Objective To investigate the effect of Aspergillus flavus cyp51 genes on antifungal susceptibility by cloning and constucting the extra copies of Aspergillus flavus cyp51 genes. Methods A.flavus cyp5l gene homologues were identified by tblastn searches inA. flavus genome database. PCR fragments composed of the 5'flanking sequence (approximately 1 000 bp) ofcyp51 ,cyp51 ORF, and its 3'flanking sequence (approximately 1 000 bp), were subcloned into shuttle plasmid pRG3-AMAl-NotI to produce recombinant plasmids. These plasmids and empty plasmid pRG3-AMA1-Notl were transformed into A.fumigatus strain AF293.1 (pyrG-) respectively to produce transformants. The Clinical Laboratory Standard Institute broth microdilution method M38-A2 and E-test method were used to assay the minimal inhibitory concentrations (MICs) of itraconazole ( ITC), voriconazole ( VRC), amphotericin B (AMB), and posaconazole (POS), or minimal effect concentration (MEC) of caspofungin (CAS), against these transformants. Results A. flavus genome contains three cyp51 gene homologues, cyp51A ,cyp51B and cyp51 C, of which the ORF size are 1 400-2 000 bp. When these genes were subcloned into shuttle plasmid pRG3-AMA1-NotI, we get plasmids pRG3-AMA1-CYP51 A, pRG3-AMA1-CYP51B and pRG3-AMA1-CYP51C. These plasmids and empty plasmid were transformed into A.fumigatus strain AF293.1 (pyrG-) to produce transformants rCYP51A, rCYP51B, rCYP51C and rpRG. The antifungal susceptibility of these A.fumigatus transformants to the antifungal drugs by broth microdilution assaying and E-test method showed that, rCYP51A and rCYP51B were cross-resistant to VRC and ITC, susceptible to both AM B and CAS; rCYP51C and rpRG were intermediate to ITC and VRC, susceptible to both A MB and CAS. Conclusion In A. fumigatus , extra copies of A.flavus ' cyp51A gene or cyp51B gene have effect on antifungal susceptibility to azoles, have no effect on AMB and CAS. Extra copy ofcyp51C has no obvious effect on all the tested drugs.%目的 通过构建黄曲霉cyp51同源基因额外拷贝株,研究这些基因对抗真菌药物敏感性的影响.方法 通过序列同源性比对,在黄曲霉基因组中找出其cyp51基因的开放读码框(ORF)及其上下游约1000bp的基因序列,PCR扩增后利用DNA重组技术将该片段克隆到穿梭质粒pRG3-AMAI-Notl;用重组后的质粒和空质粒转化烟曲霉嘧啶营养缺陷株(pyrG-) AF-293.1,并利用美国临床实验室标准化研究所(CLSI) M38-A2中的微量液基稀释法和E-test法测定转化株对两性霉素B(AMB)、伊曲康唑(ITC)、伏立康哇(VRC)、泊沙康唑(POS)和卡泊芬净(CAS)敏感性.结果 黄曲霉cyp51同源基因有3个:cyp51A,cyp5lB和cyp5lC,ORF大小约为1400-2000bp;将其克隆到穿梭质粒pRG3-AMAI-Notl产生重组质粒pRG3-AMAI-CYP51A,pRG3-AMAI-CYP51 B和pRG3-AMAI-CYP51C;连同空质粒转化AF293.1后得到阳性转化子rCYP51 A,rCYP51 B,iCYP51 C和rpRG;微量液基稀释法和E-test法显示,rCYP51 A和rCYP51 B对VRC和ITC交叉耐药,对AMB和CAS敏感;rcyp51C和rpRG对VRC和ITC中度敏感,而对AMB和CAS敏感.结论 额外拷贝黄曲霉cyp51A或cyp51B基因影响烟曲霉对唑类药物的敏感性,对AMB和CAS的敏感性没有影响;额外拷贝黄曲霉cyp51C对测试药物的敏感性没有明显影响.
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