堆肥菌株Aspergillus fumigatus Z5纤维素酶转录限制因子creA基因的克隆及其功能研究

项目名称： 堆肥菌株Aspergillus fumigatus Z5纤维素酶转录限制因子creA基因的克隆及其功能研究

项目编号： No.31201685

项目类型： 青年科学基金项目

立项/批准年度： 2013

项目学科： 植物保护学、园艺学与植物营养学

项目作者： 刘东阳

作者单位： 南京农业大学

项目金额： 25万元

中文摘要： 纤维素的分解是堆肥过程或纤维素工业利用中最重要的步骤之一，其过程主要由微生物分泌的纤维素酶来完成。纤维素酶是指能够水解纤维素及其衍生物的一系列酶的总称，主要包括葡聚糖内切酶、葡聚糖外切酶、β-葡萄糖苷酶,其表达和调控的机理尚未有完全统一的定论。目前降解物阻遏效应是一种比较认可的调控机制，在丝状真菌中这种效应是由调控基因creA所编码的产物介导的。本研究通过设计简并引物并结合highTail-PCR的方法获得高效纤维素分解菌A.fumigatus Z5的creA基因；利用双元质粒pDHt/SK定点突变creA基因，获得去阻遏效应突变体Z5-M。利用蛋白组学方法分析突变体在降解物阻遏条件下的去阻遏效应,为阐明creA基因在纤维素酶表达和调控中的作用提供重要理论依据；通过去降解物阻遏效应的突变体在农业有机废弃物资源化利用中的应用，对加快堆肥速率或提高生物酒精的产量具有重要的理论和实践意义。

中文关键词： 纤维素酶；iTRAQ；creA；基因克隆和表达；基因突变

英文摘要： Hydrolysis of cellulose is one of the most important steps in composting and cellulose industry, which is finished by cellulases. The enzymes are actually the general term of a series of enzymes responsible for the cellulose degradation process and they are endoglucanases (EGs), cellobiohydrolases (CBHs) and β-glucosidases. All of these enzymes worked synergistically to degrade the cellulose fractions. Many studies have shown that most of the cellulases are inducible, and their expression and regulation mechanism has not yet been understood. Currently, the repression effect of the degradated products is an approved regulatory mechanism, and this effect is mediated by the encoded product of gene creA in filamentous fungi. In this study, the creA gene of Aspergillus fumigatus Z5 will be cloned by high-Tail PCR based on the degenerate primers, and the site-directed mutation will be carried out by using the binary vector pDHt/SK, and the derepressed effect mutant Z5-M will be obtained. The SDS-PAGE and 2D analysis methods will be used to value the derepressed effect under the degradation repressing condition, and these results will provide important theoretical basis to clarify the effect of creA gene on the expression and regulation of cellulase. The application of the derepressed effect mutant Z5-M in the treatme

英文关键词： cellulose；iTRAQ；creA；gene cloning and expression；gene knock out