核盘菌转录因子Ss-FoxE2的基因功能研究

发布时间：2018-06-19 06:43

本文选题：核盘菌 + 转录因子　；参考：《吉林大学》2015年硕士论文

【摘要】：核盘菌（Sclerotinia sclerotiorum（Lib.）de Bary）是一种腐生性丝状病原真菌，它可以造成多种农作物的严重病害，从而引起重大经济损失。核盘菌的子囊盘是由菌核在合适的条件下萌发而成的圆盘状子实体，发育成熟的子囊盘将会释放子囊孢子，子囊孢子飞散到周围环境中侵染寄主植物，造成菌核病害的流行。在核盘菌基因组中存在4个Forkhead类转录因子的同源基因，分别为Ss-FoxE1、Ss-FoxE2、Ss-FoxE3和Ss-FoxE4，我们推断它们与核盘菌的有性生殖密切相关。因此，在本文中我们以Ss-FoxE2为研究对象，并对在核盘菌有性生殖中的作用进行了研究。本研究首先根据核盘菌基因组序列，克隆得到Ss-FoxE2上游890bp序列以及下游1024bp序列，将上下游序列分别连接到敲除载体pXEH上，成功构建了Ss-FoxE2的敲除载体，然后利用裂解酶消化核盘菌菌丝细胞壁，从而制备了质量较好的原生质体细胞，并用PEG-CaCl2介导的原生质体转化法将已构建好的敲除载体转化到核盘菌的原生质体中，以潮霉素为筛选抗性进行转化子的筛选培养，然后使用快速PCR检测方法进行初步验证，将验证正确的转化子使用Southern杂交的方法进行进一步验证。另一方面，克隆Ss-FoxE2编码基因及其上下游在内的3.4kb的片段序列，将克隆得到的序列连接到互补载体上，将构建好的互补载体转化至敲除突变体的原生质体细胞中，，通过G418抗性筛选以及PCR验证得到互补成功的转化子。将Ss-FoxE2敲除突变体和Ss-FoxE2互补突变体对照野生型菌株，进行生物学特性分析和致病性分析，结果发现，Ss-FoxE2敲除突变体与野生型菌株相比，在菌丝生长速率、菌核数量、菌核干重以及致病性上几乎没有任何差别，但是，Ss-FoxE2敲除突变体不能诱导发育形成子囊盘，这说明核盘菌转录因子Ss-FoxE2参与调控核盘菌子囊盘的形成，影响核盘菌的有性生殖，这将为菌核病的防治提供新的理论基础与技术支撑。
[Abstract]:Sclerotinia sclerotiorum Lib.de Baryis is a kind of saprophytic filamentous fungi, which can cause serious diseases of many crops and cause great economic losses. Sclerotinia sclerotiorum cotyledon is a disk-shaped fruiting body that germinates from sclerotia under suitable conditions. The mature ascomycetes will release ascospores, and ascospores will fly into the surrounding environment to infect host plants, resulting in the prevalence of sclerotinia disease in Sclerotinia sclerotiorum (Sclerotinia sclerotiorum). There are four Forkhead-like transcription factor homologous genes in the genome of Sclerotinia sclerotiorum, namely Ss-FoxE1, Ss-FoxE2, Ss-FoxE3 and Ss-FoxE4, which are closely related to the sexual reproduction of Sclerotinia sclerotiorum. Therefore, in this paper, we take Ss-FoxE2 as the research object and study the role of Ss-FoxE2 in the sexual reproduction of Sclerotinia sclerotiorum. In this study, the upstream 890bp sequence and downstream 1024bp sequence of Ss-FoxE2 were cloned according to the Sclerotinia sclerotiorum genome sequence. The upstream and downstream sequences were connected to the knockout vector pXEH, respectively, and the knockout vector Ss-FoxE2 was successfully constructed. Then the cell wall of Sclerotinia sclerotiorum was digested by lyase, and the protoplast cells with good quality were prepared. The constructed knockout vector was transformed into the protoplast of Sclerotinia sclerotiorum by PEG-CaCl2 mediated protoplast transformation. Hygromycin was used to screen and culture the transformants, and then the rapid PCR method was used to verify the transformants. The correct transformants were further verified by Southern blotting. On the other hand, the sequence of Ss-FoxE2 coding gene and its upstream and downstream 3.4kb fragments were cloned, the cloned sequence was linked to the complementary vector, and the constructed complementary vector was transformed into the protoplast cells of the knockout mutant. By G418 resistance screening and PCR validation, complementary and successful transformants were obtained. The biological characteristics and pathogenicity of Ss-FoxE2 knockout mutants and Ss-FoxE2 complementary mutants were analyzed. The results showed that Ss-FoxE2 knockout mutants had higher growth rate and sclerotia number than wild-type strains. There was almost no difference in the dry weight and pathogenicity of sclerotia, but Ss-FoxE2 knockout mutant could not induce the development of sclerotia disk, which indicated that Ss-FoxE2 was involved in regulating the formation of sclerotia cotyledon and affecting the sexual reproduction of Sclerotinia sclerotiorum. This will provide a new theoretical basis and technical support for the prevention and treatment of sclerotinia.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S432.44

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