| Autor: |
Pérez-Pérez, W. D., Carrasco-Navarro, U., García‑Estrada, C., Kosalková, K., Gutiérrez-Ruíz, M. C., Barrios-González, J., Fierro, F. |
| Rok vydání: |
2022 |
| DOI: |
10.6084/m9.figshare.19500860 |
| Popis: |
Additional file 3. Confirmation of the presence of plasmids for RNAi-mediated silencing of Pc-yap1 and Pc-rsmA and analysis of silencing in P. chrysogenum transformants. (A) Close-up of the pGpdPki-RNAi vector region with the opposite-oriented gpd and pki promoters, and the plasmids pGpdPki-RNAi/PcYap1 and pGpdPki-RNAi/PcRsmA with the inserted DNA fragments from the Pc-yap1 and Pc-rsmA genes at the NcoI site. Primers Gpd1(pki1)R and Pki1(gpd1)F are shown at the position of annealing with sequences in the gpd and pki promoters, respectively. The expected size of amplified DNA fragments in each type of transformant is indicated with double-headed arrows. (B) Agarose gel with the result of PCR amplification with primers Gpd1(pki1)R and Pki1(gpd1)F using as template DNA from the purified pGpdPki-RNAi vector (lane V), DNA from strain C1 containing the pGpdPki-RNAi vector (lane C1) and DNA from strain Wis54-1255 (lane W). (C) Results of the PCR amplification performed with the mentioned primers and DNA from eight transformants with the pGpdPki-RNAi/PcYap1 plasmid (Y1…. Y18). (D) Results of the PCR amplification performed with the mentioned primers and DNA from eight transformants with the pGpdPki-RNAi/PcRsmA plasmid (R24… R39). (E) Silencing of expression of Pc-yap1 (upper panels) and Pc-rsmA (lower panels) in selected strains containing the RNAi-silencing plasmids pGpdPki-RNAi/PcYap1 and pGpdPki-RNAi/PcRsmA, respectively. RNA was extracted from mycelium grown for 60 h in MPPY medium and used for semiquantitative RT-PCR (as described by Domínguez-Santos et al. [96]) using primers qPcYap1-F and -R for analysis of Pc-yap1 expression, qRsmA-F and -R for Pc-rsmA, and qactA-F and -R for act. The left panels show the intensity of the bands in an agarose gel loaded with the products of the RT-PCR reactions, and the right panels the densitometry analysis of the bands. The results were normalized with the bands of the constitutively expressed act gene. The parental strain Wis54-1255 (lane W) was used as reference with a value for integrated optical density (IOD) of 100. Y8: strain si-PcYap1-8; Y12: si-PcYap1-12; R24: si-PcRsmA-24; R25: si-PcRsmA-25. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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