紫孢侧耳 Ps-mnp 1基因克隆与蛋白结构预测

摘要

克隆紫孢侧耳 PS1菌株锰过氧化物酶基因 Ps-mnp 1,具有生物降解木质素的活性,可用于分析锰过氧化物酶的蛋白功能与结构,并对深入了解紫孢侧耳 Ps-mnp 1基因功能和转录调控具有重要意义.基于ITS 序列分析,明确了该菌株的分类地位.根据 GenBank 中白腐菌 MnPs 保守序列设计引物,采用染色体步移法和逆转录 PCR 法获得 Ps-mnp 1基因,GenBank 登录号为(KP189358．1).在克隆 Ps-mnp 1基因并分析蛋白结构时,采用多种现代生物信息学技术,经染色体步移法克隆的 DNA 全长序列后,利用 contigexpress 拼接软件预测 Ps-mnp 1基因的 cDNA 全长序列；使用 BioEdit 分析软件对 DNA 和 cDNA 核苷酸序列比对；通过 Augustus 网站预测 RNA 的剪接位点并在 NCBI 上进行同源序列比对校正；采用基因结构软件对比了解白腐菌 MnPs 基因的内含子/外显子的组成.序列分析表明 Ps-mnp 1的 DNA 全长序列1854 bp,具有外显子14条和内含子13条.从 Ps-mnp 1与其它白腐菌 MnPs 基因的内含子/外显子组成分析来看,紫孢侧耳和糙皮侧耳同属于侧耳属,但它们之间的 MnPs 基因结构完全不同.Ps-mnp1开放阅读框为1095 bp,起始密码子 ATG,终止密码子 TAA,编码364 aa,含有20 aa 残基构成的信号肽序列.采用 MEGA 5．1软件构建的蛋白系统进化树表明 Ps-mnp1与6株白腐菌蛋白聚类在短枝 MnPs,区分了含有5个二硫键组成的长枝 MnPs组群；此外,构建的蛋白同源模型表明,与刺芹侧耳多功能过氧化物酶相似度为72．51％,蛋白配体中结合位点有1个含铁血红素、2个钙离子、1个锰离子等,这些结合位点为不守恒.该研究为紫孢侧耳锰过氧化物酶的结构,蛋白功能 Ps-mnp1奠定了基础,并进一步为 Ps-mnp1的蛋白质工程改造提供借鉴作用.%To clone the manganese peroxidase gene Ps-mnp 1 from Pleurotus sapidus PS1 contributing a lot to the lignin biodegradation activity is important for the analysis of protein structure and functions of MnP and the under-standing of manganese peroxidase gene function and transcriptional regulation of Pleurotus sapidus .Based on the analysis of ribosomal rDNA Intenal Transcribed Spacer (ITS)sequences,the classification status of the strain PS1 was defined.Ps-mnp 1 was cloned by using the methods of Genome Walking-PCR and Reverse transcription-PCR with primers designed from some white-rot fungi which contains the conserved sequences of the known manganese peroxidase genes (GenBank No.KP189358.1).We used a variety of modern bioinformatics technology softwares toclone the manganese peroxidase gene and analyze the protein structure of Ps-mnp 1.For example,after the cloning full length DNA sequence of genome walking method,prediction of the full cDNA sequence of manganese peroxidase gene by using contigexpress splicing software;comparison the DNA and cDNA nucleotide sequences alignment analy-sis by using BioEdit software;prediction the RNA splice site through Augustus website,following the comparison and correction of Ps-mnp 1 sequences by using the the NCBI database;understanding the compositions of the intron and exon by using online software Gene Structure Display Server contrast of white rot fungus manganese peroxidase gene.The results obtained in the sequence analysis of Ps-mnp 1 from Pleurotus sapidus indicated that the full length of the DNA was 1 854 bp containing 14 exons and 13 introns.Meanwhile,exons and introns composition analysis of the genes Ps-mnp 1 from Pleurotus sapidus ,compared to other white-rot fungi manganese peroxidase genes,sug-gested that P .ostreatus and P .sapidus belonged to the same genus Pleurotus ,but the manganese peroxidase gene structures between them were entirely different.The Ps-mnp 1 gene included a signal peptide of 20 amino acids and held Open Reading Frame of 1 095 bp,with start codon ATG and stop codon TAA ,encoding 364 amino acids.The results of protein phylogenetic analysis by MEGA 5.1 software revealed that Ps-mnp1 and 6 strains of white rot fun-gus protein clustered on short branches MnPs,and differentiated 5 long branches of MnPs group formed by disulphide bond.In addition,the 3D structures of Ps-mnp1 were built using homology modelling technique,and the results showed that the binding sites of the protein ligand included 1 Fe heme,2 Ca ions and 1 Mn(II),and the those sites were non-conservation.The results also suggested that the modelling similarity was 72.51% compared with P .eryn-gii VPs.This paper could establish the basis of the study in the structure and function of the Ps-nmp1.It is further helpful to offer reference on the variation of protein engineering of the Ps-mnp1.