作 者：
刘晓晗;王峰;董爱荣;陈俏丽;刘立宏;零雅茗;王博文;丁晓霞;王世新
关键词：
粗毛纤孔菌;糖苷水解酶家族5;水曲柳;木材白色腐朽
摘 要：
【目的】克隆得到粗毛纤孔菌糖苷水解酶5基因,并对该基因进行生物信息学分析、蛋白质原核表达和酶活研究,为糖苷水解酶的利用提供依据。【方法】分离纯化粗毛纤孔菌,并于PDA斜面长期保存。应用TRIzol提取粗毛纤孔菌总RNA,通过AMV反转录系统将RNA反转录成c DNA,构建c DNA文库;应用NCBI BLAST分析并检测糖苷水解酶基因家族5阳性序列,RACE法克隆基因全长命名为Ih GH5-1并提交NCBI注册;ORF-Finder分析Ih GH5-1基因开放阅读框,推导出氨基酸序列;筛选NCBI登录的糖苷水解酶家族5同源序列,Clustal W进行保守结构域区段多序列比对;应用Mega 5.05选用WAG+G模型构建最大似然树;应用PSIPRED server对Ih GH5-1进行α螺旋和β折叠的蛋白质二级结构分析,应用SWISS-MODEL对Ih GH5-1进行三维建模,应用VMD1.8.6分析Ih GH5-1三维结构;设计原核表达引物并进行PCR扩增,将扩增得到的基因片段连接至p QE-30 UA载体并转入大肠埃希菌JM109感受态细胞,诱导表达后通过SDS-PAGE电泳检测表达量;测定糖苷水解酶活性并计算酶活。【结果】TRIzol提取的粗毛纤孔菌总RNA经分光光度计检验符合标准(OD260/OD280=2.0,OD260/OD230>1.8),c DNA文库成功构建并测序;RACE法克隆得到5'序列长度为770 bp,3'序列长度为1 562 bp且含有Poly A的序列,5'和3'序列拼接得到基因全长序列长1 727 bp,NCBI注册号为KM368321;ORF分析得到Ih GH5-1氨基酸序列含有300个氨基酸,分子质量为31.226 55 k D,等电点(p I)为9.24;结构域分析表明Ih GH5-1具有保守的催化结构域;最大似然树分析表明多数子囊菌糖苷水解酶家族5聚在一起,多数担子菌糖苷水解酶家族5聚在一起,粗毛纤孔菌Ih GH5-1蛋白质与太瑞斯梭孢壳霉和球毛壳菌等子囊菌的糖苷水解酶亲缘关系更近;蛋白质三维结构分析表明粗毛纤孔菌Ih GH5-1含有7个α螺旋、4个β折叠,三维比对表明粗毛纤孔菌Ih GH5-1三维结构与其他真菌糖苷水解酶家族5蛋白空间结构相近,进化关系与最大似然树分析相符;原核表达及酶活测定表明该基因成功表达,表达产物在65℃时相对酶活性最高。【结论】本研究可为白腐菌纤维素酶大规模的工业化应用提供前期研究基础和理论依据。
译 名：
Gene Cloning and Protein Structural Studies of a Glycoside Hydrolase Family 5 Enzyme Gene from Inonotus hispidus
作 者：
Liu Xiaohan;Wang Feng;Dong Airong;Chen Qiaoli;Liu Lihong;Ling Yaming;Wang Bowen;Ding Xiaoxia;Wang Shixin;College of Forestry,Northeast Forestry University;
关键词：
Inonotus hispidus;;glycoside hydrolase family 5;;Fraxinus mandshurica;;white rote
摘 要：
【Objective 】Inonotus hispidus is a species of white rot fungi which mainly grow in the broadleaf tree of standing forest stock,and has medicinal efficacy,therefore with high application value and development value. Glycoside hydrolase can hydrolyze the cellulose into simple sugars which can be used to produce energy substances. 【Method】In this study,we isolate and purified I. hispidus from Fraxinus mandshurica,and the fungus was preserved for long-term in the PDA cant medium. 【Result】Total RNA of I. hispidus was extracted using TRIzol reagent. RNA was reversely transcribed to c DNA was analyzed and detected by AMV reverse transcription system,and then constructed c DNA library. The glycoside hydrolase family 5 gene positive sequence by NCBI BLAST,and the full-length of glycoside hydrolase family 5gene was cloned by RACE,and named as Ih GH5- 1 then registered in NCBI. All open reading frames of Ih GH5- 1 were identifies by ORF Finder,and the amino acid sequence was deduced. The homologous sequences of glycoside hydrolase family 5 were detected from NCBI,and multiple sequence alignment was conducted by Clustal W. The maximum likelihood tree in WAG + G model was constructed by Mega 5. 05. The secondary protein structure of Ih GH5- 1 was analyzed by PSIPRED server. The three dimensional model of Ih GH5- 1 was established using SWISS-MODEL,and 3d structure of Ih GH5- 1 was analyzed with VMD1. 8. 6. The prokaryotic expression primers were design,and the gene was cloned. The fragment was connected to the p QE- 30 UA vector and transformed to E. coli JM109. Expression quantity inducible expression was detected using SDS-PAGE electrophoresis. The glycoside hydrolase activity was measured. Total extracted RNA of I. hispidus was measured by spectrophotometer( OD260/ OD280= 2. 0,OD260/ OD230> 1. 8). The c DNA library was successfully constructed and sequenced by Sangon Biotech Company. The 5' end sequence of the RACE was770 bp,and 3' end sequence( containing Poly A sequence) was 1 562 bp. The whole length of the gene from the 3‘ end to the 5 'end was 1 727 bp. Gen Bank accession number of the gene was KM368321. The amino acid sequence encoded by this gene contains 300 amino acids,the molecular weight is 31. 226 55 k D,and the isoelectric point( p I) is 9. 24.Domain structure analysis showed that Ih GH5- 1 has a conservative catalytic domain structure. The maximum likelihood tree showed that a closer relationship with the other glycoside hydrolase family 5 homologous was from fungus of Ascomycota,such as Thielavia terrestris and Chaetomium globosum. The three-dimensional comparison showed that threedimensional structure of Ih GH5- 1 had seven alpha helixs,four beta foldings. It was found that the structure was similar with other fungal glycoside hydrolase family 5 protein spatial structures,and evolutionary relationships were consistent with the maximum likelihood tree analysis. The gene expression and enzymatic assays showed that the product of this gene had the highest relative activity at 65℃.【Conclution】The gene cloning and protein structural study of Ih GH5- 1 would be a theoretical basis for industrial application of this enzyme.
