含额外拷贝ERG3B基因烟曲霉的表型分析

Phenotype Analysis of Aspergillus Fumigatus Containing Extra Copies of ERG3B Gene

目的克隆烟曲霉ERG3B基因,并构建烟曲霉ERG3B基因额外拷贝株,了解该基因对烟曲霉表型的影响。方法通过同源性比对,在烟曲霉基因组找出烟曲霉可能的ERG3基因的开放读码框(ORF),PCR扩增ERG3B基因ORF连同其上下游各约1kb的DNA片段,将该片段重组到穿梭质粒pRG-AMA1-NotI。用重组后的质粒转化嘧啶营养缺陷株烟曲霉AF293.1。观察转化子的生长速度并测定转化子对抗真菌药物敏感性。结果①烟曲霉ERG3基因有3个拷贝(ERG3A,ERG3B,ERG3C)。Erg3Ap和Erg3Bp与其他真菌的Erg3同源性高。②烟曲霉ERG3B基因被重组到了pRG-AMA1-NotI,产生质粒pERG3B。用pERG3B和空载体pRG-AMA1-NotI转化AF293.1后,分别得到转化子AF-pERG3B和AF-empty。AF-pERG3B与AF-empty的生长速度无差异;两者对伊曲康唑、伏立康唑、特比萘芬、两性霉素B、卡泊芬净、灰黄霉素的敏感性也无差异。结论额外拷贝的ERG3B基因不影响烟曲霉的生长速度及对伊曲康唑、伏立康唑、特比萘芬、两性霉素B、卡泊芬净、灰黄霉素的敏感性。

Objective To investigate the phenotype and antifungal susceptibility of Aspergillus fumigatus strain that contains extra copies of ERG3B gene to common antifungal drugs. Methods A. fumigatas ERG3B gene homologue was identified by thlastn searches in A. fumigatus genome database. A PCR fragment of ERG3B ORF sandwiched by sequences with aproximately 1 kb size at both 5-and 3-flanking sides was subcloned into a shuttle plasmid pRG-AMA1-NotI to produce a recombinant plasmid pERG3B. This plasmid and the empty plasmid pRG-AMA1-NotI were transformed into A. fumigatus strain AF293.1 （pyrG - ） resectively, to produce transformants AF-pERG3 B and AF-empty. Results There were 3 copies of ERG3 gene, ERG3A, ERG3 B and ERG3C in A. fumigatus genome. Erg3Ap and Erg3Bp had had homologous with the sterol methyltransferases in other fungi. Compared to AF-empty, radial growth and antifungal susceptibilities of AF-pERG3B containing extra copies of ERG3B gene had no difference. Conclusion Extra copies of ERG3 B gene has no effect on radial growth, and antifungal susceptibilities of A. fumigatus to itraconazole,voriconazole,terbinafine,amphotericin B,caspofungin,and grisefolvin.