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【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。
【Objective】 The objective of this study was to clone the gene of Coleus lucidum glycoside hydrolase 5 and analyze its bioinformatics, prokaryotic expression and enzyme activity in order to provide a basis for the utilization of glycoside hydrolase. 【Method】 Isolation and purification of Corynebacterium parvum and long-term preservation in PDA slant. Total RNA was extracted from Tricholoma glabrata by TRIzol and reverse transcribed into c DNA by AMV reverse transcription system. The NCBI BLAST was used to analyze and detect the glycoside hydrolase gene family 5 positive sequence. Long named Ih GH5-1 and submitted to the NCBI registration; ORF-Finder analysis Ih GH5-1 gene open reading frame, deduced amino acid sequence; Screening NCBI registered glycoside hydrolase family 5 homologous sequences, Clustal W conserved domain region Multiple sequence alignment was performed using Mega 5.05. The maximum likelihood tree was constructed using WAG + G model with Mega 5.05. The secondary structure of α-helix and β-sheet proteins of Ih GH5-1 was analyzed by PSIPRED server. SWISS-MODEL was used to detect the Ih GH5-1 3D modeling was performed and the three-dimensional structure of Ih GH5-1 was analyzed using VMD1.8.6. Prokaryotic expression primers were designed and amplified by PCR. The amplified gene fragment was ligated into pQE-30 UA vector and transformed into Escherichia coli JM109 Competent cells, after induction of expression by SDS-PAGE electrophoresis to detect the expression; glycoside hydrolase activity was measured and calculated enzyme activity. 【Result】 The total RNA extracted from TRIzol was identified by spectrophotometer (OD260 / OD280 = 2.0, OD260 / OD230> 1.8), and the c DNA library was successfully constructed and sequenced. The length of the 5 ’ The 770 bp and 3 ’sequences are 1 562 bp in length and contain the Poly A sequence. The 5’ and 3 ’sequences are spliced to obtain a full-length gene of 1 727 bp in length and the NCBI accession number is KM368321. The Ih GH5-1 amino acid sequence Which contains 300 amino acids with a molecular mass of 31.226 55 kD and an isoelectric point (pI) of 9.24. Domain analysis showed that Ih GH5-1 has a conserved catalytic domain. Maximum likelihood tree analysis showed that most Ascomycokinase glycoside hydrolase Family 5 together, the majority of the basidiomycete glycoside hydrolase family 5 together, rough hairy Ih GH5-1 protein and Clostridium terrestris and Chaetomium and other fungal asparagus glycoside hydrolase more genetic relationship The three-dimensional structure analysis of protein showed that Ih GH5-1 contained seven α-helices and four β-sheets, and the three-dimensional alignment showed that the three-dimensional structure of Ih GH5-1 was similar to other fungal glycoside hydrolase family 5 proteins The structure is similar, the evolutionary relationship is consistent with the maximum likelihood tree analysis, prokaryotic table Assaying and successful indicating that the gene expression, the expression product of the highest relative activity at 65 ℃. 【Conclusion】 This study can provide pre-research basis and theoretical basis for the large-scale industrial application of white-rot fungi cellulase.