芸薹生链格孢转录因子ZnCys_Ab0012基因功能研究

发布时间：2018-08-28 18:08

【摘要】：Zn_2Cys_6家族转录因子是锌簇家族转录因子中的C6型转录因子,是真菌比较常见的一类转录因子。该家族转录因子最早的研究是酿酒酵母中的GAL4基因,在稻瘟病菌(Magnaporthe oryzae)和禾谷镰刀菌(Fusarium graminearum)等植物病原真菌中也有比较多的研究。研究发现该类转录因子参与调控病菌的致病性、产孢量和病原菌的生长等生命过程。芸薹生链格孢(Alternaria brassicicola)是引起十字花科植物黑斑病的病原菌,给农民带来了比较严重的经济损失。目前,对于芸薹生链格孢(A.brassicicola)致病机制尚不明确,研究发现Zn_2Cys_6家族转录因子参与多种植物病原真菌的致病过程,且在芸薹生链格孢(A.brassicicola)研究较少,因此进行深入的研究就有助于我们认识该菌的致病机制。在本研究中,我们根据稻瘟病菌中Zn_2Cys_6家族MoNIT4基因,寻找到了其在芸薹生链格孢(A.brassicicola)中的同源基因ZnCys_Ab0012。据据同源重组的原理,采用原生质体转化的方法,得到了ZnCys_Ab0012基因突变体,从而对芸薹生链格孢(A.brassicicola)中ZnCys_Ab0012基因的功能进行研究。通过ZnCys_Ab0012基因缺失突变体与野生菌对比研究发现,ZnCys_Ab0012基因缺失突变体的生长速度是野生菌生长速度的1.96倍。通过对两种菌表型的观察发现,野生菌的生长以埋生菌丝为主,而ZnCys_Ab0012基因缺失突变体以气生菌丝的生长为主,且菌落颜色差异很大,ZnCys_Ab0012基因缺失突变体呈灰白色,而野生菌为深绿色。通过对于油菜的接种实验发现,ZnCys_Ab0012基因缺失突变体接种的油菜没有表现出对叶片的致病性。通过对培养的菌株进行显微镜观察,我们发现,ZnCys_Ab0012基因缺失突变体菌株在显微镜下没有观察到孢子存在。同时通过对于穿透能力以及抗逆境生长能力的研究发现,ZnCys_Ab0012基因缺失突变体与野生菌没有很明显的区别。综上所述,ZnCys_Ab0012基因能够控制病原菌的产孢量,同时对于病原菌的生长速度以及致病性造成比较明显的影响。同时对于病原菌的穿透力以及逆境生长的能力影响相对较小。
[Abstract]:Zn_2Cys_6 family transcription factors are C6 type transcription factors in zinc cluster family, and they are common transcription factors in fungi. GAL4 gene in Saccharomyces cerevisiae is the earliest study of transcription factors in this family. There are also many studies on the pathogen fungi such as (Magnaporthe oryzae) and (Fusarium graminearum) of rice blast and Fusarium graminearum. It was found that these transcription factors were involved in controlling the pathogenicity, sporulation and growth of pathogenic bacteria. Alternaria brassica (Alternaria brassicicola) is a pathogen causing black spot of cruciferous plants, which brings serious economic losses to farmers. At present, the pathogenetic mechanism of A.brassicicola in brassica is not clear. It was found that the transcription factors of Zn_2Cys_6 family were involved in the pathogenicity of many plant pathogenic fungi, and there were few studies on A.brassicicola in brassica. Therefore, in-depth study will help us to understand the pathogenic mechanism of the bacteria. In this study, based on the MoNIT4 gene of Zn_2Cys_6 family in rice blast fungus, we found the homologous gene ZnCys_Ab0012. in the brassica Alternaria (A.brassicicola). According to the principle of homologous recombination, ZnCys_Ab0012 gene mutants were obtained by protoplast transformation, and the function of ZnCys_Ab0012 gene in A.brassicicola was studied. It was found that the growth rate of the ZnCys_Ab0012 gene deletion mutant was 1.96 times faster than that of the wild strain. By observing the phenotypes of the two strains, it was found that the growth of wild bacteria was mainly buried hyphae, while that of ZnCys_Ab0012 gene deletion mutant was mainly airborne hyphae, and the colony color difference was very different, and the mutant of ZnCysAb0012 gene deletion was grayish white. Wild fungi are dark green. It was found that rape inoculated with Zn-CysAb0012 gene deletion mutant did not show pathogenicity to leaves. By microscopic observation of the cultured strain, we found that no spore was found in the strain with deletion of ZnCysAb0012 gene. At the same time, it was found that there was no significant difference between ZnCysS Ab0012 gene deletion mutants and wild bacteria through the study of penetration ability and resistance to stress growth. In conclusion, ZnCysAb0012 gene can control the sporulation of pathogenic bacteria, and has a significant effect on the growth rate and pathogenicity of pathogenic bacteria. At the same time, it had little effect on the permeability of pathogens and the ability of growth under stress.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S432.4

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