广西植物Issue(3)：393-400,8.DOI:10.11931/guihaia.gxzw201501024
紫孢侧耳 Ps-mnp 1基因克隆与蛋白结构预测
Cloning and protein structure prediction of Ps-mnp 1 from Pleurotus sapidus
摘要
Abstract
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.关键词
紫孢侧耳/锰过氧化物酶/蛋白结构/生物信息学/同源模建Key words
Pleurotus sapidus/manganese peroxidase/protein structure/bioinformatics/homology modeling分类
农业科技引用本文复制引用
尹立伟,杨春成,朱玉,伦志明,张静..紫孢侧耳 Ps-mnp 1基因克隆与蛋白结构预测[J].广西植物,2015,(3):393-400,8.基金项目
安徽省自然科学基金(AQKJ2014B008) （AQKJ2014B008）
安庆师范学院科研启动项目(044-K05000130030) （044-K05000130030）
