本文選題：藍(lán)藻 + peroxiredoxins��； 參考：《西北大學(xué)》2016年碩士論文

【摘要】：藍(lán)藻Synechococcus elongatus PCC7942生物鐘的核心振蕩器是由kaiA、kaiB和kaiC基因及其編碼產(chǎn)物構(gòu)成,這個(gè)核心振蕩器負(fù)責(zé)藍(lán)藻生物節(jié)律性時(shí)間信息的產(chǎn)生和校準(zhǔn)。而作為Kai生物鐘輸出途徑的SasA、LabA、CikA、RapA等關(guān)鍵分子則能夠通過(guò)被稱為轉(zhuǎn)錄-翻譯負(fù)反饋環(huán)路(transcriptional-translational feedback loop, TTFL)的方式,協(xié)同調(diào)控核心鐘節(jié)律性的維持,并承擔(dān)將生物鐘正確的相位信息向下游靶基因輸出的作用。過(guò)氧化還原蛋白(Peroxiredoxins, Prxs)是一種巰基特異性抗氧化蛋白,在藍(lán)藻細(xì)胞抗氧化防御方面發(fā)揮著重要的作用。藍(lán)藻Synechococcus elongatus PCC 7942的基因組共編碼6個(gè)過(guò)氧化還原蛋白家族成員,其中Prx-2Cys蛋白的氧化還原狀態(tài)周期性變化被證明是保守的非轉(zhuǎn)錄依賴生物節(jié)律性遺傳標(biāo)簽。本論文的主要工作從生理表型和分子機(jī)制兩方面探索了Prxs蛋白的抗氧化特性及其節(jié)律性表型。生理方面主要進(jìn)行了如下研究：逆境脅迫下prx-2cys基因在藍(lán)藻的抗氧化過(guò)程中的功能分析。實(shí)驗(yàn)通過(guò)比較不同逆境脅迫條件下(如：外源過(guò)氧化氫脅迫、UV-B紫外脅迫和高光脅迫)敲除系Δ2cys和野生型的生理表型和存活率,初步確定了prx-2cys基因在藍(lán)藻對(duì)抗內(nèi)源活性氧爆發(fā)過(guò)程中的貢獻(xiàn)。實(shí)驗(yàn)結(jié)果初步表明：敲除系藍(lán)藻的存活率和生長(zhǎng)速度均低于野生型藍(lán)藻株系,表明prx-2cys在藍(lán)藻抗氧化防御方面發(fā)揮著重要的作用。Prx-2Cys節(jié)律性標(biāo)簽分子機(jī)制探索方面主要進(jìn)行了如下研究：(1)藍(lán)藻Synechococcus elongatus PCC 7942總RNA提取方法的優(yōu)化。通過(guò)對(duì)藍(lán)藻總RNA提取過(guò)程中起始細(xì)胞數(shù)量、溶菌酶酶解時(shí)間、去除基因組DNA的方式、cDNA純化的方式等條件進(jìn)行優(yōu)化,找到一種適合藍(lán)藻RNA提取的方法。結(jié)果表明：當(dāng)使用用終濃度為3mg/mL的溶菌酶作用10min,起始細(xì)胞干重為1.1mg,采用試劑盒提取法獲得的RNA質(zhì)量最好。(2)應(yīng)用實(shí)時(shí)定量RT-PCR(qPCR)方法檢測(cè)生物鐘相關(guān)基因的表達(dá)模式。通過(guò)熒光實(shí)時(shí)定量檢測(cè)不同光照強(qiáng)度下生物鐘相關(guān)基因的表達(dá)模式,結(jié)果表明：在正常光照條件下,敲除prx-2cys基因不影響核心鐘基因的生物鐘節(jié)律性表型,但是影響了輸出途徑相關(guān)基因的表達(dá)模式。高光條件下,當(dāng)以野生型為模板時(shí),核心鐘基因的生物節(jié)律性表型喪失,但是敲除系的節(jié)律性表型出現(xiàn)“恢復(fù)”現(xiàn)象。無(wú)論是野生型還是敲除系,高光脅迫都影響了輸出途徑相關(guān)基因的表達(dá)模式。(3)雙敲除系載體的構(gòu)建。首先,將卡那霉素抗性表達(dá)框插入至prx-QA1、prx-QA2、 prx-QA3、prx-QB等prx家族成員基因的編碼序列中建立對(duì)應(yīng)的單基因敲除載體,并分別以上述載體轉(zhuǎn)化prx-2cys單基因敲除系藍(lán)藻△2cys(氯霉素抗性),進(jìn)而構(gòu)建雙敲除系藻株△~2A_1, △~2A_2, △~2A_3, △~2Q_B,為后續(xù)的研究提供材料。
[Abstract]:The core oscillator of the cyanobacteria Synechococcus elongatus PCC7942 clock is composed of Kai Agna kaiB and kaiC genes and their encoding products. The core oscillator is responsible for the generation and calibration of the biological rhythmic time information of cyanobacteria. As the output pathway of Kai clock, key molecules such as SasAn LabAN CikAKai RapA and so on can coordinate the maintenance of core clock rhythm by means of transcriptional-translational feedback loop, TTFL), called transcriptional-translational feedback loop, TTFL), which is called transcription-translation negative feedback loop, which is called transcription-translation negative feedback loop. And assume the role of biological clock phase information to the downstream target gene output role. Peroxiredoxins (Prxss) is a sulfhydryl specific antioxidant protein, which plays an important role in anti-oxidation defense of cyanobacteria. The genome of cyanobacteria Synechococcus elongatus PCC 7942 encodes six members of the family of reductase proteins. The periodic changes in the redox state of Prx-2Cys