南极红酵母SOD和GR基因的克隆表达及特征分析

发布时间：2018-03-06 01:20

  本文选题：南极红酵母AN5　切入点：Fe-SOD　出处：《哈尔滨工业大学》2017年硕士论文　论文类型：学位论文

【摘要】：人类活动对全球环境的影响越来越大,环境中重金属的污染日益严重。在南极地区,生物遭受高盐度、高辐射、低温、重金属等多种胁迫,导致活性氧(Reactive Oxygen Species,ROS)的积累。南极生物可以合成冷活性抗氧化酶,如超氧化物歧化酶(Superoxide Dismutases,SOD)、谷胱甘肽还原酶(Glutathione Reductase,GR)、过氧化物酶(Peroxidase,POD)和过氧化氢酶(Catalase,CAT)等,以清除活性氧ROS损伤,从而达到对重金属的积累或解毒。本文以南极红酵母AN5(Rhodotorula mucilaginosa AN5)为研究对象,对SOD和GR基因进行克隆、生物信息学分析、重组蛋白的诱导表达、纯化、蛋白特征及重金属适应性分析,初步探讨这2种基因(蛋白)在酵母逆境适应性中的作用。(1)将来自南极酵母R.mucilaginosa AN5的新型超氧化物歧化酶基因克隆,测序,然后在大肠杆菌中表达,分析目的蛋白特征。结果如下:R.mucilaginosa AN5 SOD(命名为Rm Fe SOD)基因开放阅读框(Open Reading Frame,ORF)全长为639 bp,编码212个氨基酸,预测分子量为23.52 kDa,理论等电点(isoelectric point,p I)为7.89。经凝胶过滤层析和SDS-PAGE分析,天然状态的Rm Fe SOD以同型二聚体形式存在。p H值1.0-9.0条件下,孵育1小时,重组Rm Fe SOD表现出良好的p H稳定性。50℃下孵育1小时,Rm FeSOD仍保持80%以上的活性,具有相对较高的热稳定性。通过1 mmol/L的金属离子处理,Zn2+、Cu2+、Mn2+和Fe3+能够促进酶活性,Mg2+抑制酶活性。蛋白Rm FeSOD对化合物PMSF、SDS、吐温-80、Triton X-100、DMSO、β-ME和尿素的耐受性相对较低,但对还原剂DTT表现出一定的耐受性。铜胁迫实验中,Rm Fe SOD重组大肠杆菌比非重组菌表现出更好的生长,这表明Rm Fe SOD可能在重金属的适应性中起重要作用。(2)将来自南极酵母R.mucilaginosa AN5的谷胱甘肽还原酶基因克隆,测序,然后在大肠杆菌中表达,分析目的蛋白特征。结果如下:R.mucilaginosa AN5 GR(命名为Rm GR)基因ORF全长为1500 bp,编码499个氨基酸,预测分子量为54.8 k Da,p I为6.07。重组Rm GR的最佳酶活p H和温度分别为p H 7.5和30℃。在p H值3.0-8.0条件下,孵育1小时,Rm GR表现出良好的p H稳定性。在40℃下孵育1小时,Rm GR仍保持60%以上的活性,具有相对较高的热稳定性。铜和镉胁迫实验中,Rm GR重组大肠杆菌比非重组菌表现出更好的生长,这表明Rm GR可能在重金属的适应性中起重要作用。
[Abstract]:The impact of human activities on the global environment is increasing, and the pollution of heavy metals in the environment is becoming more and more serious. In the Antarctic region, organisms suffer from various stresses, such as high salinity, high radiation, low temperature, heavy metals, etc. Antarctic organisms can synthesize cold-active antioxidant enzymes such as superoxide dismutase superoxide dismutase (SOD), glutathione reductase Glutathione reductase (Glutathione ReductaseGRN), peroxidase peroxidase (POD) and catalase catalase (catalase Cat) to remove the damage of ROS. In order to achieve the accumulation or detoxification of heavy metals, the genes of SOD and gr were cloned, bioinformatics analysis, induced expression and purification of recombinant protein from AN5(Rhodotorula mucilaginosa AN5). Protein characteristics and heavy metal adaptation analysis, the role of these two genes (proteins) in yeast stress adaptation was studied. The novel superoxide dismutase gene from Antarctic yeast R. mucilaginosa AN5 was cloned and sequenced. Then expressed in Escherichia coli and analyzed the characteristics of the target protein. The results were as follows: open Reading frame ORF of the gene Rm Fe SOD was 639 BP, encoding 212 amino acids, as follows: R. mucilaginosa AN5 SOD (named RM Fe SOD) gene open reading frame (Rm Fe SOD) was 639 BP in length, encoding 212 amino acids. The predicted molecular weight is 23.52 kDa, and the theoretical isoelectric point I) is 7.89. By gel filtration chromatography and SDS-PAGE analysis, the natural RmFe SOD is incubated in the form of homotypic dimer under the condition of 1.0-9.0 in the form of homotypic dimer, and incubated for 1 hour. The recombinant RM FeSOD showed good pH stability at 50 鈩，

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