|
|
|
| Cloning and functional identification of 14α-demethylase genes (ERG11) from two different Candida haemulonii strains with degenerate PCR combined with RACE |
| ZHANG Hao, WANG Qian, LIU Wei |
| Department of Dermatology and Venereology, Peking University First Hospital, Research Center for Medical Mycology, Beijing Key Laboratory of Molecular Diagnosis on Dermatoses, Peking University, Beijing 100034, China |
|
|
|
|
Abstract ERG11 genes from two different Candida haemulonii strains were cloned and their functions were verified for studying the mechanism of antifungal resistance. To obtain ERG11 gene, degenerate primers were designed according to the conserved sequences in Erg11 protein from the other four types of Candida spp. Partial ERG11 cDNA was amplified by degenerate polymerase chain reaction (PCR). And 5′ cDNA and 3′ cDNA were amplified by rapid amplification of cDNA ends (RACE) method. The full-length ERG11 coding sequences (CDSs) were obtained after aligning and splicing. Furthermore, ERG11 CDSs were cloned into pYES2 and transformed into Saccharomyces cerevisiae (S. cerevisiae) which is auxotrophic for uracil. The susceptibility of yeasts to fluconazole was assayed following Clinical and Laboratory Standards Institute (CLSI) M27-A3. The results showed that the complete ERG11 CDSs were obtained and identified through homologous alignment of Erg11 proteins with other Candida spp. Moreover, the susceptibility of yeasts to fluconazole was obviously reduced by overexpression of Erg11 proteins in S. cerevisiae. It is suggested that ERG11 genes can be effectively cloned by degenerate PCR combined with RACE and their functions could be preliminarily verified in pYES2 yeast expression system.
|
|
Received: 10 November 2016
Published: 25 April 2017
|
|
Corresponding Authors:
LIU Wei
|
|
|
|
|
|
|
2017, Vol. 12 
