额外拷贝ERG6基因对烟曲霉的影响

Effect of ERG6 Gene Extra Copies on Aspergillus fumigatus

通过构建烟曲霉ERG6基因额外拷贝株．研究该基因对烟曲霉生长速度、抗药物敏感性的影响。在烟曲霉基因组找出烟曲霉可能的ERG6基因的开放读码框(ORF),PCR扩增ERG6的ORF连同其上下游各约1 kb的DNA片段,利用DNA重组的方法将该片段克隆到载体pRG-AMA1-NotI。用重组后的质粒转化烟曲霉尿嘧啶营养缺陷株AF293．1。在MM和YAG培养基上观察转化子的生长速度。采用纸片扩散法和微量液基稀释法测定转化子对抗真菌药物敏感性。烟曲霉基因组中存在一个拷贝的ERG6基因,ORF大小为1,256 bp。其编码的蛋白与白念珠菌、酿酒酵母固醇甲基转移酶(Ers6p)的氨基酸相同率分别为57%和50%,相似率分别为70%和63%。烟曲霉中ERG6基因被成功克隆到了pRG-AMA1-Not I,产生了质粒pERG6。用pERG6和空载体pRG-AMA1-Not I转化AF293．1后,分别得到转化子AF-pERG6和AF-empty。AF-pERG6在MM和YAG培养基上的生长速度均比AF-empty慢。AF-pERG6和AF-empty对伊曲康唑、伏力康唑、特比萘芬、两性霉素B、卡泊芬净、灰黄霉素的敏感性没有差异。ERG6基因额外拷贝不影响烟曲霉对伊曲康唑、伏力康唑、特比萘芬、两性霉素B、卡泊芬净、灰黄霉素的敏感性,但是能使烟曲霉的生长速度减慢。

To investigate the role of ERG6 gene in the growth rate and antifungal susceptibility, Aspergillus fumigatus strain with extra copies of ERG6 gene was constructed. Open reading frame （ORF） of putative ERG6 gene was searched in A. fumigatus genome. A PCR fragment, ERG6 ORF sandwiched by its flanking sequences （about 1 kb respectively）, was amplified and was then subcloned into vector pRG-AMA1- NotI to produce a recombinant plasmid pERG6, which was further transformed into uracil anxotroph A.fumigatus strain AF293. 1 to produce the transformant AF-pERG6. In the same time, empty plasmid pRG-AMA1-NotI was also transformed into A.fumigatus strain AF293. 1 to produce the transformant AF-empty as a control Radial growth of the transformants was tested on minimal medium （MM） and YAG medium. Antifungal susceptibilities of thase resulting transformants, AF-pERG6 and AF-empty, to the common antifungal agents were performed by using both disk diffusion and broth micredillution methods. A.fumigatus genome contains a ERG6 gene, of which the ORF size is 1 256 bp. Comparing to Gandida albicans and Saechromyces cerivisiae sterol methyltransferase, A. fumigatus Erg6p had 57% and 50% identity, and had 70% and 63% similarity in amino acid sequences, respectively. Radial growth of transformant AF-pERG6 was dower than that of transformant AF-empty. The antifungal susceptibilties of transformant AF-pERG6 to the antifungal drugs itraconasole, voriconazole, terbinaline, amphotericin B, caspofungin and grisefolvin were same to that of transformant AF-empty. In A. fumigatus, extra copies of ERG6 gene have no effect on antifungal susceptibilities to itraconazole, voriconazole, terbinafine, amphotericin B, caspofungin and grisefolvin. Radial growth of A. fumigatus harboring extra copies of ERG6 gene becomes slower compared to the control.