一色齿毛菌锰过氧化物酶（MnP）活性检测与MnP1基因的克隆

doi:10.11707/j.1001-7488.20140315

摘要/Abstract

摘要：

锰过氧化物酶（MnP）广泛存在于白腐菌中，是降解木质素的主要酶系。首先对采自长白山的一色齿毛菌菌株CB1降解木质素的过氧化物酶进行检测，结果表明一色齿毛菌可产生MnP，但不产生木质素过氧化物酶（LiP）；Mn²⁺不是其产生MnP的必要因子，在LNAS培养基中加入木屑为底物的条件下，一色齿毛菌MnP最大酶活为18.4 U·L^-1。在酶活检测的基础上，为了深入研究MnP降解木质素的功能，根据已知的MnP基因保守区序列设计引物，以一色齿毛菌菌株CB1的基因组DNA为模板，采用染色体步移技术，克隆到该菌株一个编码MnP1蛋白的全长DNA基因（GenBank登录号JQ782578.1），命名为 Cu-mnp1 。该基因DNA全长4 268 bp，含有16个外显子和15个内含子；其启动子区域含有TATA-Box，CAAT-Box，SP1，AP1，HSE，XRE等多个顺式作用元件；其cDNA基因（GenBank登录号JQ782579.1）编码区长度为1 092 bp，编码363个氨基酸。BLAST比对分析表明，在cDNA水平上Cu-mnp1与猴头菇菌株CB1的mnp2基因的相似性最高为70%。蛋白结构分析表明，Cu-MnP1含有4个二硫键属于短MnP。

关键词: 一色齿毛菌（单色下皮黑）, 木质素降解酶, 锰过氧化物酶, 基因克隆, 染色体步移

Abstract:

Manganese peroxidase (MnP) widely exists in white-rot fungi and it is the main enzyme to degrade lignin. The ligninolytic peroxidase activity of Cerena unicolor stain CB1 collected from Changbai Mountain was firstly detected, result showed that C. unicolor could produce MnP, but no lignin peroxidase (LiP) excreted, and Mn²⁺was not the essential factor for C. unicolor to produce MnP. The highest MnP activity was gained when sawdust was added to LNAS culture solution with Mn²⁺ substrate which was 18.4 U·L^-1. On the basis of MnP activity determination, a MnP full length DNA gene termed Cu-mnp1 (GenBank accession No. JQ782578.1) was cloned in order to study the function of MnP to degrade lignin furtherly. Forward and reverse primers for PCR and nested gene-specific primers for genome walking PCR were designed based on the conserved sequences of the known MnP genes and amplified DNA fragment, all the PCR reactions were conducted by the genomic DNA of the C. unicolor stain CB1 as the template. The full length DNA gene Cu-mnp1 was 4 268 bp containing 16 exons and 15 introns, its promoter region contained diverse cis-acting elements such as TATA-Box, CAAT-Box, SP1, AP1, HSE, and XRE. Its cDNA gene contained 1 092 bp coding region encoding 363 amino acids. BLAST analysis indicated that Cu-mnp1 in the cDNA level showed the highest similarity with mnp2 gene (GenBank accession No. JQ248599.1)from Hericium erinaceum strain CB1 which was 70%. Protein structure analysis showed that Cu-Mnp1 contained 4 disulfide bonds belonging to short MnP.

Key words: Cerena unicolor, ligninolytic enzyme, manganese peroxidase (MnP), gene cloning, genome walking

中图分类号:

S718.81

于存, 池玉杰. 一色齿毛菌锰过氧化物酶（MnP）活性检测与MnP1基因的克隆[J]. 林业科学, 2014, 50(3): 109-116.

Yu Cun, Chi Yujie. Detection on Manganese Peroxidase Activity and Cloning of Cu-mnp1 of Cerena unicolor[J]. Scientia Silvae Sinicae, 2014, 50(3): 109-116.

