The Founction Of RTA2 And Its Expression Mechanism Regulated By CaN Signal Pathway In C. Albicans

Candida albicans antifungal resistance are the main reasons for treatment failure, The calcineurin pathway has also been reported to participate in drug resistance in C. albicans. But the regulatory mechanism of RTA2 remains unclear. Ura-Blaster gene knockout strategy was used to delete RTA2 gene in cnaΔ/Δmutant strain (DSY2101) and crz1Δ/Δmutant strain (MKY59). First of all, line the RTA2 gene knockout plasmid, with the lined knockout plasmid transfecting Ura-host strain DSY2101, MKY59, PCR method and phenotypic screening were used to verify deletion strains during the continuous operation of gene knock-out. We use liquid-based micro-dilution method in our study of wild strains of CAF2-1, CNA (encoding calcineurin catalytic subunit) deletion strain DSY2091, CNA and RTA2 both deletion strains DJY201, CNA reply strains DSY2115, CRZ1 ( encoding calcineurin pathway transcription factor Crz1p) deletion strains DSY2195, CRZ1 and both RTA2 missing strain MJY201 and CRZ1 reply strain MKY268 by adding extracellular calcium as well as the removal of calcium ions on the antifungal activity of fluconazole. The results showed that: In wild-type strain CAF2-1, by adding 2.5mmol / L CaCl2 can significantly reduce the antifungal activity of fluconazole; at CNA deletion strains DSY2091, CNA and RTA2 both deletion strain DJY201, CRZ1 deletion strain DSY2195, CRZ1 and RTA2 both deletion strains MJY201, add 2.5 mmol / L CaCl2 for the antifungal activity of fluconazole effects of very small; at CNA reply strain DSY2115 and CRZ1 reply strain MKY268, by adding 2.5mmol / L CaCl2 also be able to reduce the antifungal activity of fluconazole; In conclusion, our findings suggest that RTA2 is responsible for the development of calcineurin-mediated azole resistance in C. albicans.Subject to the bioinformatics prediction of CaN signaling pathway target gene regulatory elements in 5ˊ-GCTGT-3ˊ for the target sequence, To address the importance of this region in calcium regulation, different RTA2 promoter lengths were fused to the RLUC reporter system and transformed in crz1Δ/Δmutant strain,rta2Δ/Δmutant strain.The results showed that, RTA2 expression is regulated by CRZ1p in the presence of calcium,and the CDRE in RTA2 was between -785bp and -455bp with respect to ATG..