2. Materials and methods
2.2. Methods
2.2.2. DNA methods
2.2.2.10. Aspergillus nidulans strain construction
2.2.2.10.1. Construction of laeA, laeA complementation and other strains in laeA
DNA sequence informations were obtained from AspGD (Cerqueira et al., 2014).
Strains used in this study contain a recyclable marker module (Krappmann et al., 2005). In order to generate the laeA deletion construct the 5’ UTR region was amplified from wild-type FGSC A4, veA+ genomic DNA with primers BD45/BD46 and the 3’ UTR was amplified with the primers BD47/BD48. The two amplicons were fused with the recyclable phleo cassette (the phleo cassette contains a bleomycin (Bm) resistance- encoding gene ble) using a seamless cloning reaction (Gene Art® seamless cloning and assembly kit, Invitrogen life technologies, A13288). The deletion cassette was integrated into a pBluescript SK (+) vector. The linear deletion cassette was excised from the vector using a PmeI restriction cutting site and transformed into a nkuA (AGBB52) and in a lysA; nkuA (AGB1092) parental strain. For complementation of laeA the laeA genomic locus was amplified from wild-type FGSC A4, veA+ genomic DNA (primers BD45/BD76) fused together with a recyclable phelo marker and with the 3’ UTR using a seamless cloning reaction.
In order to construct the nptA::gfp cassette a two-step cloning strategy was used (Mounts et al., 1989). The 5’ UTR plus the sequence coding region of nptA was amplified with BD104/BD105 and the 3’ UTR was amplified with the primers BD108/BD109. The gfp (green fluorescent protein) sequence was amplified using BD99 and BD100. The gfp sequence was fused with the coding sequence of nptA by fusion PCR. The fragment was integrated in the plasmid pJG229 containing a recyclable phleo cassette using a SwaI restriction cutting site. In the second step, the 3’ UTR was integrated into the plasmid using an Eco74I restriction cutting site. In order to construct the rfeA::gfp cassette the 5’ UTR plus the gene rfeA was amplified with the primers BD111/BD112. The 3’ UTR was amplified with the primers BD113/BD114.
The amplicons were fused using the two-step cloning strategy explained above. Linear cassettes of nptA::gfp and rfeA::gfp were transformed into a lysA; nkuA (AGB1092) and a laeA lysA; nkuA (AGB1074) strain. All strains were confirmed by Southern hybridization (Figure 9-13).
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Figure 9. Restriction map and Southern hybridization before and after the marker was recycled for the deletion strain laeA; nkuA (AGB1073).
The following restriction enzymes were used: SacI and EcoRV.
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Figure 10. Restriction map and Southern hybridization before and after the marker was recycled for the complementation strain of laeA (laeA::laeA; nkuA, AGB1075).
The following restriction enzymes were used: EcoRV and PvuI.
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Figure 11. Restriction map and Southern hybridization before and after the marker was recycled for lysine auxotrophic laeA strain (lysA; laeA; nkuA, AGB1074).
The following restriction enzymes were used: SacI and EcoRV.
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Figure 12. Restriction map and Southern hybridization before and after the marker was recycled for the complementation strain lysA; laeA (lysAlaeA::laeA;nkuA, AGB1076).
The following restriction enzymes were used: EcoRV and PvuI.
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Figure 13. Restricition map and Southern hybridization to confirm nptA::gfp and rfeA::gfp strains Strains: lysA;nptA::gfp;nkuA (AGB1082), lysA;laeA;nptA::gfp;nkuA (AGB1083). Strains:
lysA;rfeA::gfp;nkuA (AGB1084), lysA;laeA;nptA::gfp;nkuA (AGB1085). The following restriction enzymes were used: SmaI and HindIII. The strains contained a recyclable phleo cassette and were already recycled
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2.2.2.10.2. Construction of xptB::gfp strain
The xptB (xanthone prenyltransferase) gene was fused with gfp. The xptB::gfp cassette was constructed using a two-step cloning strategy (Mounts et al., 1989). In order to construct the xptB::gfp cassette the 5’ UTR plus the coding sequence of the gene xptB was amplified with the primers BD119/BD120(xptB) (Figure 14). The 5‘ UTR plus the coding sequence of the gene xptB were fused with the sequence of gfp. This fusion fragment was integrated into the plasmid pJG229 containing a recyclable phleo cassette using a SwaI restriction cutting site. The 3’ UTR was amplified with the primers BD121(xptB)/BD122(xptB). Next the 3’ UTR was integrated into the plasmid pJG299 using an Eco74I restriction cutting site.
