核盘菌转录因子Ss-FoxE2互作蛋白的验证

发布时间：2018-04-30 07:49

本文选题：核盘菌 + Forkhead　；参考：《吉林大学》2017年硕士论文

【摘要】：核盘菌(Sclerotinia sclerotiorum(Lib.)de Bary)是一种多寄主的死体营养型的植物病原真菌。核盘菌菌丝聚集形成的菌核能够抵御各种物理,化学,以及微生物的破坏,在土壤当中可以保持活性达八年以上,在条件适宜的情况下菌核又可以萌发产生子囊盘,子囊盘释放出的子囊孢子作为侵染源又会进行新一轮的病害循环,因此菌核病是一种防治十分困难的病害,每年给农业的生产带来巨额的经济损失。在实验室的前期研究中发现,将Ss-Fox E2转录因子敲除之后,与野生型相比其对菌丝及菌核的发育,致病性方面无影响,但是在有性生殖阶段无法产生子囊盘,表明Ss-Fox E2转录因子与核盘菌的有性发育相关;通过酵母双杂交实验筛选得到了9个可能与转录因子Ss-Fox E2互作的候选蛋白。本研究在此基础之上,通过酵母双杂交回转验证,双分子荧光互补实验验证候选互作蛋白,并试图通过染色质免疫共沉淀技术分析转录因子Ss-Fox E2下游调控的靶序列,从而初步建立起转录因子Ss-Fox E2的互作调控网络,主要的研究结果如下:通过将候选基因的开放阅读框构建到酵母双杂交AD载体上,与连有转录因子Ss-Fox E2的诱饵载体共转化到酵母菌株中,其中有6个共转质粒可以激活下游报告基因,且无自激活情况发生,证明得到了6个可以与转录因子Ss-Fox E2中Forkhead结构域互作的蛋白。之后又将9个候选基因的开放阅读框构以及Ss-Fox E2的开放阅读框构分别建到双分子荧光互补载体p SAT4-c YFP/p SAT4-n YFP上,在激发光的照射下,有4个互作蛋白可以与转录因子Ss-Fox E2产生相互作用并发出黄色荧光,因此,通过酵母双杂交回转验证及双分子荧光互补实验的验证,筛掉具有假阳性的互作蛋白,共验证得到4个与转录因子Ss-Fox E2互作的蛋白。染色体免疫共沉淀是研究纤转录因子Ss-Fox E2转录过程中与启动子区互作所涉及的主要技术。该项技术难度较大、质量控制要求高,本研究对Ch IP实验中的关键控制点进行摸索,其中超声破碎条件则摸索为在20%的功率下,使用2mm直径超声波探头,2s工作,2s间歇的工作状态下,超声总时间8-10分钟时,染色体DNA断裂程度可以达到500-1000bp之间;同时制备了ELISA效价达到1:50k,可以与转录因子Ss-Fox E2特异性结合的多克隆抗体,为稳定实验条件,获取可靠数据和后续深入研究奠定基础。以上各研究结论为下一步Ss-Fox E2互作蛋白的功能研究以及调控网络的深入研究提供了基础。
[Abstract]:Sclerotinia sclerotiorum (Lib.) de Bary is a plant pathogenic fungus of the dead body type of multiple hosts. The sclerotia formed by the accumulation of mycelium of Sclerotinia mycelium can resist the destruction of various physical, chemical and microorganism, and can keep the activity for more than eight years in the soil, and the sclerotium can germinate under the condition of suitable conditions. The sac disk and the conidia released from the cysts of the cysts will carry out a new round of disease cycle as the source of infection, so Sclerotinia is a very difficult disease, which brings huge economic losses to the agricultural production every year. In the previous study of the laboratory, the Ss-Fox E2 transcription factor was knocked out and compared with the wild type. There was no influence on the development and pathogenicity of mycelium and sclerotium, but it was impossible to produce a sac in the sexual reproduction stage, indicating that the Ss-Fox E2 transcription factor was related to the sexual development of the fungus, and 9 possible candidate proteins interacting with the transcriptional factor Ss-Fox E2 were screened by yeast two hybrid experiment. The parent double hybrid gyration verification, the double molecular fluorescence complementary experiment verifies the candidate interaction protein, and attempts to analyze the target sequence regulated by the transcription factor Ss-Fox E2 downstream by chromatin immunoprecipitation technology, and thus initially establish the transcription factor Ss-Fox E2 interaction control network. The main results are as follows: through the opening of candidate genes, The frame was constructed on the yeast two hybrid AD vector, and the bait carrier, which had the transcription factor Ss-Fox E2, was transformed into the yeast strain. 6 of the co rotation plasmids could activate the downstream reporter gene, and there was no self activation. It was proved that 6 proteins that could interact with the Forkhead domain of the transcription factor E2 were obtained. After that, 9 of the proteins were found to be interacted with the Forkhead domain in the transcription factor E2. The open reading frame of the candidate genes and the open reading frame of Ss-Fox E2 were built on the double molecule fluorescent complementary carrier P SAT4-c YFP/p SAT4-n YFP. Under stimulated luminescence, 4 interacting proteins could interact with the transcription factor Ss-Fox E2 and emit yellow fluorescein. Therefore, the yeast two hybrid gyration was used to verify and be verified by the yeast two hybrid gyration. It was verified by the double molecular fluorescence complementary experiment that 4 proteins interacting with the transcription factor Ss-Fox E2 were screened out with the false positive interaction protein. The chromosome immunoprecipitation was the main technology involved in the interaction of the Ss-Fox E2 in the transcription factor of fibrin transcription factor and the promoter region. The technique was difficult and the quality control was high. In this study, the key control points in the Ch IP experiment were explored, in which the ultrasonic breakage conditions were found to be at 20% power, using the 2mm diameter ultrasonic probe, 2S work, and 2S intermittent working state. When the total ultrasonic time was 8-10 minutes, the chromosome DNA fracture degree could reach 500-1000bp, and ELISA titer reached 1:50k at the same time. The polyclonal antibody that can be specifically associated with the transcription factor Ss-Fox E2 is the basis for stabilizing the experimental conditions, obtaining reliable data and further research. The conclusions of the above studies provide the basis for the further research on the function of the next Ss-Fox E2 interaction protein and the in-depth study of the regulatory network.

【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S432.44

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