猴头菌锰过氧化物酶1基因在构巢曲霉的异源转化与表达

为提高猴头菌菌株CB1锰过氧化物酶（MnP）基因的表达产量,采用PEG/CaCl2介导的原生质体转化方法,将携带有He-mnp1的重组质粒pLB01/He-mnp1转入到构巢曲霉尿嘧啶尿苷营养缺陷菌株TN02A7的原生质体中,获得了转化子菌株TN02A7-He-mnp1,并在乙醇脱氢酶启动子alcA（p）控制下实现了异源表达。将TN02A7-He-mnp1、TN02A7、构巢曲霉野生型菌株WJA01、猴头菌菌株CB1在相同的木质素环境下进行培养并检测MnP酶活性,结果表明：转化子菌株TN02A7-He-mnp1在0.05 g.L-1血红素的情况下、诱导96 h后酶活性最高为38.31 U.L-...

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Bibliographic Details
Published in林业科学研究 Vol. 26; no. 4; pp. 480 - 487
Main Author 尹立伟 池玉杰
Format Journal Article
LanguageChinese
Published 东北林业大学林学院,黑龙江哈尔滨150040 2013
安庆师范学院生命科学学院,安徽安庆246011%东北林业大学林学院,黑龙江哈尔滨,150040
Subjects
构巢曲霉
猴头菌
表达
转化
锰过氧化物酶
锰过氧化物酶
expression
Hericium erinaceum
Aspergillus nidulans
猴头菌
表达
转化
transformation
构巢曲霉
manganese peroxidase
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ISSN1001-1498

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Summary:为提高猴头菌菌株CB1锰过氧化物酶（MnP）基因的表达产量,采用PEG/CaCl2介导的原生质体转化方法,将携带有He-mnp1的重组质粒pLB01/He-mnp1转入到构巢曲霉尿嘧啶尿苷营养缺陷菌株TN02A7的原生质体中,获得了转化子菌株TN02A7-He-mnp1,并在乙醇脱氢酶启动子alcA（p）控制下实现了异源表达。将TN02A7-He-mnp1、TN02A7、构巢曲霉野生型菌株WJA01、猴头菌菌株CB1在相同的木质素环境下进行培养并检测MnP酶活性,结果表明：转化子菌株TN02A7-He-mnp1在0.05 g.L-1血红素的情况下、诱导96 h后酶活性最高为38.31 U.L-1,比不添加血红素的酶活力高8.64倍,但比猴头菌菌株CB1的酶活力低,而TN02A7与WJA01始终无MnP酶活性,说明基因He-mnp1已经成功地被转化到TN02A7-He-mnp1中,并在木质素环境下得到表达,血红素是重组MnP基因异源表达的限制性因素之一。本文为生产MnP和提高MnP产量提供了新的途径。
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The recombinant plasmid pLB01 / He-mnp1 which contains a gene encoding for manganese peroxidase（He-mnp1） from Hericium erinaceum CB1 was transformated into protoplasts of auxotrophic stain TN02A7 of Aspergillus nidulans by means of protoplast transformation method mediated by PEG / CaCl2so as to enhance MnP production.A transformant stain TN02A7-He-mnp1 of A.nidulans was gained,the gene He-mnp1 was expressed under the control of alcohol dehydrogenase alcA（p） promoter.The transformant stain TN02A7-He-mnp1,auxotrophic stain TN02A7,wild stain of A.nidulans WJA01,and H.erinaceum CB1 were cultured under the same lignin condition and detected the MnP activity.The results showed that TN02A7-He-mnp1 could produce MnP activity in the absence and presence of heme,but the MnP activity was up to 38.31 U.L-1 on 96h with 0.05 g.L-1 heme which was 8.64 times higher than that without heme but less than that of H.erinaceum CB1,whereas TN02A7 and WJA01 could not produce MnP activity at any time,indicating that the gen
ISSN:1001-1498