The xptB::gfp cassette was transformed into a lysA; nkuA (AGB1092) parental strain and integration of the xptB::gfp cassette was confirmed by diagnostic PCR (Figure 15). In order to perform the diagnostic PCR to confirm the integration of the cassette the 5’ UTR plus the coding sequence and the sequence of gfp was amplified using genomic DNA with the primers xptB::gfp BD119/BD107. Additionally, the gfp sequence was amplified with the primers BD106/BD107.
Figure 14. Schematic illustration of the two-step cloning strategy.
The sequence of the 5’ UTR fused with the coding sequence of the xptB and with gfp was first integrated into the plasmid pJG229 containing a recyclable phleo cassette using a SwaI cutting site. Next the 3’
UTR was integrated into the plasmid pJG229 using an Eco74I restriction cutting site.
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2.2.2.10.3. Construction of xptC::gfp strain
The xptC (oxidoreductase) gene was fused with gfp. In order to construct the xptC::gfp cassette the 5’ UTR plus the coding sequence of the gene xptC was amplified with the primers BD113(xptC)/BD114(xptC). The sequence of xptC was fused with gfp. The 3’
UTR was amplified with the primers BD115/BD116. The amplicons were fused using the two-step cloning strategy mentioned above. The fragments were integrated into the plasmid pJG229 using a SwaI and an Eco74I restriction cutting site. The xptC::gfp cassette was transformed into a nkuA (AGB552) parental strain and integration of the xptC::gfp cassette was confirmed by diagnostic PCR (Figure 16). In order to perform the diagnostic PCR to confirm the integration of the cassette the 5’ UTR plus the coding sequence and the sequence of gfp was amplified using genomic DNA with the primers BD114/BD107. Additionally, the gfp sequence was amplified with the primers BD106/BD107.
Figure 15. Diagnostic PCR to confirm the integration of the xptB::gfp cassette.
The 5’ UTR plus the coding sequence of xptB and the sequence of gfp was amplified using genomic DNA (xptB::gfp; nkuA lysA, AGB1086) with the primers BD119/BD107. The amplified product has a size of 4.2 kb. As negative control genomic DNA of nkuA lysA was used. In order to amplify the gfp sequence the primers BD106 and BD107 were used. The amplified product has a size of 0.7 kb.
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2.2.2.10.4. Construction of AN8434::gfp strain
In order to construct the AN8434::gfp cassette the 5’ UTR plus the gene AN8434 was amplified with the primers: BD121/BD122 .The 3’ UTR was amplified with the primers BD123/BD124. The amplicons were fused using the two-step cloning strategy mentioned above. First the 5’ UTR and the coding sequence of AN8434 was fused with the sequence of gfp. The fragment was integrated into the plasmid pJG229 containing a recyclable phleo cassette using a SwaI restriction cutting site. The 3’ UTR was integrated into the plasmid pJG229 using an Eco74I restriction cutting site. The AN8434::gfp cassette was transformed into a nkuA (AGB552) parental strain.
Integration of the AN8434::gfp was confirmed by diagnostic PCR (Figure 17). In order to perform the diagnostic PCR to confirm the integration of the cassette the 5’ UTR plus the coding sequence and the sequence of gfp was amplified using genomic DNA with the primers AN8434::gfp (BD121/BD107). Additionally, the gfp sequence was amplified with the primers BD106/BD107. As negative control genomic DNA of the parental strain nkuA was used.
Figure 16. Diagnostic PCR to confirm the integration of xptC::gfp cassette.
