IADR Abstract Archives
Candida albicans SAP9: a gene with novel function in hyphal development in Candida albicans
Objectives: To investigate the role of C. albicans Sap9 in yeast-to-hyphal morphological transition
Methods: In this study, SAP9 null mutant strain (Dsap9::hisG/Dsap9::hisG-URA3-hisG) was created by URA blaster method. Ura- phenotype strain was selected in synthetic complete glucose medium containing 0.1% 5-fluoroorotic acid, and complemented by URA3. Hyphal development of fungal cells (1×106 cfu/ml) was evaluated in both liquid and solid hyphal-inducing media, including Lee’s medium (pH 7.0), RPMI-1640, YPD+10% fetal bovine serum (FBS) and Spider’s medium at 37℃. To analyze the mRNA expression of hyphal-specific genes, fungal cells were prepared in the YPD containing 10% FBS at 37 ℃. Total RNA was extracted by SV Total RNA Isolation System and was reverse transcribed into cDNA using Superscirpt II. Gene expression was determined by Fast SYBR Green Master Mix, with the program: 95 ℃ incubation for 20s, and then 40 cycles of 95 ℃ for 1s and 60 ℃ for 20s. Student’s t test (P value < 0.05) was used for statistical analysis.
Results: Compared with wild type SC5314 strain, SAP9 null mutant strain was defective in the hyphal development in both liquid and solid hyphal-inducting media. Gene expression analysis indicated that the expression level of transcription factor gene EFG1 was down-regulated by about 40% (P<0.05).
Conclusions: C. albicans SAP9 might play a pivotal role in hyphal development in C. albicans via the cellular events mediated by EFG1.
Methods: In this study, SAP9 null mutant strain (Dsap9::hisG/Dsap9::hisG-URA3-hisG) was created by URA blaster method. Ura- phenotype strain was selected in synthetic complete glucose medium containing 0.1% 5-fluoroorotic acid, and complemented by URA3. Hyphal development of fungal cells (1×106 cfu/ml) was evaluated in both liquid and solid hyphal-inducing media, including Lee’s medium (pH 7.0), RPMI-1640, YPD+10% fetal bovine serum (FBS) and Spider’s medium at 37℃. To analyze the mRNA expression of hyphal-specific genes, fungal cells were prepared in the YPD containing 10% FBS at 37 ℃. Total RNA was extracted by SV Total RNA Isolation System and was reverse transcribed into cDNA using Superscirpt II. Gene expression was determined by Fast SYBR Green Master Mix, with the program: 95 ℃ incubation for 20s, and then 40 cycles of 95 ℃ for 1s and 60 ℃ for 20s. Student’s t test (P value < 0.05) was used for statistical analysis.
Results: Compared with wild type SC5314 strain, SAP9 null mutant strain was defective in the hyphal development in both liquid and solid hyphal-inducting media. Gene expression analysis indicated that the expression level of transcription factor gene EFG1 was down-regulated by about 40% (P<0.05).
Conclusions: C. albicans SAP9 might play a pivotal role in hyphal development in C. albicans via the cellular events mediated by EFG1.