东方肉座菌EU7-22纤维素酶基因的克隆及序列分析  

Cloning and Sequence Analysis of Cellulase Genes from Hypocrea orientalis EU7-22

作　　者：龙传南[1] 成奕瑾[1] 邬小兵[2] 刘健[1] 龙敏南[1,2] 

机构地区：[1]厦门大学能源研究院,厦门361005 [2]厦门大学生命科学学院,厦门361005

出　　处：《生物技术通报》2012年第11期110-117,共8页Biotechnology Bulletin

基　　金：国家重点基础研究发展计划("973"计划)(2010CB732201);中央高校基本科研业务费专项资金项目(201112G026);国家自然科学基金项目(31170067)

摘　　要：东方肉座菌EU7-22与XC-9、里氏木霉、康宁木霉、黑曲霉、斜卧青霉进行产纤维素酶比较,结果表明菌株EU7-22具有较高的产纤维素酶能力及完整的纤维素酶系。根据里氏木霉和绿色木霉的外切葡聚糖酶,内切葡聚糖酶及β-葡萄糖苷酶相关基因序列,设计引物PCR扩增出菌株EU7-22 cbhⅠ、cbhⅡ、egⅠ、egⅡ及bglⅠ。基因序列经NCBI Blast分析表明,cbhⅠ与绿色木霉cbh1基因(FJ871063)同源性最高达99%;cbhⅡ与康宁木霉cbh2基因(DQ504304)同源性最高达99%;egⅠ与长枝木霉eg1基因(GU144298)同源性最高达99%;egⅡ与绿色木霉eg2基因(EF602036)同源性最高达99%;bglⅠ与菌株Trichodermasp.SSLbgl基因(FJ040193)同源性最高达100%。5种纤维素酶基因编码的相应氨基酸序列与其他木霉纤维素酶的氨基酸序列相似性也非常高。对上述纤维素酶基因编码的相应蛋白的分子量、等电点、N-糖基化位点、信号肽序列进行分析;对纤维素结合区及糖基水解酶家族特征结构区进行了定位;用SWISS-Model模拟了酶蛋白的三级结构。The fungi of Hypocrea orientalis EU7-22, Hypocrea orientalis XC-9, Trichoderma reesei, Trichoderma koningii, Aspergillus niger and Penicillium decumbens were investigated to produce cellulase. The results indicated that the strain EU7-22 had highly cellulase activities in comparison with other fnngi, and possessed of integrated cellulase system. According to the reported cellobiohydrolase, endoglucanase and [3-glucosidase gene sequences of T. reesei and Trichoderma viride, the primers were designed and five cellulase genes （ cbh I, cbh Ⅱ, eg I , eg Ⅱ, bgl I ） were successfully cloned by PCR. By conducting sequence alignment analysis with NCBI Blast, it was found that the homology of same cellulase genes between strain EU7-22 and other Trichoderma were： 99% to cbhl （ FJ871063 ） from T. viride;99% to cbh2 （ DQ504304 ） from T. koninqii ; 99% to egl （ GUI44298 ） from Trichoderma longibrachiatum ; 99% to eg2 （ EF602036 ） from T. viride ; 100% to bgl （ FJ040193 ） from Trichoderma sp. SSL. Furthermore, the corresponding amino acid sequences were also quite similar. The molecular weight, isoelectrie point, N-glycosylation sites and signal peptide sequence of these cellulase genes encoding the corresponding protein were analyzed. The cellulose- binding domain and conserved domains of glycosyl hydrolases family were confirmed. By using SWISS-Model, the tertiary structure of cellulase proteins were predicted and simulated.

关 键 词：东方肉座菌 纤维素酶 基因 同源性 

分 类 号：Q78[生物学—分子生物学]