The 5’ UTR plus the coding sequence of xptC and the sequence of gfp was amplified using genomic DNA (xptC::gfp;nkuA AGB1088) with the primers BD114/BD107. The amplified product has a size of 4.2 kb. In order to amplify the gfp sequence the primers BD106 and BD107 were used. The amplified product has a size of 0.7 kb. As negative control genomic DNA of nkuA (AGB552) was used and no amplified products were observable.
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2.2.2.10.5. Construction of AN8435::gfp strain
In order to construct the AN8435::gfp strain cassette the 5’ UTR plus the gene AN8435 was amplified with the primers BD125/BD127. This sequence AN8435 was fused with the sequence of gfp. The 3’ UTR was amplified with the primers BD128/BD129. The amplicons were fused using the two-step cloning strategy. The fragments were integrated into the plasmid pJG229 using a SwaI and an Eco74I restriction cutting site.
The AN8435::gfp cassette was transformed into a nkuA (AGB552) parental strain and integration of AN8435::gfp was confirmed by diagnostic PCR (Figure 18). In order to perform the diagnostic PCR to confirm the integration of the cassettes the 5’ UTR plus the coding sequence and the sequence of gfp was amplified using genomic DNA with the primers AN8435::gfp (BD125/BD107). Additionally, the gfp sequence was amplified with the primers BD106/BD107. As negative control genomic DNA of the parental strain nkuA was used.
Figure 17. Diagnostic PCR to confirm the integration of the AN8434::gfp cassette.
The 5’ UTR plus the coding sequence AN8434 and the sequence of gfp was amplified using genomic DNA (AN8434::gfp;nkuA AGB1089) with the primers BD121/BD107. The amplified product has a size of 4.5 kb. The sequence of gfp was amplified using the primers BD106/BD107. The amplified product has a size of 0.7 kb. As negative control genomic DNA of nkuA (AGB552) was used and no amplified products were observable.
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2.2.2.10.6. Construction of mphA, mphA complementation and other strains in mphA
The mphA (maltose permease-like protein of Hülle cells) knockout plasmid and knockout strain was constructed as mentioned above using the seamless cloning and assembly strategy. The 5’ UTR and the 3’ UTR region was amplified from the wild-type FGSC A4, veA+ genomic DNA using the primers BD83/BD84 and BD85/BD86.
The linear deletion cassette was excised from the vector using a PmeI restriction cutting site and transformed into a nkuA (AGBB52) parental strain. In order to construct the gfp fusion plasmids the C- terminus of mphA was tagged. Therefore, the 5’ UTR plus the coding sequence of mphA was amplified with the primers BD83/BD98.
The gfp sequence was amplified using the primers BD99/BD100. As mentioned above 3’ UTR was amplified using the primers BD85/BD86. The complementation of mphA;
nkuA was performed with and without gfp. The mphA::gfp cassette was transformed into a laeA;nkuA (AGB1073) strain. All strains were confirmed by Southern hybridization (Figure 19-21).
Figure 18. Diagnostic PCR to confirm the integration of the AN8435::gfp cassette.
The 5’ UTR plus the coding sequence of AN8435 and the sequence of gfp was amplified using genomic DNA (AN8435::gfp;nkuA AGB1090) with the primers BD125/BD107. The amplified product has a size of 5.4 kb. The sequence of gfp was amplified using the primers BD106/BD107. The amplified product has a size of 0.7 kb. As negative control genomic DNA of nkuA (AGB552) was used and no amplified products were observable.
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Figure 19. Restriction map and Southern hybridization before and after the marker was recycled for the nkuA; mphA (AGB1077) strain.
The following restriction enzymes were used: SspI, NheI, HindIII and BstXI.
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Figure 20. Restriction map and Southern hybridization before and after the marker was recycled for the complementation strain (nkuA mphA::mphA::gfp, AGB1078) plus the mphA::gfp;nkuA
(AGB1079) strain.
BamHI was used as a restriction enzyme.
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Figure 21. Restricition map and Southern hybridization to confirm the complementation strain (mphA::mphA, nkuA AGB1080) and the laeA;mphA::gfp;nkuA (AGB1081) strain.
NheI and EcoRV were used as restriction enzymes. The strains contained a recyclable phleo cassette and the recyclable phleo cassette was already recycled.
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