参考文献

闫洪波. 2009. 偏肿拟栓菌锰过氧化物酶cDNA基因克隆及在毕赤酵母中的表达.哈尔滨:东北林业大学博士学位论文.
(Yan H B. 2009. Molecular cloning of cDNA gene encoding a manganese peroxidase from pseudotrametes gibbosa and heterologous expression of this gene in pichia pastoris. Haerbin: PhD thesis of Northeast Forestry University. [in Chinese] )
尹立伟,池玉杰,王雪童.2010.灰树花的系统发育分析和主要木质素降解酶的测定.林业科学研究,23(4):574-580.
(Yan L W, Chi Y J, Wang X T. 2010. Phylogenetic analysis and detection on the major ligninolytic enzymes system of grifola frondosa. Forest Research, 23(4):574-580. [in Chinese] )
Alic M,Akileswaran L,Gold M H.1997.Characterization of the gene encoding manganese peroxidase isozyme 3 from Phanerochaete chrysosporium.Biochimica et Biophysica Acta,1338(1):1-7.
Deguchi T, Kitaoka Y, Kakezawa M, et al.1998.Purification and characterization of a nylon-degrading enzyme. Appl Environ Microbiol,64(4):1366-1371.
Giardina P, Palmieri G, Fontanella B, et al.2000.Manganese peroxidase isoenzymes produced by Pleurotus ostreatus grown on wood sawdust.Archives of Biochemistry and Biophysics,376(1):171-179.
Hilden K, Martinez A T, Hatakka A, et al.2005. The two manganese peroxidases Pr-MnP 2 and Pr-MnP 3 of Phlebia radiata, a lignin-degrading basidiomycete,are phylogenetically and structurally divergent. Fungal Genetics and Biology,42(5):403-419.
Janusz G, Kucharzyk K H, Pawlik A, et al.2013.Fungal laccase, manganese peroxidase and lignin peroxidase: gene expression and regulation.Enzyme and Microbial Technology, 52(1):1-12.
Johansson T, Nyman P O, Cullen D.2002.Differential regulation of mnp 2 , a new manganese peroxidase-encoding gene from the ligninolytic fungus Trametes versicolor PRL 572.Appl Environ Microbiol,68(4):2077-2080.
Kirk T K, Cullen D. 1998. Enzymology and molecular genetics of wood degradation by white-rot fungi//Young R A, Akhtar M.Environmentally friendly technologies for the pulp and paper industry. John Wiley and Sons, New York, 273-307.
Kuwahara M, Glenn J K, Morgan M A, et al. 1984.Separation and characterization of two extracelluar H₂O-dependent oxideses from ligninolytic cultures of Phanerochaete chrysosporium. FEBS Lett,169(2):247-250.
Lankinen P,Hildén K,Aro N,et al.2005.Manganese peroxidase of Agaricus bisporus: grain bran-promoted production and gene characterization.Appl Microbiol Biotechnol, 66(4): 401-407.
Li D, Li N, Ma B, et al.1999.Characterization of genes encoding two manganese peroxidases from the lignin-degrading fungus Dichomitus squalens. Biochimica et Biophysica Acta,1434(2):356-364.
Lobos S, Larrondo L, Salas L, et al. 1998.Cloning and molecular analysis of a cDNA and the Cs-mnp 1 gene encoding a manganese peroxidase isoenzyme from the lignin-degrading basidiomycete Ceriporiopsis subvermispora.Gene, 206(2):185-193.
Maeda Y,Kajiwara S,Ohtaguchi K.2001.Manganese peroxidase gene of the perennial mushroom Elfvingia applanata: cloning and evaluation of its relationship with lignin degradation.Physics in Medicine and Biology,23(2):103-109.
Morgenstern I, Robertson D L, Hibbett D S.2010.Characterization of three mnp genes of Fomitiporia mediterranea and report of additional class Ⅱ peroxidases in the order Hymenochaetales.Appl Environ Microbiol,76(19):6431-6440.
Nagai M,Sakamoto Y,Nakade K,et al.2007.Isolation and characterization of the gene encoding a manganese peroxidase from Lentinula edodes.Mycoscience,48(2):125-130.
Pease E A, Andrawis A, Tien M. 1989.Manganese-dependent peroxidase from Phanerochaete chrysosporium. Primary structure deduced from cDNA sequence.Journal of Biological Chemistry,264(23):13531-13535.
Sundaramoorthy M, Kishi K, Gold M H, et al. 1994.The crystal structure of manganese peroxidase from Phanerochaete chrysosporium at 2.06-A resolution. J Biol Chem, 269(52): 32759-32767.
Tello M,Corsini G, Larrondo L F, et al.2000.Characterization of three new manganese peroxidase genes from the ligninolytic basidiomycete Ceriporiopsis subvermispora.Biochimica et Biophysica Acta, 1490(1/2): 137-144.

