文献类型: 中文期刊
作者机构: 1.中国农业科学院饲料研究所农业农村部饲料生物技术重点开放实验室
关键词: 里氏木霉;CRISPR/Cas9;RNAi;纤维素酶;CBH1;EG2
期刊名称: 中国农业科技导报
ISSN: 1008-0864
年卷期: 2020 年 22 卷 012 期
页码: 50-57
收录情况: 北大核心 ; CSCD
摘要: 丝状真菌里氏木霉(Trichoderma reesei)产生的纤维素酶主要包括纤维二糖水解酶和内切葡聚糖酶.在使用里氏木霉表达外源基因时,它们会形成较高的纤维素酶背景,而且其表达还可能会导致对细胞转录、翻译和分泌等相关资源的占用.应用CRISPR/Cas9技术,在体外组装Cas9/gRNA复合物并转化里氏木霉以定点敲除主要的纤维二糖水解酶cbh1基因,同时结合RNAi干扰技术来沉默主要的内切葡聚糖酶eg2基因,以期一步构建里氏木霉低纤维素酶背景的表达系统.获得了一株纤维素酶表达显著下降的11号转化子SUS6.该转化子中,cbh1的表达完全消失,而eg2基因的表达水平较出发菌株SUS5降低了98%.以该转化子为宿主表达外源基因NfBgl3A,两个代表性转化子的β-葡萄糖苷酶酶活最高分别为172.4和79.3 U·mE-1,远高于以出发菌株(高纤维素酶背景)为宿主表达β-葡萄糖苷酶酶活(两个代表转化子分别为11.6和31.9 U·mL-1).因此,构建低纤维素酶背景的菌株作为表达宿主,还可明显的提高某些异源基因的表达水平.
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