食用菌学报2025，Vol.32Issue(3)：1-12,12.DOI:10.16488/j.cnki.1005-9873.2025.03.001
利用CRISPR/Cas9基因编辑与杂交技术靶向创制灵芝基因位点纯合突变株的方法
CRISPR/Cas9-Assisted Hybridization Strategy for Generating Homozygous Mutants at Targeted Locus in Ganoderma lucidum
摘要
Abstract
Using the auxotrophic screening marker gene ura3 as the target,CRISPR/Cas9 ribonucleoprotein(RNP)-mediated gene editing was carried out on dikaryotic Ganoderma lucidum strain G0119(cultivar'Hunong Lingzhi No.1')and its mating-compatible monokaryotic strains L1 and L2.The editing efficiencies between the monokaryotic and dikaryotic strains were compared.The edited monokaryotic strains of L1 and L2 were crossed to generate dikaryotic strains with homozygous mutations in ura3.The results showed that seven edited strains of G0 119 were obtained.Crossing of these edited G0119 strains with edited strains of L1 and L2 showed that all seven edited G0 119 strains were heterozygous:six of them were edited in their L1-type nuclei,and the remaining one was edited in its L2-type nuclei.There were 10 and 7 monokaryotic edited strains of L1 and L2,respectively,and the editing efficiency of L1 nuclei was 100％.Crossings of monokaryotic edited strains L1-2×L2-1,both having a C-base deletion in ura3,and monokaryotic edited strains L1-5×L2-3,both containing an A-base insertion in ura3,generated two dikaryotic homozygous ura3 disrupted strains.This is a showcase of obtaining dikaryotic G.lucidum mutants with homozygous mutations in targeted genes through CRISPR/Cas9 ribonucleoprotein-mediated gene editing and single hybridization.This strategy also provided a new approach for phenotypic analysis of dikaryotic G.lucidum strains by enabling precise genetic manipulation of targeted genes.关键词
灵芝/双核体/基因位点纯合菌株/CRISPR/Cas9/单单杂交Key words
Ganoderma lucidum/dikaryon/homozygous strain of targeted gene editing/CRISPR/Cas9/mon-mon crossing引用本文复制引用
田佳琳,董蓓蓓,唐传红,周帅,冯娜,冯杰,张劲松,谭贻..利用CRISPR/Cas9基因编辑与杂交技术靶向创制灵芝基因位点纯合突变株的方法[J].食用菌学报,2025,32(3):1-12,12.基金项目
国家重点研发计划(2023YFD1600501) （2023YFD1600501）
