基于CRISPR／Cas9系统的金针菇基因组编辑载体构建  

Construction of Flammulina velutipes genome editing vector by using CRISPR/Cas9 system

作　　者：罗润[1,2] 林俊芳[1,2,3] 郭丽琼[1,2,3] 叶志伟[1,2] 郭天芬[1,2] 云帆 

机构地区：[1]华南农业大学食品学院,华南农业大学食品生物技术研究所,广东广州510640 [2]广东省微生态制剂工程技术研究中心,广东广州510640 [3]广州市澳键丰泽生物科技有限公司,广东广州510640

出　　处：《食品工业科技》2016年第20期230-234,共5页Science and Technology of Food Industry

基　　金：国家自然科学基金项目(31372116,31572178);广东省科技计划项目(2014B020205003,2014B050505018)

摘　　要：采用高效基因编辑系统CRISPR/Cas9,构建金针菇基因Mads-8敲除载体及转化体系。以转录组数据为依据,通过分析基因Mads-8序列信息,选择高效的靶位点序列,同时加入筛选标记基因hph构建过渡载体sgRNA-T,通过菌落PCR鉴定和测序来验证靶位点序列是否正确插入。以表达载体pg Fvs-cas9为框架,酶切连接后构建重组敲除载体pg Fvs-Cas9-gRNA,PCR和酶切鉴定敲除载体。再以PEG介导的金针菇原生质体转化表达载体,在潮霉素抗性平板上筛选拟转化子,以拟转化子基因组DNA为模板扩增Cas9基因。结果显示,靶位点序列成功插入敲除载体且序列正确,重组质粒成功转化进金针菇的基因组。对金针菇基因组敲除载体的构建,为后续金针菇相关基因的功能验证及育种研究提供载体材料及理论依据。High-efficiency gene editing system CRISPR/Cas9 system was employed to construct a Flammulina velutipes Mads-8 gene knockout vector and transformation system. Based on the transcriptome data, efficient targeted point sequence was selected by analyzing the sequence information of gene Mads-8 to construct transition vecter sgRNA-T with hph as selection marker gene. The target sequence of vecter sgRNA-T was comfirmed by colony PCR and DNA sequencing.Recombinant vector pgFvs-Cas9-gRNA was constructed using expression vector pgFvs-cas9 as the frame and verified by PCR methods and digestion with restriction enzyme. Recombinant vector infected Flammulina velutipes protoplast with PEG mediating.Hygromycin resistance plate was used to screen the transformants, and the Cas9 gene was verified by PCR amplifying using genomic DNA as template.Results showed that targeted point sequence was successfully inserted into the recombinant vector followed by the transformation into the genome of Flammulina velutipes. In this work, a complete method of Flammulina velutipes genome knockout and transformation was established.This method will provide theoretical foundation for the following gene functional identification and breeding research in Flammulina velutipes.

关 键 词：金针菇 CRISPR/Cas9 转录组 MADS—box PEG转化 

分 类 号：TS255.1[轻工技术与工程—农产品加工及贮藏工程]