香菇中CRISPR/Cas9基因编辑体系的构建  

Construction of CRISPR/Cas9 gene editing technology in Lentinula edodes

作　　者：付阳 宋春艳[1] 董晶晶 刘建雨 姜宁[1] 章炉军[1] 于海龙[1] 尚晓冬[1] 谭琦[1] FU Yang;SONG Chunyan;DONG Jingjing;LIU Jianyu;JIANG Ning;ZHANG Lujun;YU Hailong;SHANG Xiaodong;TAN Qi(National Research Center of Edible Fungi Biotechnology and Engineering,Institute of Edible Fungi,Shanghai Academy of Agricultural Sciences,Shanghai 201403,China;College of Food Science&Technology,Shanghai Ocean University,Shanghai 201306,China)

机构地区：[1]上海市农业科学院食用菌研究所国家食用菌工程技术研究中心,上海201403 [2]上海海洋大学食品学院,上海201306

出　　处：《菌物学报》2024年第8期23-39,共17页Mycosystema

基　　金：国家重点研发计划(2023YFD1201600);上海市农业科学院“卓越团队”建设计划(沪农科卓[2022]001)。

摘　　要：香菇Lentinula edodes是世界第二大食药用菌,具有较高的食用、药用、经济和生态学研究价值,探索其本身具备优良性状的调控机制,同时对可能参与其中的功能基因进行反义遗传学研究是十分必要的。本研究对已知的功能基因利用CRISPR/Cas9基因编辑技术进行编辑,确定该技术能在香菇中工作。预测并筛选出一个香菇U6snRNA启动子,以乳清苷-5'-磷酸脱羧酶编码基因pyrG为目的基因设计CRISPR/Cas9技术的重要工作元件sgRNA,对异源cas9基因依据香菇基因组密码子偏好性进行优化,构建了一个同时携带sgRNA和cas9基因的双元表达载体。利用农杆菌介导的香菇菌丝遗传转化技术将该载体转入香菇单核体中对pyrG基因进行编辑,成功获得pyrG基因发生碱基缺失的香菇单核体突变株,经功能筛选后确定其为尿嘧啶营养缺陷型单核体菌株。本研究成功构建了一个能同时表达CRISPR/Cas9技术重要组件的双元表达载体,证明了CRISPR/Cas9能够在香菇中发挥编辑目的基因的功能,为进一步开展香菇重要功能基因的研究奠定了良好的基础。Lentinula edodes is edible and medicinal mushroom with the second largest yield and output value in the world.It is necessary to explore the regulatory mechanism of qualified characteristics and the important functional genes related to characteristics in L.edodes.In this study,a known functional gene is edited by using CRISPR/Cas9 technology,and CRISPR/Cas9 is confirmed to work in L.edodes.By predicting and screening a U6 snRNA promoter in L.edodes,sgRNA was designed using a pyrG encoding gene of orotidine-5ʹ-monophosphate decarboxylase as the target gene for CRISPR/Cas9.According to the codon preference of L.edodes genome,the cas9 gene that is the core component of CRISPR/Cas9 system is optimized.A binary expression vector with both sgRNA and cas9 gene was constructed.The vector was transformed into the monokaryon by Agrobacterium-mediated genetic transformation of L.edodes mycelium to make the mutation of pyrG gene.The L.edodes monokaryon mutant with base deletion in pyrG gene was obtained successfully and the mutant was determined by functional screening as an uracil trophic deficiency strain.In this study,a binary expression vector was successfully constructed,which could simultaneously express the two important components of CRISPR/Cas9,and the purpose of using CRISPR/Cas9 to edit functional genes was realized.

关 键 词：香菇 CRISPR/Cas9基因编辑技术 pyrG基因 尿嘧啶营养缺陷型 功能基因 

分 类 号：S646.12[农业科学—蔬菜学]