烟曲霉Bem46基因荧光定位菌株构建及对极性生长相关基因影响作用初探

目的 构建以eGFP作为荧光标记的烟曲霉Bem46基因定位菌株,观察其在烟曲霉不同形态中的定位情况,初步明确该基因对极性生长相关基因Bem1、Bud5表达的影响。方法 运用分子生物学方法将烟曲霉Bem46基因克隆入质粒pUCGH,构建荧光定位载体。原生质体法转化烟曲霉获得该基因定位菌株并验证。激光共聚焦显微镜观察绿色荧光在烟曲霉孢子、芽管以及菌丝状态下分布情况。RT-PCR观察对照菌株Ku80和ΔBem46中极性生长相关基因Bem1、Bud5的表达量。结果 通过全基因克隆成功获得烟曲霉Bem46基因定位菌株并经PCR验证。激光共聚焦显微镜下绿色荧光在孢子中弥散分布,在孢子即将发芽时荧光在孢子一侧聚集,并随着芽管形成,荧光在极性生长的一侧延伸分布于整个菌丝中,在菌丝形成侧枝膨大处可见荧光明显聚集。RT-PCR结果表明ΔBem46中基因Bem1、Bud5表达量均较对照菌株有所下降。结论 烟曲霉Bem46基因可能同孢子发芽、菌丝发生侧枝的极性生长相关;且该基因缺陷可影响极性生长相关基因Bem1、Bud5表达。

Construction of Bem46 fluorescent localization strain and investigation of the effect on polar growth gene in Aspergillus fumigatus

Aspergillus fumigatus is an environmental filamentous fungus that can cause life?threatening disease in immunocompromised individuals. BEM46 proteins are evolutionarily conserved, but their functions remain elusive. We constructed a localization strain of Bem46 gene in A. fumigatus with eGFP as the reporter gene was constructed and the distribution of green fluorescent protein in different morphology of A. fumigatus was observed. The expression of Bem1 and Bud5 gene related with polar growth between ΔBem46 and Ku80 was also compared. The Bem46 gene of A. fumigatus was cloned into plasmid pUCGH by molecular biological method and the vector of fluorescent localization strain was constructed. The six transformations of Bem46 localization strains were obtained by the protoplast transformation method and were identified by different PCR. The fluorescence was diffused in the spore and gathered on one side of the spore when the spore was about to germinate by laser confocal microscopy. With the formation of the germ tube, the fluorescence extended on the polar growth side and gradually developed into hypha, and then distributed in the whole hypha. The obvious fluorescence accumulation was found on the tips of the hypha branch. The expression of Bem1 and Bud5 gene in ΔBem46 was lower than the control strain by RT?PCR. In conclusion, it was speculated that Bem46 gene of A. fumigatus may be related to the polar growth of spore budding and the tips of lateral branches of mycelium. The defection of Bem46 gene may affect the expression of Bem1 and Bud5 genes.