烟曲霉Bem46基因敲除株构建及其功能初步研究

Construction of Bem46 knockout strain and the role of the Bem46 gene in Aspergillus fumigatus

目的构建烟曲霉Bem46基因敲除株,初步明确Bem46基因在烟曲霉生长出芽和各种压力传导中的作用,以及对细胞壁形成的影响。方法采用生物信息学方法查找烟曲霉中Bem46基因,设计引物,并以pyrG作为筛选标记构建烟曲霉Bem46基因敲除株(ΔBem46)。观察含不同压力物质培养基上对照菌株和ΔBem46径向生长速度,观察两菌株发芽状况以及在含有细胞壁抑制剂培养基上的生长状况。结果通过序列比对在烟曲霉基因组中找到了Bem46基因,其编号为Afu7g04660,由1 116bp碱基组成,编码311个氨基酸。使用原生质体法得到烟曲霉ΔBem46并经PCR和Southernblot确认。经观察在含有山梨醇作为渗透压力来源的培养基上ΔBem46生长明显较对照菌株快。显微镜下观察到在GMM液体培养基中,ΔBem46发芽速度慢于对照株。结论烟曲霉Bem46基因涉及由山梨醇引起的渗透压压力传导,并且该基因对促进孢子发芽可能有作用。

The aims of this study are to construct the Bem46 knockout strain of Aspergillus fumigatus,investigate the effect of the Bem46 in the growth and germination,and observe the sensitivity of the oxidative stress,the osmotic stress,the temperature and the response to caspofungin and nikkomycin.The Bem46 gene was identified by sequence alignment in A.fumigatus genome database.The Bem46 knockout strain was construction by the protoplast.The growth was observed and compared between Ku80 and ΔBem46on different culture medium,such as GMM,GMM containing hydrogen peroxide,formamide,sodium chloride and sorbitol.The growth diameter was measured under the different temperatures.The germination rate was observed and compared by microscope.Results showed that the Bem46 gene,Afu7g04660,contained 1 116 bp bases pair and encoding 311 amino acids.Six transformants were obtained by gene cloning and protoplast method and only one was confirmed by PCR and Southernblot.TheΔBem46grew significantly faster than the control strain on the medium containing sorbitol.And there were no visible difference betweenΔBem46and Ku80 on the other medium.The germination rate of theΔBem46was more retarded than the Ku80 in GMM liquid medium.In conclusion,the Bem46 gene plays a role in osmotic stress in A.fumigatus and involved in osmotic pressure induced by sorbitol.And there is no visible effect in oxidative stress.The Bem46 gene has a positive effect on spore germination.