利用强启动子对纤维二糖酶基因在里氏木霉中进行异源表达  

Heterologous Expression of Cellobiase Gene in Trichoderma reesei using Strong Promoters

作　　者：杨何宝 郭世奇 尹守亮 杨明 耿晓然 YANG He-bao;GUO Shi-qi;YIN Shou-liang;YANG Ming;GENG Xiao-ran(Zhongrong Technology Co.,Ltd,Tangshan 063000,China;College of Life Sciences,North China University of Science and Technology,Tangshan 063000,China;Hebei Zhaohuihengmei Testing Technology Co.,Ltd,Tangshan 063000,China)

机构地区：[1]中溶科技股份有限公司,河北唐山063000 [2]华北理工大学生命科学学院,河北唐山063000 [3]河北兆惠恒美检测技术有限公司,河北唐山063000

出　　处：《纤维素科学与技术》2022年第4期39-48,67,共11页Journal of Cellulose Science and Technology

摘　　要：为提高里氏木霉中纤维二糖酶的表达量,构建了含有不同强启动子的纤维二糖酶基因重组质粒,以根癌农杆菌介导的方法将表达元件整合到里氏木霉基因组中,获得了高效表达纤维二糖酶基因的里氏木霉优势菌株。采用反转录的方法从黑曲霉RNA中克隆得到纤维二糖酶基因(cb)的编码序列,并以启动子Pcbh1和Pxyn1控制cb基因的表达,通过根癌农杆菌介导转化,成功将表达元件整合到里氏木霉基因组中。通过对不同启动子控制下的阳性转化子进行发酵筛选,纤维二糖酶活性最高比对照分别提高了376%和323%。通过水解纤维素实验,重组菌株表达的纤维素酶能更好的对纤维素进行水解,水解率比对照菌株分别高出了29.73%和26.78%。该研究成果对解决里氏木霉表达纤维二糖酶低的问题具有一定的指导意义。In order to increase the expression of cellobiase in Trichoderma reesei, the target gene expression elements containing different promoters were integrated into the genome of Trichoderma reesei by Agrobacterium tumefaciens mediated method, and dominant strains of Trichoderma reesei with high expression of cellobiase gene were obtained. The coding sequence of the cellobiase gene(cb) was cloned from Aspergillus niger RNA by reverse transcription method, and the cb gene expression was controlled by the promoters Pcbh1 and Pxyn1. The expression elements were successfully integrated into the genome of Trichoderma reesei through Agrobacterium tumefaciens mediated transformation. By screening the positive transformants controlled by different promoters,the cellobiase activity was increased by 376% and 323% respectively compared with the control. Through the cellulose hydrolysis experiment, the cellulase expressed by the recombinant strain can better hydrolyze cellulose,and the hydrolysis rate is 29.73% and 26.78% higher than that of the control strain respectively. The research results have certain guiding significance for solving the problem of low expression of cellobiase by Trichoderma reesei.

关 键 词：启动子 纤维二糖酶 里氏木霉 根癌农杆菌 异源表达 

分 类 号：Q815[生物学—生物工程]