猴头菌MnP1基因全长cDNA克隆及生物信息学分析

doi:10.11707/j.1001-7488.20150508

摘要/Abstract

摘要：

【目的】克隆猴头菌CB1锰过氧化物酶(MnP)基因,用于分析He-mnp1基因及蛋白序列、基因的结构与功能,为研究猴头菌MnPs基因功能、转录调控和构建优良工程菌株提供参考。【方法】根据GenBank中已报道的白腐菌MnPs基因cDNA序列的保守区域设计引物,采用简并PCR、逆转录RT-PCR、cDNA末端的快速扩增(RACE)等方法扩增出全长cDNA基因序列,命名为He-mnp 1(GenBank登录号为HM116841.3),并对其猴头菌He-mnp 1基因进行生物信息学的分析。通过NCBI数据库进行BLAST同源搜索; ORF Finder查找该基因的完整开放阅读框(ORF); 利用Expasy数据库和BioEdit软件预测He-mnp1蛋白质的理化特性及氨基酸的组成; 同时进行亲/疏水性及跨膜区的分析; 采用SignalP 4.1软件进行蛋白信号肽的预测; 并利用Clustal W和MEGA 5.1软件对He-mnp1蛋白序列同源性比对和构建白腐菌MnPs系统发育树; 利用数据库Conserved Domain Database(CDD)蛋白保守结构域的预测,查看He-mnp1血红素、基质及锰、钙等结合位点; 采用PredictProtein软件和SWISS-MODEL软件进行He-mnp1蛋白二级结构的预测和同源三维建模。【结果】该基因He-mnp 1的cDNA全长1 279 bp,完整开放阅读框1 080 bp,起始密码子ATG,终止密码子TAA,5'端非翻译区有68个核苷酸,3'端非翻译区有131个核苷酸,编码蛋白359 aa; 生物信息学分析表明,猴头菌He-mnp1氨基酸组成中丙氨酸含量最高,无酪氨酸,分子量为38.18 kDa,等电点为 4.35,He-mnp1的蛋白有明显的亲水区,存在81-105、121-141间有2处疏水区域,此蛋白为亲水蛋白。He-mnp1蛋白质多肽前体包含1个18 aa的信号肽及1个5 aa的中间前导短肽。【结论】蛋白系统进化分析表明He-mnp1分布在第2组群,与平菇侧耳、冬生多孔菌、变色栓菌的MnPs亲缘关系最为接近。He-mnp 1基因存在1个保守结构域,分析显示为Class Ⅱ类的真菌血红素过氧化物酶蛋白家族。He-mnp1蛋白二级结构预测α-螺旋占30.99%,β-折叠占3.38%,无规则卷曲占65.63%,属于稳定蛋白。He-mnp1蛋白三维建模,结果得到1个Fe血红素,2个Ca²⁺、1个Mn²⁺的结合位点及组氨酸残基等。

关键词: 猴头菌, 锰过氧化物酶, 基因克隆, 生物信息学, 序列分析

Abstract:

【Objective】 The cDNA gene sequences of Manganese peroxidase (MnP) were isolated from H. erinaceum CB1, and used for analyzing structure and function of the He-mnp 1.【Method】Degenerate primers were designed according to conservative domain of white-rot fungi MnPs gene cDNA sequences reported in GenBank, the full-length cDNA gene sequence was obtained by using the methods of PCR, Reverse transcription-PCR and Rapid Amplification of cDNA Ends (RACE) and named as He-mnp 1 (GenBank No. HM116841.3), and the bioinformatics of He-mnp 1 gene were analyzed. BLAST homology search was conducted through the NCBI database; ORF Finder was used to look up the complete open reading frame of the gene; The Expasy database and BioEdit software were used to predict physicochemical properties and amino acid composition of He-mnp 1 protein, and analyze the hydrophilicity/hydrophobicity and transmembrane region; The SignalP 4.1 software was used to predict protein signal peptide; The clustal W with MEGA 5.1 software was adopted to complete the He-mnp1 protein sequence homology alignment and to construct the phylogenetic trees of white rot fungi MnPs, respectively. The CDD database was used to predict protein conserved domains, and check the He-mnp1 heme, substrate and manganese, calcium binding site etc. The PredictProtein software and SWISS-MODEL software were used to complete the He-mnp1 protein secondary structure prediction and to construct homologous 3D modeling, respectively.【Result】The full-length cDNA of He-mnp 1 was 1 279 bp, the ORF of 1 080 bp with starting codon of ATG and stopping codon of TAA, including 5'UTR of 68 bps and 3'UTR of 131 bps and encoded 359 amino acids. Bioinformatics analysis showed that the He-mnp1 protein has the highest content of Ala, without Tyr, and the Mw 38.18 is kDa, with the pI of 4.35. The He-nmp1 protein has an obvious hydrophilic region and two hydrophobic regions in the area of 81-105 and 121-141, and belongs to hydrophilic protein. He-mnp1 protein precursor polypeptides consists of a 18 aa signal peptide and a 5 aa the intermediate leader peptide.【Conclusion】Protein phylogenetic analysis revealed that He-mnp1 is distributed in Group II, and has closely evolutionary relationship to MnPs of Pleurotus ostreatus, Polyporus brumalis, and Trametes versicolor. He-mnp 1 has a conserved domain, and belongs to Class II fungal heme-dependent peroxidase superfamily, predicting that the protein secondary structure accounts for α-helix of 30.99%, β-sheet of 3.38% and random coil of 65.63%, and it is a stable protein. He-mnp1 protein 3D modeling showed that there are 1 Fe heme, 2 Ca²⁺, 1 Mn²⁺ binding sites and the histidine residues.

Key words: Hericium erinaceum, manganese peroxidase, genomic clone, bioinformatics analysis, sequence analysis

中图分类号:

S718.81

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.

Yin Liwei, Chi Yujie. Cloning and Bioinformatics Analysis of MnP 1 cDNA Gene from Hericium erinaceum[J]. Scientia Silvae Sinicae, 2015, 51(5): 68-77.

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