一色齿毛菌锰过氧化物酶2基因克隆及生物信息学分析

Cloning and Bioinformatics Analysis of MnP2 Gene from Cerrena unicolor

锰过氧化物酶(manganese peroxidase,Mn P)是由一系列同功酶组成的木质素降解酶。我们前期工作克隆了一色齿毛菌(Cerrena unicolor)Mn P1基因序列。在此基础上,本研究采用简并PCR、染色体步移和RACE等技术对C.unicolor mnp2基因(Cu-mnp2)序列进行克隆。同时,采用生物信息学软件对Cu-mnp2的基因结构、Cu-Mn P2的蛋白质结构及多物种Mn Ps蛋白质序列的系统进化关系进行分析。克隆得到3 053 bp的Cu-mnp2 DNA序列(Gen Bank:JX270806.1)和1 429 bp的Cu-mnp2 c DNA序列(Gen Bank:JQ782580.1)。序列分析结果显示,Cu-mnp2 DNA序列包含14个外显子和13个内含子,启动子区域包含TATA-BOX、SP1和AP1等作用元件;Cu-mnp2 c DNA序列包含71 bp的5'UTR、230 bp的3'UTR以及1 128 bp的开放阅读框(ORF)。Cu-mnp2 ORF序列的BLAST比对结果表明,Cu-mnp2与Trametes versicolor FP-101664 SS1 mnp序列覆盖度为53%,序列相似性为65%;与Heterobasidion irregulare mnp、C.unicolor mnp1等c DNA序列都有较高的序列相似性。Cu-mnp2的ORF编码(Gen Bank:AFK91530.1)由340个氨基酸残基组成的多肽链(CuMn P2)。Cu-Mn P2蛋白质序列的BLAST比对和蛋白质三维结构均显示,Cu-Mn P2包含Mn2+、Ga2+、血红素及芳香底物结合位点。对包含Cu-Mn P1、Cu-Mn P2蛋白质序列在内的多物种Mn Ps蛋白质序列的系统发育分析表明,多物种的Mn Ps分为两大类群,分别为包含4个二硫键的短Mn Ps和包含5个二硫键的长Mn Ps。其中,Cu-Mn P1与Cu-Mn P2均属于短Mn Ps,Cu-Mn P2与Trametes versicolor Mr P的蛋白质序列亲缘关系最近。通过Cu-mnp2基因的克隆和序列分析,对继续研究C.uniclor的Mn P同工酶基因结构和功能奠定基础。

Manganese peroxidase（ Mn P） is composed of a series of isozymes of ligninolytic enzymes. In the previous work,we cloned the Mn P1 gene sequence of Cerrena unicolor. In this study, using degenerate PCR,genome walking and rapid amplification of c DNA end（ RACE）,a mnp2 gene was cloned from C. unicolor. The Cu-mnp2 gene structure,Cu-Mn P2 protein structure and the phylogenetic relationships compared with muti-species Mn Ps were analyzed by bioinformatics software. The result showed that a 3 053 bp of Cu-mnp2 DNA sequences（ Gen Bank： JX270806. 1） and a 1 429 bp of Cumnp2 c DNA sequences（ Gen Bank： JQ782580. 1） were cloned,respectively. The sequence analysis revealed that the Cu-mnp2 DNA sequence contained 14 exons and 13 introns; promoter region containsthe TATA-BOX,SP1 and AP1 components; Cu-mnp2 c DNA sequence contains 71 bp 5＇ UTR,230 bp3＇ UTR and an 1 128 bp ORF. The Cu-mnp2 c DNA ORF BLAST sequence results showed that the sequence coverage between Cu-mnp2 and Trametes versicolor FP-101664 SS1 mnp is 53%. In addition,the sequences of Cu-mnp2 and Heterobasidion irregulare TC 32-1 mnp,C. unicolor mnp1 and other white rot fungus mnps c DNA sequences also have a high sequence similarity. Cu-mnp2 has a complete open reading frame（ ORF） encoding a 340 amino acid sequence（ Gen Bank： AFK91530. 1）. Amino acid sequence BLAST ratio and protein 3D structure showed that Cu-Mn P2 amino acid contained binding sites for Mn2 ＋,Ga2 ＋,heme and aromatic substrate. Phylogenetic analysis based on the amino acid sequence of a multi species Mn Ps,including Cu-Mn P1 ~ 2,showed that different species of Mn Ps were divided into two groups,short Mn Ps containing 4 disulfide bonds and long Mn Ps containing 5 disulfide bonds,respectively. Cu-Mn P1 and Cu-Mn P2 both belong to short Mn Ps. The amino acid sequence of Cu-Mn P2 was the most closely related to Trametes versicolor mrp recently.