两种紫茉莉小热休克蛋白基因的克隆及其融合基因在酿酒酵母中的遗传转化

发布时间：2019-02-21 09:56

【摘要】：伴随着经济的快速的发展,我国的石油开采量日益增加,随之而来的环境污染问题日渐显著。近年来,植物修复技术被越来越多的应用在修复石油污染土壤等领域,但关于植物是如何耐受石油等多环芳烃类污染物的具体机理仍不明确,本研究以抗逆植物紫茉莉为供试材料,根据前期差异蛋白质组实验得到的的石油胁迫下差异蛋白的表达谱,发现石油污染组中两种小热休克蛋白(small heat shock protein, sHsp)Hsp18.2与Hsp17.7与清洁对照组相比表达量显著上调,并通过RACE(Rapid-amplification of cDNA ends)法获得这两种蛋白的编码框序列,利用融合PCR及2A肽技术获得共表达融合序列,并成功将上述3种ORF转入酿酒酵母(Saccharomyces cerevisiae),以期研究其表达变化对酿酒酵母细胞抗石油胁迫能力的影响,并借此推测石油污染条件下紫茉莉较强耐受能力的机理,同时也为今后快速筛选石油污染修复植株提供分子理论依据。本研究结果如下所示：1、通过RACE技术获得紫茉莉两种小热休克蛋白Hsp18.2与Hsp17.7的完整开放阅读框,分别长为为483 bp和474 bp,编码161和158个氨基酸,分子量分别约为18.2 kDa与17.7 kDa,均含有保守的ACD结构域(a-crystallin)。2、依据2A肽原理及融合PCR法获得了HSP18.2与HSP17.7的胞内共表达序列,片段长度1091 bp,融合片段可以通过一个完整的开放阅读框同时表达HSP18.2-Flag与HSP17.7-HA。3、成功构建出三种含有BamHI和Xbal双酶切位点的酿酒酵母胞内表达载体pYES2-HSP18.2-Flag、pYES2-HSP17.7-HA及pYES2-HSP-fusion,且均经过双酶切鉴定及测序检测。4、成功将上述三种酿酒酵母胞内表达载体转入酵母菌株Saccharomyces cerevisiae,并通过SC-U筛选培养基获得阳性克隆,为进一步对紫茉莉小热休克蛋白HSP18.2与HSP17.7的功能机制的研究提供了实验材料。
[Abstract]:With the rapid development of economy, the oil exploitation in China is increasing day by day, and the environmental pollution problem is becoming more and more obvious. In recent years, phytoremediation technology has been used more and more in the field of remediation of petroleum contaminated soil. However, the mechanism of how plants tolerate polycyclic aromatic hydrocarbons (PAHs) such as petroleum is still unclear. In this study, the stress resistant plant Mirabilis was used as the experimental material. According to the differentially expressed protein profiles obtained from the preliminary differential proteome experiment, two small heat shock proteins (small heat shock protein,) were found in the oil-contaminated group. SHsp) Hsp18.2 was significantly up-regulated compared with Hsp17.7 compared with the clean control group. The coding frame sequences of the two proteins were obtained by RACE (Rapid-amplification of cDNA ends) method) and coexpressed fusion sequences were obtained by fusion PCR and 2A peptide techniques. The three kinds of ORF were successfully transferred into Saccharomyces cerevisiae (Saccharomyces cerevisiae), in order to study the effect of their expression changes on the resistance to oil stress of Saccharomyces cerevisiae cells. At the same time, it also provides molecular theory basis for rapid screening of oil contaminated remediation plants in the future. The results of this study are as follows: 1. The complete open reading frames of two small heat shock protein (Hsp18.2) and Hsp17.7 were obtained by RACE technique. The length of Hsp18.2 and Hsp17.7 were 483 bp and 474 bp, encoding 161 and 158 amino acids, respectively. The molecular weight of HSP18.2 and HSP17.7 were 18.2 kDa and 17.7 kDa, respectively, containing conserved ACD domain (a-crystallin). 2. According to 2A peptide principle and fusion PCR method, the intracellular coexpression sequences of HSP18.2 and HSP17.7 were obtained, and the length of the fragments was 1091 bp,. The fusion fragment can simultaneously express HSP18.2-Flag and HSP17.7-HA.3, through a complete open reading frame, and successfully construct three intracellular expression vectors pYES2-HSP18.2-Flag, containing double restriction sites of BamHI and Xbal in Saccharomyces cerevisiae. Both pYES2-HSP17.7-HA and pYES2-HSP-fusion, were identified by double enzyme digestion and sequenced. 4. The above three expression vectors were successfully transferred into yeast strain Saccharomyces cerevisiae, and positive clones were obtained by SC-U screening medium. The experimental materials were provided for further study on the functional mechanism of small heat shock protein (HSP18.2) and small heat shock protein (HSP17.7) in Mirabilis.
【学位授予单位】：沈阳农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q943.2

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