通过构建烟曲霉bem46过表达菌株研究其在极性生长中的作用

Study of the role of bem46 gene of Aspergillus fumigatus in polar growth by constructing an overexpression strain

目的构建烟曲霉bem46基因过表达株,探究bem46基因过表达情况下对烟曲霉极性生长及与其他极性基因的相互作用关系。方法原生质体法构建烟曲霉bem46过表达株;激光共聚焦显微镜观察烟曲霉bem46过表达时的绿色荧光定位;观察记录烟曲霉Ku80、烟曲霉bem46基因缺陷株(Δbem46)和烟曲霉bem46过表达株的发芽率;记录烟曲霉Ku80、Δbem46和过表达株菌落径向生长情况;光学显微镜下观察乳酸酚棉蓝染色的烟曲霉bem46过表达菌落生长情况;实时荧光定量PCR方法测定烟曲霉Ku80、Δbem46和过表达株中有关极性生长的基因的相对表达量。结果利用原生质体法成功构建烟曲霉bem46的过表达株;激光共聚焦显微镜观察到bem46过表达时绿色荧光大量聚集在孢子头部位,而菌丝中几乎无荧光显示;相比于Ku80正常状态的发芽情况,bem46过表达株的发芽时间提前,而Δbem46株的发芽时间滞后;Ku80株、Δbem46和过表达株的径向生长差异无统计学意义;经过乳酸酚棉蓝染色以及显微镜下的观察,发现Ku80株、Δbem46和过表达株的孢子头形态有明显的差异,bem46过表达时可明显刺激孢子头膨大;通过测定烟曲霉Δbem46和bem46过表达株中极性生长基因的相对表达量,发现不论bem46基因在烟曲霉中缺失还是过表达,烟曲霉中cdc24、bem1、bem3、bud5、rsr1以及rasb基因的相对表达量均明显降低,cdc24表达量变化不大。结论bem46基因可以促进烟曲霉孢子萌发,影响烟曲霉极性生长过程中的孢子和菌丝形态;在相关基因的信号转导通路中,bem46基因可能参与cdc 42有关的极性生长信号转导通路。

Objective To construct an overexpression strain of bem46 gene of A.fumigatus,and explore the polar growth of A.fumigatus and its interaction with other polar genes under the condition of bem46 gene overexpression.Methods The overexpression strain of bem46 gene was constructed by protoplast method.The green fluorescence localization of A.fumigatus bem46 was observed by laser confocal microscope.The germination rates of A.fumigatus Ku80,Δbem46 and over-expressed strains were observed and recorded.The radial growth of A.fumigatus Ku80,Δbem46 and over-expressed strains was recorded.The growth of over-expressed colonies of A.fumigatus bem46 stained with lactic acid phenol cotton blue was observed under optical microscope.Real-time fluorescence quantitative PCR was used to determine the relative expression of genes related to polar growth in A.fumigatus Ku80,Δbem46 and overexpressed strains.Results The overexpression strain of A.fumigatus bem46 was successfully constructed by protoplast method.Laser confocal microscope observed that when bem46 was overexpressed,a large amount of green fluorescence gathered in the spore head,but there was almost no fluorescence in the mycelium.Compared with the germination of Ku80,the germination time of the overexpressed strain of bem46 gene was earlier than that ofΔbem46 strain.There was no significant difference in radial growth among Ku80 strain,Δbem46 strain and overexpressed strain.After dyeing and observation under microscope,it was found that the spore heads of Ku80 strain,Δbem46 strain and overexpressed strain were obviously different,and the overexpressed strain could obviously stimulate the spore heads to expand.By measuring the relative expression levels of polar growth genes in A.fumigatusΔbem46 and the overexpression strains,it was found that the relative expression levels of cdc24,bem1,bem3,bud5,rsr1 and rasb genes in A.fumigatus were significantly reduced,and the expression level of cdc24 was not changed.Conclusion The bem46 gene can promote the spore germination of A.fumigatus and affect the spore and mycelium morphology during the polar growth of A.fumigatus.In the signal transduction pathway of related genes,bem46 gene may participate in the polar growth signal transduction pathway related to cdc24.