烟曲霉Bem46基因敲除株的构建及其在烟曲霉极性生长过程中的作用初探

发布时间：2018-05-18 23:40

  本文选题：烟曲霉 + Bem46　； 参考：《山西医科大学》2017年硕士论文

【摘要】：目的:细胞的极性生长是指从细胞的某一区域非对称性的生长出另一种特定的结构或形状。这种独特的结构对细胞的功能至关重要,并协助细胞间的相互作用。烟曲霉等曲霉菌属是典型的极性生长生物体,主要表现为其菌丝尖端的不断生长。本文将构建烟曲霉Bem46基因敲除株,并在配制含不同抗真菌药物的培养基上,观察烟曲霉Bem46基因缺陷株与对照株的生长差异,以期发现新的药物靶点;以及构建烟曲霉Bem46基因荧光定位株,观察其在烟曲霉菌丝中的定位情况;对比对照株及敲除株发芽状况并检测部分Bem46极性生长传导通路中的相关基因表达量情况来探索Bem46基因在烟曲霉极性生长过程中的作用。方法: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)。在基础培养基接种相同数量的对照株与缺陷株孢子,观察二者生长有无差异。配制五种药物(两性霉素B、卡泊芬净、伊曲康唑、伏立康唑、他克莫司)不同浓度的平皿,浓度分别为0、0.1、0.3、0.5、0.8、1.0、2.0、5.0mg/L,点种相同数量的对照株及敲除株的孢子,37℃培养72h,测量并记录直径,实验重复三次。将Bem46基因与增强型绿色荧光蛋白(EGFP)及潮霉素筛选转化子相连,转入烟曲霉,潮霉素筛选后得到Bem46荧光定位株,在共聚焦显微镜下观察Bem46在烟曲霉极性生长过程中的定位情况。在倒置显微镜下对比敲除株与对照株的出芽差异,然后分别提取二者的RNA,使用实时荧光定量PCR方法检测极性生长相关基因Bem1、Bud5在对照株及敲除株中的表达差异。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1116bp碱基组成,编码311个氨基酸。经PCR和Southernblot验证得到Bem46基因敲除株。在五种药物不同浓度平皿上,对照株与敲除株生长直径经重复测量方差分析并无明显差异。经原生质体转化法及PCR验证成功得到Bem46基因荧光定位株,在共聚焦显微镜下观察,荧光在菌丝内呈弥散分布,并没有观察到特异性的定位区域,但在分支处有聚集现象。敲除株与定位株的出芽率经重复测量方差分析,可见敲除株出芽明显滞后于对照株。极性生长相关基因Bem1、Bud5经实时荧光定量PCR检测,在敲除株中的表达量均低于对照株。结论:烟曲霉Bem46基因并不能作为两性霉素B、卡泊芬净、伊曲康唑、伏立康唑、他克莫司五种药物的药物靶点;Bem46基因并不能直接构成极性生长的相关结构,如菌丝隔膜或菌丝尖端等;但Bem46基因确实参与烟曲霉的极性生长过程,该基因可能有促进孢子发芽的作用,并且它的缺失导致极性生长相关基因表达下调,极有可能影响了极性生长传导通路,为研究烟曲霉生长机制提供了新思路。
[Abstract]:Objective: polar growth of cells refers to the asymmetric growth of a cell from one region of another specific structure or shape. This unique structure is essential for cell function and facilitates intercellular interaction. Aspergillus such as Aspergillus fumigatus is a typical polar growth organism, which is mainly characterized by the continuous growth of its hyphae tip. In this paper, the Bem46 gene knockout strain of Aspergillus fumigatus was constructed, and the growth difference between Bem46 gene deficient strain of Aspergillus fumigatus and control strain was observed on the medium containing different antifungal drugs, in order to find new drug target. The fluorescent locus of Bem46 gene of Aspergillus fumigatus was constructed and its localization in Aspergillus fumigatus filaments was observed. To explore the role of Bem46 gene in polar growth of Aspergillus fumigatus, the germinating status of control plants and knockout plants was compared and the expression of related genes in some Bem46 polar growth conduction pathways was detected. Methods: Bem46 gene in Aspergillus fumigatus was identified by bioinformatics, primers were designed, and pyrG was used as screening marker to construct Bem46 knockout plant (螖 Bem46C) of Aspergillus fumigatus. The spores of the control and defective strains were inoculated in the basic culture medium to observe the difference between them. Five kinds of drugs (amphotericin B, carpofensin, itraconazole, volconazole, tacrolimus) were prepared with different concentrations of 0 0. 1 ~ 0. 3 ~ 0. 5 ~ 0. 0 mg 路L ~ (-1) and 0. 8% 0. 0 mg 路L ~ (-1). The spores of the control and knockout plants were cultured at 37 鈩，

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