RNA-silencing in Penicillium chrysogenum and Acremonium chrysogenum: validation studies using beta-lactam genes expression.
In this work we report the development and validation of a new RNA interference vector (pJL43-RNAi) containing a double-stranded RNA expression cassette for gene silencing in the filamentous fungi Penicillium chrysogenum and Acremonium chrysogenum. Classical targeted gene disruption in these fungi is very laborious and inefficient due to the low frequency of homologous recombination. The RNAi vector has been validated by testing the attenuation of two different genes of the beta-lactam pathway; pcbC in P. chrysogenum and cefEF in A. chrysogenum. Quantification of mRNA transcript levels and antibiotic production showed knockdown of pcbC and cefEF genes in randomly isolated transformants of P. chrysogenum and A. chrysogenum, respectively. The process is efficient; 15 to 20% of the selected transformants were found to be knockdown mutants showing reduced penicillin or cephalosporin production. This new RNAi vector opens the way for exploring gene function in the genomes of P. chrysogenum and A. chrysogenum.
Ullán
Ricardo V
RV
Institute of Biotechnology (INBIOTEC), Avda. Real no. 1, 24006 León, Spain.
Godio
Ramiro P
RP
Teijeira
Fernando
F
Vaca
Inmaculada
I
García-Estrada
Carlos
C
Feltrer
Raúl
R
Kosalkova
Katarina
K
Martín
Juan F
JF
D000164:Acremonium / Q000145:classification / Q000235:genetics* / Q000378:metabolism; D002511:Cephalosporins / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D005821:Genetic Techniques; D009154:Mutation; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism; D010406:Penicillins / Q000378:metabolism*; D010408:Penicillium chrysogenum / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000235:genetics / Q000378:metabolism; D014170:Transformation, Genetic; D047090:beta-Lactams / Q000378:metabolism
D002511:Cephalosporins; D005656:Fungal Proteins; D010406:Penicillins; D012330:RNA, Double-Stranded; D047090:beta-Lactams; D010088:Oxidoreductases; C038370:isopenicillin N synthetase