虎奶菇Mn-SOD提取、鉴定及基因的克隆  

Extraction,Identification and Gene Cloning of Mn-SOD from Pleurotus tuber-regium

作　　者：杨加亮 田云恒 马爱民[1] YANG Jia-liang;TIAN Yun-heng;MA Ai-min(College of Food Science and Technology,Huazliong Agricultural University,Wuhan 430070,China)

机构地区：[1]华中农业大学食品科学技术学院,湖北武汉430070

出　　处：《食品工业科技》2020年第15期150-157,共8页Science and Technology of Food Industry

基　　金：国家自然科学基金项目(31772375)。

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。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 H2 O2.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 H2 O2 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.

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 

分 类 号：TS201.3[轻工技术与工程—食品科学]