蓝藻prx基因抗氧化功能及其与Kai生物种关联关系研究

发布时间：2018-05-30 17:42

本文选题：蓝藻 + peroxiredoxins　；参考：《西北大学》2016年硕士论文

【摘要】：蓝藻Synechococcus elongatus PCC7942生物钟的核心振荡器是由kaiA、kaiB和kaiC基因及其编码产物构成,这个核心振荡器负责蓝藻生物节律性时间信息的产生和校准。而作为Kai生物钟输出途径的SasA、LabA、CikA、RapA等关键分子则能够通过被称为转录-翻译负反馈环路(transcriptional-translational feedback loop, TTFL)的方式,协同调控核心钟节律性的维持,并承担将生物钟正确的相位信息向下游靶基因输出的作用。过氧化还原蛋白(Peroxiredoxins, Prxs)是一种巯基特异性抗氧化蛋白,在蓝藻细胞抗氧化防御方面发挥着重要的作用。蓝藻Synechococcus elongatus PCC 7942的基因组共编码6个过氧化还原蛋白家族成员,其中Prx-2Cys蛋白的氧化还原状态周期性变化被证明是保守的非转录依赖生物节律性遗传标签。本论文的主要工作从生理表型和分子机制两方面探索了Prxs蛋白的抗氧化特性及其节律性表型。生理方面主要进行了如下研究：逆境胁迫下prx-2cys基因在蓝藻的抗氧化过程中的功能分析。实验通过比较不同逆境胁迫条件下(如：外源过氧化氢胁迫、UV-B紫外胁迫和高光胁迫)敲除系Δ2cys和野生型的生理表型和存活率,初步确定了prx-2cys基因在蓝藻对抗内源活性氧爆发过程中的贡献。实验结果初步表明：敲除系蓝藻的存活率和生长速度均低于野生型蓝藻株系,表明prx-2cys在蓝藻抗氧化防御方面发挥着重要的作用。Prx-2Cys节律性标签分子机制探索方面主要进行了如下研究：(1)蓝藻Synechococcus elongatus PCC 7942总RNA提取方法的优化。通过对蓝藻总RNA提取过程中起始细胞数量、溶菌酶酶解时间、去除基因组DNA的方式、cDNA纯化的方式等条件进行优化,找到一种适合蓝藻RNA提取的方法。结果表明：当使用用终浓度为3mg/mL的溶菌酶作用10min,起始细胞干重为1.1mg,采用试剂盒提取法获得的RNA质量最好。(2)应用实时定量RT-PCR(qPCR)方法检测生物钟相关基因的表达模式。通过荧光实时定量检测不同光照强度下生物钟相关基因的表达模式,结果表明：在正常光照条件下,敲除prx-2cys基因不影响核心钟基因的生物钟节律性表型,但是影响了输出途径相关基因的表达模式。高光条件下,当以野生型为模板时,核心钟基因的生物节律性表型丧失,但是敲除系的节律性表型出现“恢复”现象。无论是野生型还是敲除系,高光胁迫都影响了输出途径相关基因的表达模式。(3)双敲除系载体的构建。首先,将卡那霉素抗性表达框插入至prx-QA1、prx-QA2、 prx-QA3、prx-QB等prx家族成员基因的编码序列中建立对应的单基因敲除载体,并分别以上述载体转化prx-2cys单基因敲除系蓝藻△2cys(氯霉素抗性),进而构建双敲除系藻株△~2A_1, △~2A_2, △~2A_3, △~2Q_B,为后续的研究提供材料。
[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.
【学位授予单位】：西北大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q949.2

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