′-ATGCGGCCGCAGCTGGAAGCTCTTCCGCTA-3′ (NotI) | |
| AflaF1b | 5′-GCGCGCATGAGGGAGAT-3′ | cyp51A gene of A. flavus |
| AflaR1b | 5′-CAATGCATGAGGTTCCAGATCA-3′ | |
| ALF1-TaqManb | FAM-5′-TCATTAACGAGCGCCGCAAGAACC-3′-TAMRA | |
| AflaF2b | 5′-ATTCGACTCGACATTTGCTGAA-3′ | cyp51B gene of A. flavus |
| AflaR2b | 5′-GCATCACGCTTGCGGTTAT-3′ | |
| ALF2-TaqManb | FAM-5′-CATGATCTCGACATGGGTTTTGCCC-3′-TAMRA | |
| AflaF3b | 5′-GTGACAACGTCCCGGAGAAG-3′ | cyp51C gene of A. flavus |
| AflaR3b | 5′-ATCAGCAGCGTGATCATGATG-3′ | |
| ALF3-TaqManb | FAM-5′-ATGCAGTGCACCTACAAGAACGGACAGC-3′-TAMRA | |
| AflaActinFb | 5′-TGGTTCCAATCTACGAAGGTTTC-3′ | actin gene of A. flavus |
| AflaActinRb | 5′-ATCTCGTGCTCGGCAGATGT-3′ | |
| ALFActin-TaqManb | FAM-5′-CTATGCCACACGCTATCGCTCGGATG-3′-TAMRA | |
| AflaCYP51CF2c | 5′-TCGAGCTCCACGTACACCAAACACTGCT-3′ (SacI) | cyp51C gene of A. flavus |
| AflaCYP51CR2c | 5′-AACCCGGGAGCTGGAAGCTCTTCCGCTA-3′ (SmaI) | |
| F-161d | 5′-AGCACAATCCACTATGGAACGGATCCGTAC-3′ | Recombinant plasmid pDHt/sk-3357C-161 |
| R-161d | 5′-GTTCCATAGTGGATTGTGCTGCCGATGAAT-3′ | |
| F-788d | 5′-CTGCCCATGCTCGGATGCGTGCTATCTACAT-3′ | Recombinant plasmid pDHt/sk-3357C-788 |
| R-788d | 5′-CACGCATCCGAGCATGGGCAGCATCGCGTTT-3′ | |
| F-1325d | 5′-CCGGTGGGAAACACAGGCAACTCAGGAAAA-3′ | Recombinant plasmid pDHt/sk-3357C-1325 |
| R-1325d | 5′-TTGCCTGTGTTTCCCACCGGTGCGGGTCCCA-3′ | |
| F-1337d | 5′-CACAGGCACCTCAGGAAAACGATAAGGATGA-3′ | Recombinant plasmid pDHt/sk-3357C-1337 |
| R-1337d | 5′-CGTTTTCCTGAGGTGCCTGTGTTTCCCACCG-3′ |
a
Primers used in cloning of the genes cyp51A, cyp51B, and cyp51C for sequencing analysis and construction of the A. flavus transformants with extra copies of these genes from A. flavus.
b
Primers and probes used for real-time PCR assaying.
c
Primers used for amplification of cyp51C for both gene replacement and site-directed mutagenesis.
d
Primers used for construction of plasmids pDHt/sk-3357C-161, pDHt/sk-3357C-788, pDHt/sk-3357C-1325, and pDHt/sk-3357C-1337 during site-directed mutagenesis experiment.
e
Underlining indicates the sites for the restriction enzymes indicated in parentheses or site-directed mutations (single underlined bases). FAM, 6-carboxyfluorescein; TAMRA, 6-carboxytetramethylrhodamine.