protein have been proved to be conserved non-transcription-dependent biological rhythmic genetic tags. The main work of this thesis is to explore the antioxidant characteristics and rhythmic phenotypes of Prxs protein from physiological phenotypes and molecular mechanisms. Physiological studies were carried out as follows: the function of prx-2cys gene in the antioxidant process of cyanobacteria was analyzed under stress. The physiological phenotypes and survival rates of the knockout lines 螖 2cys and wild type under different stress conditions (such as exogenous hydrogen peroxide stress UV-B UV stress and high light stress) were compared. The contribution of prx-2cys gene to the resistance of cyanobacteria to endogenous reactive oxygen species was preliminarily confirmed. The results showed that the survival rate and growth rate of knockout cyanobacteria were lower than that of wild cyanobacteria. The results showed that prx-2cys played an important role in anti-oxidative defense of cyanobacteria. Prx-2Cys rhythmic labeling molecular mechanism was studied as follows: 1) the extraction method of total RNA from cyanobacteria Synechococcus elongatus PCC 7942 was optimized. By optimizing the initial cell number, lysozyme enzymatic hydrolysis time and the way of purification of genomic DNA during the total RNA extraction of cyanobacteria, a suitable method for extracting RNA from cyanobacteria was found. The results showed that when the lysozyme with final concentration of 3mg/mL was used for 10 min and the dry weight of the initial cell was 1.1 mg, the best quality of RNA was obtained by using kit extraction method. The expression pattern of clock related genes was detected by real-time quantitative RT-PCRnqPCR. The expression patterns of clock related genes in different light intensity were detected by real-time fluorescence quantitative analysis. The results showed that knockout of prx-2cys gene did not affect the circadian rhythm of the core clock gene under normal illumination. But it affects the expression pattern of genes related to the output pathway. Under high light conditions, the biological rhythmic phenotype of the core clock gene was lost when the wild type was used as the template, but the rhythmic phenotype of the knockout line was "restored". Both wild type and knockout line, high light stress affected the construction of double knockout line vector. Firstly, the kanamycin resistant expression frame was inserted into the coding sequence of prx family members such as prx-QA1, prx-QA2, prx-QA3, prx-QB, and the corresponding single-gene knockout vector was established. The prx-2cys single-gene knockout line 2cys2 (chloramphenicol resistant strain) was transformed into the above vectors, and then the double knockout strain, 2A1, 2A2, 2A3, 2QBwere constructed, which provided materials for further research.
【學(xué)位授予單位】：西北大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：Q949.2

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