碳阻遏因子CRE对匍枝根霉产纤维素酶调控作用的研究  

The Role of Carbon Metabolism Repressor CRE in the Regulation of Cellulase Produced by Rhizopus stolonifer

作　　者：孟庆婷[1] 汤斌[1] 

机构地区：[1]安徽工程大学微生物发酵安徽省工程技术研究中心,芜湖241000

出　　处：《中国生物工程杂志》2016年第3期31-37,共7页China Biotechnology

基　　金：国家自然科学基金资助项目(31270135)

摘　　要：为研究匍枝根霉(Rhizopus stolonifer)TP-02中碳代谢阻遏因子CRE对其产纤维素酶的调控特性,从其基因组DNA中扩增得到CRE全长基因,并利用重叠PCR(overlapping PCR)技术将潮霉素B抗性基因(hyg B)插入其中,既破坏cre正常转录又提供了筛选标记。通过电转化将p UCmT-cre-hyg B导入匍枝根霉萌发孢子,经抗性筛选得到△CRE突变株。利用RT-q PCR对此突变株纤维素酶基因转录水平进行研究,发现eg、bg、cbh1和cbh2的转录均有所提高,分别为48.75%、26%、5.6%和38.6%。同时,△CRE突变株的纤维素酶在表达水平上也均高于原菌,其中内切葡聚糖苷酶酶活提高了58.62%。CRE的破坏在一定程度上减弱了碳阻遏效应,其对纤维素酶的调控具有特异性。其中,对内切酶的调控最为显著。此外,△CRE突变株在3%糖浓度下仍高产纤维素酶,这为后续酶系优化及产业化生产提供了一定的依据。In order to study the control characteristic of carbon metabolism repressor CRE to the cellulases produced by Rhizopus stolonifer TP-02,a full-length gene cre was extracted from the genomic DNA. An antibiotic resistance gene hyg B was inserted into the cre by overlapping PCR, which not only broken the normal transcription of cre but also provided a selective maker for the work follow-up. The p UCm-T-cre-hyg B was transformed into the germinal spores of R. stolonifer TP-02 by electroporation,and then a mutant △CRE was screened by the hygromycin resistance. RT-q PCR was utilized to analyze the transcription features of cellulases secreted by the mutant. It was found that the transcription of eg,bg,cbh1 and cbh2 are improved than the wild type,the values are 48. 75%,26%,5. 6% and 38. 6%,respectively. Meanwhile,the mutant was preceded in the expression level cellulases,in which the activity of endoglucanases was improved 58. 62%. CRE has a specificity in the control of cellulases genes,expecially to the endoglucanase gene eg. Destruction of CRE could weaken the effect of carbon repression. In addition,the mutant △CRE could maintain a high yields of cellulases within the 3% sugar concentration,which may lay a foundation for the subsequent enzyme system optimization and industrial production.

关 键 词：碳阻遏因子 纤维素酶 匍枝根霉 转录调控 

分 类 号：Q78[生物学—分子生物学]