Abstract:

【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 cDNA was analyzed and detected by AMV reverse transcription system, and then constructed cDNA library. The glycoside hydrolase family 5 gene positive sequence by NCBI BLAST, and the full-length of glycoside hydrolase family 5 gene was cloned by RACE, and named as IhGH 5-1 then registered in NCBI. All open reading frames of IhGH 5-1 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 IhGH5-1 was analyzed by PSIPRED server. The three dimensional model of IhGH5-1 was established using SWISS-MODEL, and 3d structure of IhGH5-1 was analyzed with VMD1.8.6. The prokaryotic expression primers were design, and the gene was cloned. The fragment was connected to the pQE-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 cDNA library was successfully constructed and sequenced by Sangon Biotech Company. The 5' end sequence of the RACE was 770 bp, and 3' end sequence (containing PolyA sequence) was 1 562 bp. The whole length of the gene from the 3' end to the 5' end was 1 727 bp. GenBank 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 kD, and the isoelectric point (pI) is 9.24. Domain structure analysis showed that IhGH5-1 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 three-dimensional structure of IhGH5-1 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 IhGH 5-1 would be a theoretical basis for industrial application of this enzyme.

Key words: Inonotus hispidus, glycoside hydrolase family 5, Fraxinus mandshurica, white rote