核盘菌转录因子Ss-FoxE2的表达及其互作蛋白的筛选

发布时间：2018-05-17 16:37

本文选题：核盘菌 + Forkhead　；参考：《吉林大学》2016年硕士论文

【摘要】：核盘菌是一种重要的丝状植物病原真菌,因其寄主范围广,生活史复杂,防治困难,每年都会给农业生产带来重大的经济损失,菌核病的防治问题已经受到了世界各地科学工作者的密切关注。核盘菌生活史包含了无性和有性两个发育阶段,无性阶段组织主要为菌丝以及由菌丝聚集形成的特殊结构——菌核组成。而有性发育阶段的起点在于菌核萌发形成子囊盘。在有性发育阶段由子囊盘产生的子囊孢子数量惊人,远距离传播能力强,因此子囊盘产生的子囊孢子作为侵染原在病害流行等方面扮演了重要角色。本实验室前期工作发现,对核盘菌其中一个属于Forkhead转录因子家族基因Ss-Fox E2进行敲除后,突变体无法正常形成子囊盘,由此我们认为Ss-Fox E2对于核盘菌子囊盘的发育十分重要。因此本研究在此基础之上,围绕基因Ss-Fox E2的表达特征以及与其互做蛋白的筛选等方面展开工作,以期待明确转录因子Ss-Fox E2在子囊盘发育过程中所起到的作用,主要研究结果如下:通过对核盘菌基因组数据库进行搜索以及对候选目标的结构域进行预测分析,找到核盘菌中一共存在4个Forkhead家族转录因子,它们分别为:Ss-Fox1、Ss-Fox E2、Ss-Fox3和Ss-Fkh1,并且通过序列比对和系统进化分析,明确了它们所具有的氨基酸序列特征以及系统进化关系;利用反转录PCR(RT-PCR)方法,成功克隆得到Ss-Fox E2基因的开放阅读框(ORF),该基因编码了449个氨基酸,含有一个内含子;利用荧光定量PCR技术(q RT-PCR)对核盘菌各发育阶段组织中Ss-Fox E2基因的表达含量进行了检测,结果显示Ss-Fox E2的相对表达含量在子囊盘时期组织中明显高于菌丝期和菌核各时期组织,并且随子囊盘的发育成熟,其含量不断升高。该结果证明Ss-Fox E2基因与子囊盘的发育具有相关性;将Ss-Fox E2蛋白与绿色荧光蛋白(e GFP)融合表达,利用烟草叶片表皮细胞瞬时表达技术对Ss-Fox E2进行了亚细胞定位分析,结果显示Ss-Fox E2蛋白定位在细胞核当中;利用p ET28a系统在大肠杆菌Rosetta(DE3)中对Ss-Fox E2进行原核表达分析,结果显示Ss-Fox E2大部分以包涵体形式表达,但通过镍柱纯化大量上清,最终可以得到一定量纯化蛋白,能够满足后期其他实验需要;成功构建基因Ss-Fox E2全长诱饵载体p GBKT7::Ss-Fox E2,对诱饵蛋白自激活性检测发现全长Ss-Fox E2蛋白独自能够激活下游报告基因的表达,具有自激活性即转录因子Ss-Fox E2具有转录激活活性;为了避免Ss-Fox E2全长蛋白的自激活性,我们利用无自激活性的Ss-Fox E2蛋白Forkhead结构域部分作为诱饵蛋白在核盘菌均一化c DNA文库中进行其互作蛋白的筛选,最终经过两次复筛以及一次回转验证,我们得到6个与Ss-Fox E2结构域部分互作的候选蛋白。以上各研究结论为下一步Ss-Fox E2互作蛋白的确认及更深入开展功能、调控网络研究提供了基础。
[Abstract]:Sclerotinia sclerotiorum is an important pathogen of filamentous plants. Because of its wide host range, complicated life history and difficult control, Sclerotinia sclerotiorum brings great economic losses to agricultural production every year. The prevention and cure of sclerotinia has been paid close attention by scientists all over the world. The life cycle of Sclerotinia sclerotiorum consists of two stages: asexual and sexual. The asexual phase is mainly composed of hyphae and a special structure formed by the aggregation of hyphae-sclerotia. The starting point of sexual development stage is that sclerotia germinates and forms oocyst disc. At the stage of sexual development, the number of ascospores produced by ascomyst disk is amazing, and the ability of long distance transmission is strong. Therefore, ascospores produced by oocystis play an important role in the epidemic of disease. Our previous work found that after knockout of one of the genes belonging to Forkhead transcription factor family Ss-Fox E2, the mutant could not normally form the cotyledon disc, so we think that Ss-Fox E2 is very important for the development of sclerotia cotyledon. On the basis of this, this study focused on the expression characteristics of Ss-Fox E2 gene and the screening of proteins with Ss-Fox E2, in order to clarify the role of transcription factor Ss-Fox E2 in the development of the oocytes, and so on, in order to elucidate the role of the transcription factor Ss-Fox E2 in the development of the ovariectoma. The main results are as follows: by searching the genome database of Sclerotinia sclerotiorum and predicting the domain of candidate target, four Forkhead family transcription factors were found in Sclerotinia sclerotiorum. They are: Ss-Fox1, Ss-Fox E2Ss-Fox3 and Ss-Fkh1, respectively. By sequence alignment and phylogenetic analysis, the amino acid sequence characteristics and phylogenetic relationships between them are determined. The open reading frame of Ss-Fox E2 gene was cloned successfully, which encodes 449 amino acids and contains an intron. The expression of Ss-Fox E2 gene in the tissues of Sclerotinia sclerotiorum was detected by fluorescence quantitative PCR technique. The results showed that the relative expression of Ss-Fox E2 was significantly higher than that in hyphae and sclerotia tissues in the oocyst disc stage, and increased with the development and maturation of the sclerotia. The results showed that Ss-Fox E2 gene was related to the development of ovariectoma, and the expression of Ss-Fox E2 protein and green fluorescent protein (GFP) were fused, and the subcellular localization of Ss-Fox E2 was analyzed by transient expression technique of tobacco leaf epidermis cells. The results showed that the Ss-Fox E2 protein was located in the nucleus, the prokaryotic expression of Ss-Fox E2 was analyzed by p ET28a system in E. coli Rosettade3. The results showed that most of Ss-Fox E2 was expressed in the form of inclusion body, but a large number of supernatants were purified by nickel column. Finally, a certain amount of purified protein can be obtained, which can meet the needs of other experiments in the later stage. The full-length decoy vector p GBKT7::Ss-Fox E2 of Ss-Fox E2 was successfully constructed. The detection of the self-activation of decoy protein showed that the full-length Ss-Fox E2 protein alone could activate the expression of downstream reporter gene, that is, the transcription factor Ss-Fox E2 had transcriptional activation activity. In order to avoid the autoactivation of Ss-Fox E2 full-length protein, the Forkhead domain of Ss-Fox E2 protein was used as bait protein to screen the interacting proteins in the homogenized c DNA library of Sclerotinia sclerotiorum. Finally, six candidate proteins interacting with Ss-Fox E2 domain were obtained after two double sieves and one rotation test. These results provide a basis for the further identification of Ss-Fox E2 interaction protein and the further development of its function and regulatory network.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S432.44

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