摘要
利用CRISPR-Cas9基因编辑技术在Lager型啤酒酵母(Saccharomyces pasteurianus)中建立CRISPRCas9基因敲除系统。将质粒pMY中毕赤酵母基因表达系统中常用GAP启动子替换为酿酒酵母基因表达系统中常用PGK1启动子,增强外源基因表达量。设计酿酒酵母(S.cerevisiae)基因组及真贝酵母(S.eubayanus)基因组中目的基因TRP1小向导RNA。增加向导RNA结构中锤头状核酶和肝炎三角洲病毒核酶结构,提高gRNA转录水平。Lager型啤酒酵母Pilsner中TRP1基因被敲除,成功构建Lager型啤酒酵母中CRISPR-Cas9基因敲除系统。
To establish a CRISPR-Cas9 gene deletion system in lager yeast(Saccharomyces pasteurianus)using the technology of CRISPR-Cas9 gene editing.In plasmid pMY,the GAP promoter commonly used in Pichia pastoris was replaced with the PGK1 promoter which commonly used in S.cerevisiae to enhance the expression level of exogenous genes.The small guide RNA(sgRNA)of the target gene TRP1 in the S.cerevisiae genome and the S.eubayanus genome was designed separately due to its genetic differences.The guide RNA(gRNA)structure was combined with Hammerhead ribozyme(HH)and Hepatitis Delta Virus ribozyme(HDV)to increase the transcription level of gRNA.Finally,TRP1 was knocked out in lager yeast Pilsner.The CRISPR-Cas9 gene knockout system was successfully constructed in lager yeast.
作者
李梦琦
张可心
郑飞云
钮成拓
刘春凤
李崎
王金晶
LI Mengqi;ZHANG Kexin;ZHENG Feiyun;NIU Chengtuo;LIU Chunfeng;LI Qi;WANG Jinjing(The Key Laboratory of Industrial Biotechnology,Ministry of Education,School of Biotechnology,Jiangnan University,Wuxi Jiangsu 214122,China;Laboratory of Brewing Science and Technology,Jiangnan University,Wuxi Jiangsu 214122,China)
出处
《东北农业大学学报》
CAS
CSCD
北大核心
2020年第3期36-44,96,共10页
Journal of Northeast Agricultural University
基金
国家自然科学基金项目(31771963)
江苏省研究生科研与实践创新计划项目(KYCX19_1868)。