[1]	任健, 殷雨晴, 张慧慧, 陈典, 王可新, 王勇. 马占相思纤维素合酶基因AmCesA1的克隆及分析[J]. 林业科学, 2018, 54(8): 79-87.
[2]	张芹, 徐宗大, 赵凯, 李晓伟, 张罗沙, 张启翔. 梅花花青素苷调控基因PmMYB1的分离及功能分析[J]. 林业科学, 2018, 54(10): 64-72.
[3]	徐嘉娟, 李火根. 鹅掌楸LcPAT8基因的克隆及功能初步分析[J]. 林业科学, 2017, 53(9): 45-54.
[4]	张玉龙, 池玉杰, 冯连荣. 偏肿革裥菌锰过氧化物酶酶学性质和染料脱色[J]. 林业科学, 2017, 53(9): 73-80.
[5]	张玉龙, 池玉杰, 冯连荣. 偏肿革裥菌锰过氧化物酶的分级纯化[J]. 林业科学, 2017, 53(8): 64-70.
[6]	游韩莉, 袁义杭, 李长江, 张凌云. 青杄MYB转录因子基因PwMYB20的克隆及表达分析[J]. 林业科学, 2017, 53(5): 23-32.
[7]	王凤娟, 李伟庆, 牟志美, 王彦文, 刘庆信, 高绘菊. Paraconiothyium variable GHJ-4木质素降解酶的酶学性质[J]. 林业科学, 2017, 53(1): 94-100.
[8]	孙世娜, 池玉杰, 于存, 李月. 构巢曲霉转化子菌株产MnP条件优化及对3种染料的脱色[J]. 林业科学, 2016, 52(4): 75-82.
[9]	罗群凤, 胥猛, 冯源恒, 徐嘉娟, 李火根. 北美鹅掌楸LtCHS基因的克隆及生物信息学与组织表达特征分析[J]. 林业科学, 2015, 51(5): 37-45.
[10]	尹立伟, 池玉杰. 猴头菌MnP1基因全长cDNA克隆及生物信息学分析[J]. 林业科学, 2015, 51(5): 68-77.
[11]	王建勇, 谭晓风, 陈鸿鹏, 曾艳玲, 龙洪旭, 梅芳芳, 刘凯. 油茶丙二酰单酰CoA:ACP转酰基酶基因的克隆与原核表达[J]. 林业科学, 2015, 51(3): 148-154.
[12]	于存, 池玉杰. 一色齿毛菌产MnP的条件优化及对染料的脱色[J]. 林业科学, 2014, 50(7): 121-127.
[13]	谷振军, 章怀云, 张党权, 谢耀坚, 何含杰, 陈丽莉, 彭宽, 刘果, 杨丹. 赤桉CCoAOMT亚家族的基因克隆及可变剪接分析[J]. 林业科学, 2014, 50(5): 62-69.
[14]	唐欣, 王瑞辉, 杨秀艳, 朱建峰, 刘正祥, 倪建伟, 张华新. 唐古特白刺液泡膜Na⁺/H⁺逆向运输蛋白基因NtNHX1的克隆与表达分析[J]. 林业科学, 2014, 50(3): 38-44.
[15]	胡美美, 池玉杰. 偏肿革裥菌Lg-mnp2基因在构巢曲霉中的表达[J]. 林业科学, 2014, 50(2): 139-143.