Figure 1The results of RNA slot blot hybridization of fluconazole-susceptible, -SDD, and-resistant Candida glabrata strains. (A) C. glabrata-susceptible (D-31), -SDD (D-284), -resistant (D-116) strains, and 26s ribosomal RNA (quantity control). (B) Results of RNA expression from fluconazole resistant-induced isolate (MIC 1, 8: susceptible, MIC 16: SDD, MIC 64: resistant). (C) Lane 1: 1-kb marker, PCR product of ERG3 (Lane 2–4) and ERG11 (Lane 5–7) genes. Lane 2 and Lane 5: C. glabrata D-31 strain (susceptible), Lane 3 and Lane 6: C. glabrata D-284 strain (SDD), Lane 4 and Lane 7: C. glabrata D-116 strain (resistant). The PCR product was sequenced. SDD = susceptible dose dependent; MIC = minimal inhibitory concentration; PCR = polymerase chain reaction.
Figure 2Differently expressed genes were screened by the reverse transcription-PCR method using GeneFishing DEG kits. RNA was purified from three Candida glabrata strains (1: susceptible, 2: susceptible dose dependent, 3: resistant). The DEGs were cloned and sequenced. A total of 258 genes showed higher or lower expression in the resistant strain compared to the susceptible strain. The PCR product with numbers is selected for the sequencing. PCR = polymerase chain reaction; DEG = differentially expressed gene.
Table 1Base substitution and amino acid changes of ERG3 and ERG11 genes from Candida glabrata fluconazole-susceptible, -susceptible dose-dependent, and -resistant strains
|
Erg3
|
Erg11
|
|
Base substitution |
Amino acid change |
Base substitution |
Susceptible (C. glabrata D-31) |
T435C |
Glu139Ala |
T768C |
S-DD (C. glabrata D-284) |
T317A |
Leu106Gln |
T768C |
T435C |
Glu139Ala |
T834C |
|
A1593G |
Resistant (C. glabrata D-116) |
G423A |
Glu139Ala |
C588T |
T435C |
|
T768C |
|
C918T |
Table 2Identification of differently expressed genes from reverse transcription-polymerase chain reaction
Increased expression |
Decreased expression |
Saccharomyces cerevisiae
|
Saccharomyces cerevisiae
|
Tryptophanyl-tRNA synthetase
|
DNA-dependent ATPase/DNA helicase B |
Carnitine O-acetyltransferase |
GTP cyclohydrolase II |
Peroxisomal long-chain fatty acid import protein 2 |
Prohibitin antiproliferative protein |
Fumarate hydratase |
Methionine aminopeptidase |
Manganese transporter |
Aldehyde dehydrogenase |
Transcriptional regulation mediator |
Oxidoreductase |
General transcriptional adaptor or co-activator |
prohibitin-like protein |
Component of actin cortical patches |
|
Negative regulator of ras-cAMP pathway |
Candida glabrata
|
AAC2 ADP/ATP carrier protein |
ATP-binding cassette transporter |
Involved in cell wall biogenesis and architecture |
|
Gamma-glutamylphosphate reductase |
|
Acetyl-CoA synthetase |
|
|
|
Candida glabrata
|
|
Carnitine acetyltransferase gene |
|
ATP-binding cassette transporter |
|
|
|
Saccharomyces kluyveri
|
|
Putative citrate synthase gene |
|