Department of Laboratory Medicine, Shanghai EastHospital, Tongji University School of Medicine, Shanghai, 200123, China.
J Antibiot (Tokyo). 2020 May;73(5):309-313. doi: 10.1038/s41429-020-0283-8. Epub 2020 Feb 14.
To study differences in SRB1 gene expression between Candida albicans fluconazole-resistant strains and fluconazole-sensitive strains, and to identify new antifungal drug treatment targets. We studied 30 fluconazole-resistant and 47 fluconazole-sensitive C. albicans strains. The strains were routinely cultured, and total RNA was extracted, reverse transcribed into cDNA and analyzed with real-time PCR amplification with 18S rRNA used as an internal reference gene. The expression levels of the two groups were analyzed in Light Cycler system software version 3.0, and independent Student's t test was performed in SPSS 19.0 statistical software. P < 0.05 was considered to indicate a statistical difference. DNAMAN multiple sequence alignment analysis was used to randomly analyze SBR1 related sequences of four resistant strains and four sensitive strains. An evolutionary tree was constructed with the maximum likelihood method in Mega6.0 software. The mean SRB1 gene expression in the drug-resistant group was 0.75138 × 10, and that in the sensitive group was 1.6664 × 10. Independent Student's t test indicated a statistically significant difference (T = -3.972, P = 0.000, P < 0.05). DNAMAN multiple sequence alignment analysis showed that the sequence identity of the CDS in the eight strains was 75.17%, and that of sequences 1000 bp upstream of the CDS was 96.35%. Cluster analysis showed that the CDS and sequences 1000 bp upstream of the CDS showed no significant differences between groups. At the mRNA level, the SRB1 gene expression in fluconazole-resistant C. albicans was lower than that in fluconazole-sensitive strains, thus suggesting that the gene may be associated with drug resistance and that the regulatory mechanism leading to this difference is complex.
为了研究白色念珠菌氟康唑耐药株与氟康唑敏感株之间 SRB1 基因表达的差异,寻找新的抗真菌药物治疗靶点。我们研究了 30 株氟康唑耐药株和 47 株氟康唑敏感株白色念珠菌。常规培养菌株,提取总 RNA,逆转录为 cDNA,用实时 PCR 扩增,以 18S rRNA 为内参基因进行分析。在 Light Cycler 系统软件版本 3.0 中分析两组的表达水平,在 SPSS 19.0 统计软件中进行独立 Student's t 检验。P<0.05 表示有统计学差异。采用 DNAMAN 多序列比对分析随机分析 4 株耐药株和 4 株敏感株 SBR1 相关序列。在 Mega6.0 软件中采用最大似然法构建进化树。耐药组 SRB1 基因的平均表达量为 0.75138×10,敏感组为 1.6664×10。独立 Student's t 检验表明差异有统计学意义(T=-3.972,P=0.000,P<0.05)。DNAMAN 多序列比对分析显示 8 株菌 CDS 序列的同源性为 75.17%,CDS 上游 1000bp 序列的同源性为 96.35%。聚类分析显示 CDS 和 CDS 上游 1000bp 序列在两组间无明显差异。在 mRNA 水平,氟康唑耐药白色念珠菌 SRB1 基因表达低于氟康唑敏感株,提示该基因可能与耐药性相关,导致这种差异的调控机制复杂。