Extraction, Identification and Gene Cloning of Mn-SOD from Pleurotus tuber-regium
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摘要: 以虎奶菇菌丝为材料,提取、鉴定了虎奶菇锰超氧化物歧化酶,克隆虎奶菇锰超氧化物歧化酶基因。用液氮研磨、超声、硫酸铵沉淀和透析等方法提取虎奶菇Mn-SOD;WST-8 法测定虎奶菇 Mn-SOD 酶活力;H2O2 鉴定虎奶菇 SOD 的类型;用同源克隆、cDNA 末端快速扩增技术和融合引物与巢式 PCR 等方法从虎奶菇中克隆锰超氧化物歧化酶基因。结果表明,虎奶菇 Mn-SOD 酶活力为 1.66 个酶活力单位。酶经过 H2O2处理后仍然有活性,与未处理无明显差异,表明虎奶菇中 SOD 主要是 Mn-SOD。克隆获得了虎奶菇锰超氧化物歧化酶基因PtMn-SOD,其DNA序列全长1025 bp,开放阅读框全长为663 bp,编码220个氨基酸。序列分析与系统进化树表明,PtMn-SOD与属于侧耳属的糙皮侧耳(登录号:MH645359.1)关系较近。预测该蛋白质分子量为 24.54 kDa,蛋白等电点为 7.86。PtMn-SOD 蛋白定位于线粒体,在线粒体中发挥功效。
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关键词:
- 虎奶菇 /
- 锰超氧化物歧化酶 /
- 酶活 /
- 同源克隆 /
- 融合引物与巢式 PCR
Abstract: The manganese superoxide dismutase of Pleurotus tuber-regium was extracted and identified. The manganese superoxide dismutase (Mn-SOD) gene of P. tuber-regium was cloned from the mycelium of P.tuber-regium. Mn-SOD of P.tuber-regium was extracted by liquid nitrogen grinding, ultrasound, ammonium sulfate precipitation and dialysis. Mn-SOD activity of P. tuber-regium was determined by WST-8 method. SOD type of P. tuber-regium was identified by H2O2. Mn-SOD gene was cloned from P. tuber-regium by homologous cloning, rapid amplification of cDNA ends, fusion primers and nested PCR. Results showed that the activity of Mn-SOD of P. tuber-regium was 1.66 U. There was no significant difference between the enzyme treated with H2O2 and untreated, indicating that the main SOD in P. tuber-regium was Mn-SOD. PtMn-SOD was cloned and obtained. Its DNA sequence was 1025 bp in total, 663 bp in open reading frame and 220 amino acids were encoded. Sequence analysis and phylogenetic tree showed that PtMn-SOD was closely related to Pleurotus ostreatus (accession number:MH645359.1). It was predicted that the molecular weight of the protein was 24.54 kDa and the isoelectric point of the protein was 7.86. PtMn-SOD protein located in mitochondria and played a role in mitochondria.
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