基于CRISPR/Cas9方法构建‘沪农灵芝1号’基因编辑系统  

Construction of a CRISPR/Cas9-Based Genome Editing System in Ganoderma lucidum‘Hunong No.1’Cultivar

作　　者：张志刚 张劲松[2] 邹根 唐传红[2] 冯杰[2] 鲍大鹏[2] 陈建波[1] 谭贻 ZHANG Zhigang;ZHANG Jingsong;ZOU Gen;TANG Chuanhong;FENG Jie;BAO Dapeng;CHEN Jianbo;TAN Yi(College of Life Sciences,Shanghai Normal University,Shanghai 200234,China;Institute of Edible Fungi,Shanghai Academy of Agricultural Sciences,Key Laboratory of Edible Fungal Resource s and Utilization(South),Ministry of Agriculture and Rural Affairs,P.R.China,National Engineering Research Center of Edible Fungi,Key Laboratory of Agricultural Genetics and Breeding of Shanghai,Shanghai 201403,China)

机构地区：[1]上海师范大学生命科学学院,上海200234 [2]上海市农业科学院食用菌研究所,农业农村部南方食用菌资源利用重点实验室,国家食用菌工程技术研究中心,上海市农业遗传育种重点实验室,上海201403

出　　处：《食用菌学报》2023年第2期9-18,共10页Acta Edulis Fungi

基　　金：上海市现代农业产业技术体系(沪农科产字2022第9号);上海市农业科学院卓越团队建设计划(G2022003)。

摘　　要：构建密码子优化的cas 9表达质粒pMD-EXP-cas 9,通过聚乙二醇(polyethylene glycol,PEG)介导转化灵芝(Ganoderma lucidum)菌株‘沪农灵芝1号’(‘Hunong No.1’)单核菌株L1,得到含有cas9完整表达盒的菌株L1-cas9。利用T7启动子构建靶向ura3基因的表达质粒psgRNA-1和psgRNA-2,体外转录后得到sgRNA 1和sgRNA 2。通过PEG介导的转化,将sgRNA 1和sgRNA 2转化进L1-cas9的原生质体中,得到ura3基因被破坏的突变体,首次在‘沪农灵芝1号’菌株单核体中构建成簇的规律间隔的短回文重复序列及其相关蛋白9(clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9,CRISPR/Cas9)基因编辑系统,ura3基因的编辑效率为4个突变体(107个原生质体)。利用0.006%曲拉通X-100优化PEG介导的转化系统,可使ura3基因编辑效率提高至18个以上突变体(107个原生质体),本研究的结果为灵芝基因功能的研究和分子育种提供更高效的方法。A codon-optimized cas9 expression plasmid pMD-EXP-cas9 was constructed,and then transformed to monokaryotic strain L1 of Ganoderma lucidum‘Hunong No.1’cultivar by polyethylene glycol(PEG)mediated transformation to yield L1-cas9 that contained the complete expression cassette of cas9.Using T7 promoter,expression plasmids psgRNA-1 and psgRNA-2 targeting ura3 were constructed,and then sgRNA 1 and sgRNA 2 were obtained by in vitro transcription.Through PEG-mediated transformation,sgRNA 1 and sgRNA 2 were transformed into protoplasts of L1-cas9.For the first time,a CRISPR/Cas9 gene editing system was constructed in G.lucidum‘Hunong No.1’cultivar,and the gene editing efficiency for ura3 was four mutants per 107 protoplasts.Using 0.006%Triton X-100,the PEG-mediated transformation system was optimiz ed,and the editing efficiency increased to greater than 18 mutants per 107 protoplasts.This study provided an efficient tool for studying gene function and molecular breeding in G.lucidum.

关 键 词：‘沪农灵芝1号’ 基因破坏 成簇的规律间隔的短回文重复序列及其相关蛋白9(CRISPR/Cas9) 曲拉通X-100 

分 类 号：S567.31[农业科学—中草药栽培]