本文選題：南極紅酵母AN5　切入點：Fe-SOD　出處：《哈爾濱工業(yè)大學》2017年碩士論文　論文類型：學位論文

【摘要】：人類活動對全球環(huán)境的影響越來越大,環(huán)境中重金屬的污染日益嚴重。在南極地區(qū),生物遭受高鹽度、高輻射、低溫、重金屬等多種脅迫,導致活性氧(Reactive Oxygen Species,ROS)的積累。南極生物可以合成冷活性抗氧化酶,如超氧化物歧化酶(Superoxide Dismutases,SOD)、谷胱甘肽還原酶(Glutathione Reductase,GR)、過氧化物酶(Peroxidase,POD)和過氧化氫酶(Catalase,CAT)等,以清除活性氧ROS損傷,從而達到對重金屬的積累或解毒。本文以南極紅酵母AN5(Rhodotorula mucilaginosa AN5)為研究對象,對SOD和GR基因進行克隆、生物信息學分析、重組蛋白的誘導表達、純化、蛋白特征及重金屬適應性分析,初步探討這2種基因(蛋白)在酵母逆境適應性中的作用。(1)將來自南極酵母R.mucilaginosa AN5的新型超氧化物歧化酶基因克隆,測序,然后在大腸桿菌中表達,分析目的蛋白特征。結果如下:R.mucilaginosa AN5 SOD(命名為Rm Fe SOD)基因開放閱讀框(Open Reading Frame,ORF)全長為639 bp,編碼212個氨基酸,預測分子量為23.52 kDa,理論等電點(isoelectric point,p I)為7.89。經凝膠過濾層析和SDS-PAGE分析,天然狀態(tài)的Rm Fe SOD以同型二聚體形式存在。p H值1.0-9.0條件下,孵育1小時,重組Rm Fe SOD表現出良好的p H穩(wěn)定性。50℃下孵育1小時,Rm FeSOD仍保持80%以上的活性,具有相對較高的熱穩(wěn)定性。通過1 mmol/L的金屬離子處理,Zn2+、Cu2+、Mn2+和Fe3+能夠促進酶活性,Mg2+抑制酶活性。蛋白Rm FeSOD對化合物PMSF、SDS、吐溫-80、Triton X-100、DMSO、β-ME和尿素的耐受性相對較低,但對還原劑DTT表現出一定的耐受性。銅脅迫實驗中,Rm Fe SOD重組大腸桿菌比非重組菌表現出更好的生長,這表明Rm Fe SOD可能在重金屬的適應性中起重要作用。(2)將來自南極酵母R.mucilaginosa AN5的谷胱甘肽還原酶基因克隆,測序,然后在大腸桿菌中表達,分析目的蛋白特征。結果如下:R.mucilaginosa AN5 GR(命名為Rm GR)基因ORF全長為1500 bp,編碼499個氨基酸,預測分子量為54.8 k Da,p I為6.07。重組Rm GR的最佳酶活p H和溫度分別為p H 7.5和30℃。在p H值3.0-8.0條件下,孵育1小時,Rm GR表現出良好的p H穩(wěn)定性。在40℃下孵育1小時,Rm GR仍保持60%以上的活性,具有相對較高的熱穩(wěn)定性。銅和鎘脅迫實驗中,Rm GR重組大腸桿菌比非重組菌表現出更好的生長,這表明Rm GR可能在重金屬的適應性中起重要作用。
[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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