导　　师： 薛乐勋;关方霞

学科专业： 071009

授予学位： 硕士

作　　者： ;

机构地区： 郑州大学

摘　　要： 背景 过氧化物酶（Peroxiredoxins，Prdxs）是一类在生物体内广泛存在并且具有较高表达量的抗氧化蛋白，可以催化还原生物体内的H2O2、氢过氧化物以及过亚硝酸盐，避免细胞受到损伤。过氧化物酶分子量在22~27kDa之间，目前已知在哺乳动物中存在六种亚型（Prdx1~Prdx6），根据过氧化物酶活性位点上半胱氨酸残基的位置和数目不同，可以将其分为三类：1、典型2-Cys Prdxs；2、非典型2-Cys Prdxs；3、1-Cys Prdxs。Prdx1属于典型的2-Cys Prdxs，是Prdxs家族中分布最为广泛的成员。该蛋白除了具有抗氧化的作用外，还具有多种其他功能，参与多种生理过程包括细胞生长、细胞增殖、细胞分化及细胞凋亡等。此外，近年来有研究表明Prdx1在多种肿瘤中异常高表达，参与肿瘤发生发展过程中的信号调节。因此，Prdx1作为多种信号通路中的调节分子，在细胞信号通路中发挥的作用日益受到关注，成为研究的热点。 目的 探究杜氏盐藻Prdx1（Dunaliella salina Peroxiredoxins1，DsPrdx1）在细胞内的分子作用机制，筛选杜氏盐藻中能够与Prdx1相互作用的蛋白，并通过回交实验和体外实验对两者的相互作用进行验证。 方法 1、利用酵母双杂交的方法筛选杜氏盐藻Prdx1的互作蛋白 设计上下游引物，利用PCR扩增杜氏盐藻Prdx1的开放阅读框，酶切鉴定和测序鉴定正确后，将扩增的杜氏盐藻Prdx1的开放阅读框与酵母双杂交诱饵表达载体pGBKT7进行连接，构建酵母双杂交诱饵质粒pGBKT7-Prdx1。用PEG//LiAc法将所构建的诱饵质粒pGBKT7-Prdx1分别转入酵母菌Y187与AH109中，进行诱饵质粒的自激活和毒性检测，鉴定诱饵质粒pGBKT7-Prdx1是否适用于酵母双杂交系统。 以pGBKT7-Prdx1为诱饵，从杜氏盐藻cDNA表达文库中筛选与杜氏盐藻Prdx1相互作用的蛋白。经过营养缺陷型培养基和α-半乳糖苷酶的多次筛选，获得阳性克隆。 2、相互作用的验证 回交实验：提取酵母双杂交阳性克隆的猎物质粒，利用PEG//LiAC的方法与诱饵质粒共转化到酵母细胞AH109中，通过营养缺陷型和α-半乳糖苷酶的筛选作用，进行验证。 体外实验：提取杜氏盐藻总RNA，将提取的杜氏盐藻总RNA与已纯化的杜氏盐藻Prdx1蛋白混合，冰上孵育15min进行凝胶阻滞实验以及Prdx1保护实验，对两者的相互作用进行验证。 结果 1、利用酵母双杂交的方法筛选杜氏盐藻Prdx1的互作蛋白 获得正确的杜氏盐藻Prdx1开放阅读框并成功构建酵母双杂交诱饵质粒pGBKT7-Prdx1。随后将诱饵质粒pGBKT7-Prdx1转化至酵母菌株Y187和AH109中，经过酵母双杂交诱饵质粒自激活检测和毒性实验检测，所构建的诱饵质粒pGBKT7-Prdx1对两株酵母菌AH109和Y187没有自激活作用，也没有毒性，可以用于酵母双杂交系统。 利用酵母双杂交的方法，以pGBKT7-Prdx1为诱饵质粒从杜氏盐藻cDNA文库中筛选相互作用蛋白，经过筛选得到168个阳性克隆，其中包括杜氏盐藻28sRNA。Prdx1作为一种过氧化物酶，它的功能除了清除生物体内过量的过氧化物，保护细胞免受损伤外，还可以间接或者直接的作为信号分子，参与多种与氧化有关的生理过程，但是目前对于Prdx1是否在RNA的代谢过程中起作用的研究非常少。由于28s RNA不容易分离与纯化，因此本实验选择了总RNA作为后续实验的研究对象。 2、相互作用的验证 回交实验：提取酵母双杂交猎物质粒pGADT7-RNA并将其与酵母诱饵质粒pGBKT7-Prdx1共转入酵母菌AH109中，转化后的酵母菌在营养缺陷型培养基上生长良好，并在X-α-gal的筛选下呈蓝色，说明杜氏盐藻Prdx1与RNA在酵母菌株中存在相互作用。 体外实验：构建原核表达载体pET28/(a/)+-Prdx1，表达杜氏盐藻Prdx1蛋白并进行纯化。提取杜氏盐藻总RNA，将杜氏盐藻总RNA与已纯化的杜氏盐藻Prdx1蛋白混合孵育，进行凝胶阻滞实验与Prdx1保护实验。最后，两个实验均证明杜氏盐藻Prdx1与杜氏盐藻RNA存在相互作用。 结论 以杜氏盐藻Prdx1为诱饵蛋白筛选杜氏盐藻cDNA酵母文库，发现杜氏盐藻Prdx1蛋白与RNA之间存在相互作用，经过后续的回交实验和体外实验，进一步验证了两者之间的相互作用。除了两者之间具有相互作用的关系，通过Prdx1保护试验证明Prdx1对RNA具有与较强的保护作用，提示Prdx1可能参与了RNA的代谢过程并对RNA起保护作用。 BackgroundPeroxiredoxins （Prdxs） are antioxidant proteins that are distributed ubiquitously inhigh level, they can protect cells from damage by catalyzing peroxide reduction ofH2O2, peroxynitrite and hydroperoxides. The molecular weight of Prdxs is between22kDa and27kD. Prdxs possess six mammalian isoforms which can classified intothree subgroups based on the number and position of conserved Cys residues:1.Typical2-Cys Prdxs;2. Atypical2-Cys Prdxs;3.1-Cys Prdxs. Peroxiredoxin1/(Prdx1/) is the most extensive distributed member of the Prdxs family and belongs tothe typical2-Cys Prdxs. Prdx1is known to be involved in both antioxidant and avariety of biological processes including cell growth, cell proliferation, celldifferentiation and cell apoptosis. Moreover, there are also some evidences showedthat the expression of Prdx1is aberrant increased in many kinds of tumors and Prdx1modulates cell signaling of the development of tumors. Therefore, as the molecularmediator, the function of Prdx1has received attention. Objective To explore the molecular mechanism of Prdx1in Dunaliella salina, this studyscreened the proteins that could interact with Prdx1in Dunaliella salina and verifiedthis interaction through cotransformation experiment and in-vitro experiments. Methods 1. Screening proteins that can interact with Prdx1in Dunaliella