本文選題：核盤菌 + Forkhead　； 參考：《吉林大學(xué)》2016年碩士論文

【摘要】：核盤菌是一種重要的絲狀植物病原真菌,因其寄主范圍廣,生活史復(fù)雜,防治困難,每年都會給農(nóng)業(yè)生產(chǎn)帶來重大的經(jīng)濟損失,菌核病的防治問題已經(jīng)受到了世界各地科學(xué)工作者的密切關(guān)注。核盤菌生活史包含了無性和有性兩個發(fā)育階段,無性階段組織主要為菌絲以及由菌絲聚集形成的特殊結(jié)構(gòu)——菌核組成。而有性發(fā)育階段的起點在于菌核萌發(fā)形成子囊盤。在有性發(fā)育階段由子囊盤產(chǎn)生的子囊孢子數(shù)量驚人,遠距離傳播能力強,因此子囊盤產(chǎn)生的子囊孢子作為侵染原在病害流行等方面扮演了重要角色。本實驗室前期工作發(fā)現(xiàn),對核盤菌其中一個屬于Forkhead轉(zhuǎn)錄因子家族基因Ss-Fox E2進行敲除后,突變體無法正常形成子囊盤,由此我們認為Ss-Fox E2對于核盤菌子囊盤的發(fā)育十分重要。因此本研究在此基礎(chǔ)之上,圍繞基因Ss-Fox E2的表達特征以及與其互做蛋白的篩選等方面展開工作,以期待明確轉(zhuǎn)錄因子Ss-Fox E2在子囊盤發(fā)育過程中所起到的作用,主要研究結(jié)果如下:通過對核盤菌基因組數(shù)據(jù)庫進行搜索以及對候選目標(biāo)的結(jié)構(gòu)域進行預(yù)測分析,找到核盤菌中一共存在4個Forkhead家族轉(zhuǎn)錄因子,它們分別為:Ss-Fox1、Ss-Fox E2、Ss-Fox3和Ss-Fkh1,并且通過序列比對和系統(tǒng)進化分析,明確了它們所具有的氨基酸序列特征以及系統(tǒng)進化關(guān)系;利用反轉(zhuǎn)錄PCR(RT-PCR)方法,成功克隆得到Ss-Fox E2基因的開放閱讀框(ORF),該基因編碼了449個氨基酸,含有一個內(nèi)含子;利用熒光定量PCR技術(shù)(q RT-PCR)對核盤菌各發(fā)育階段組織中Ss-Fox E2基因的表達含量進行了檢測,結(jié)果顯示Ss-Fox E2的相對表達含量在子囊盤時期組織中明顯高于菌絲期和菌核各時期組織,并且隨子囊盤的發(fā)育成熟,其含量不斷升高。該結(jié)果證明Ss-Fox E2基因與子囊盤的發(fā)育具有相關(guān)性;將Ss-Fox E2蛋白與綠色熒光蛋白(e GFP)融合表達,利用煙草葉片表皮細胞瞬時表達技術(shù)對Ss-Fox E2進行了亞細胞定位分析,結(jié)果顯示Ss-Fox E2蛋白定位在細胞核當(dāng)中;利用p ET28a系統(tǒng)在大腸桿菌Rosetta(DE3)中對Ss-Fox E2進行原核表達分析,結(jié)果顯示Ss-Fox E2大部分以包涵體形式表達,但通過鎳柱純化大量上清,最終可以得到一定量純化蛋白,能夠滿足后期其他實驗需要;成功構(gòu)建基因Ss-Fox E2全長誘餌載體p GBKT7::Ss-Fox E2,對誘餌蛋白自激活性檢測發(fā)現(xiàn)全長Ss-Fox E2蛋白獨自能夠激活下游報告基因的表達,具有自激活性即轉(zhuǎn)錄因子Ss-Fox E2具有轉(zhuǎn)錄激活活性;為了避免Ss-Fox E2全長蛋白的自激活性,我們利用無自激活性的Ss-Fox E2蛋白Forkhead結(jié)構(gòu)域部分作為誘餌蛋白在核盤菌均一化c DNA文庫中進行其互作蛋白的篩選,最終經(jīng)過兩次復(fù)篩以及一次回轉(zhuǎn)驗證,我們得到6個與Ss-Fox E2結(jié)構(gòu)域部分互作的候選蛋白。以上各研究結(jié)論為下一步Ss-Fox E2互作蛋白的確認及更深入開展功能、調(diào)控網(wǎng)絡(luò)研究提供了基礎(chǔ)。
[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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S432.44

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