salina by yeast two-hybrid system. The open reading frame /(ORF/) of Prdx1gene was obtained by PCR and then wasinserted into pGBKT7vector after being identified by DNA sequencing. Therecombinant plasmid pGBKT7-Prdx1was transformed into yeast strains Y187andAH109respectively by using PEG//LiAc method. Then the transcriptional activationand toxicity of the bait plasmid pGBKT7-Prdx1were tested to identify if the baitprotein was suitable to be used in the yeast two-hybrid system.The vector pGBKT7-Prdx1was used as the bait to screen the proteins that caninteract with Prdx1from the cDNA library of Dunaliella salina. The positivecolonies were obtained after being screened by auxotroph culture andα-galactosidase activity. 2. Identification of the interaction Cotransformation experiment: The prey plasmid of the positive colony was extractedand then was cotransformed into the yeast strain AH109with the bait plasmid. Theinteraction was identified by auxotroph culture and α-galactosidase activity.In-vitro experiments: The total RNA of Dunaliella salina were extracted andincubated with Prdx1to test the interaction by agarose gel shift assay and protectionof Prdx1-nucleic acid complexes. Results 1. Screening proteins that can interact with Prdx1in Dunaliella salina by Yeasttwo-hybrid system. The ORF of Prdx1was obtained and the bait plasmid pGBKT7-Prdx1wasconstructed successfully. The fusion plasmid pGBKT7-Prdx1was transformed intoyeast strains Y187and AH109respectively. After being tested by the transcriptionalactivation and toxicity assay, the bait protein was both inactive and nontoxic.Therefore, the constructed plasmid can be used in the yeast two-hybrid system.The yeast two-hybrid system was used to screen the interacting proteins from thecDNA library of Dunaliella salina. After screening,168positive colonies wereobtained including Dunaliella salina28s RNA. As a member of Peroxiredoxins, thefunctions of Prdx1involve in both eliminating peroxide and regulating multiplebiological processes as the molecular mediator. However, studies about if Prdx1 involve in the RNA metabolism. Because it is very difficult to isolate28s RNA, thusRNA was selected as the research object. 2. Identification of the interactionCotransformation experiment: Extracting prey plasmid pGADT7-RNA andcotransforming the prey plasmid into the yeast strain AH109with the bait plasmidpGBKT7-Prdx1. The cotransformation can survive on the auxotrophic base andexpress the blue phenotype, the results showed that Prdx1can interact with RNA inthe yeast strains. In-vitro experiment: The vector pET28/(a/)+-Prdx1was constructed and transformedinto E.coli BL21to express Prdx1protein. Then the protein was purified by Ni-IDASefinose Kit. The total RNA of Dunaliella salina were extracted and incubated withpurified Prdx1protein to conduct agarose gel shift experiment and protection ofPrdx1-nucleic acid complexes. The test also identified the interaction between Prdx1and RNA. Conclusion Prdx1of Dunaliella salina was used as the bait protein to screen the cDNA library ofDunaliella salina. After screening, the cotransformation experiment and the in-vitroexperiment were used to identify the interaction. Protection of Prdx1-nucleic acidcomplexes suggested that Prdx1can protect RNA in RNA metabolism.

关 键 词： 杜氏盐藻 过氧化物酶 酵母双杂交 蛋白纯化 蛋白相互作用

分 类 号： [Q946]

领　　域： [生物学]