利用真空渗透和CRISPR/Cas9系统获得非转基因菜薹突变体  

Production of Marker-free Mutants of Brassica campestris Mediated by CRISPR/Cas9 Through Vacuum Infiltration

作　　者：宗梅[1] 韩硕[1] 郭宁[1] 段蒙蒙 刘凡[1] 王桂香[1] ZONG Mei;HAN Shuo;GUO Ning;DUAN Meng-meng;LIU Fan;WANG Gui-xiang(Vegetable Research Institute of Beijing Academy of Agriculture and Forestry Sciences/Key Laboratory of Biology and Genetic Improvement of Horticultural Crops(North China),Ministry of Agriculture and Rural Affairs,P.R.China/Beijing Key Laboratory of Vegetable Germplasm Improvement,Beijing 100097)

机构地区：[1]北京市农林科学院蔬菜研究所,农业农村部华北地区园艺作物生物学与种质创制重点实验室,蔬菜种质改良北京市重点实验室,北京100097

出　　处：《生物技术通报》2022年第10期159-163,共5页Biotechnology Bulletin

基　　金：北京市农林科学院创新能力建设项目(KJCX20200401,KJCX20200205,KJCX20200113);国家自然科学基金项目(31972401)。

摘　　要：通过CRISPR/Cas9基因编辑系统获得非转基因菜薹突变体,实现CRISPR/Cas9体系在菜薹原位转化中的应用,为无选择标记的基因编辑技术应用提供参考。以‘49菜心’为试材,番茄红素脱氢酶基因(PDS)为靶基因,采用真空渗透原位转化方法,转化菜薹25株。结果表明,在2032株原位转化种子苗中鉴定出3株发生PDS基因编辑,其中1株有外源转化载体插入,PDS发生杂合编辑,表型与野生型一致。另外2株具有矮化和失绿表型,且基因组无外源载体片段插入,PDS发生不同类型的敲除突变。CRISPR/Cas9通过不依赖组织培养的原位转化可以在菜薹中实现基因编辑,并能直接获得无外源插入的非转基因编辑突变体。The objective is to acquire non-transgenic Brassica campestris mutants via CRISPR/Cas9 gene editing system,for achieving the application of CRISPR/Cas9 in B.campestris in planta transformation method and providing reference for application of marker-free gene editing technique.Using‘49 Caixin’as plant material and dehydrogenase gene(PDS)as target gene,total of 25 plants were transformed by in planta transformation via vacuum-infiltration method.The results showed that 3 of the 2032 transformed seedlings were identified to have PDS gene editing,one of which was detected to have exogenous vector insertion and heterozygous editing of PDS gene,and the phenotype was consistent with that of the wild type.The other two seedlings had a dwarf,albino phenotype,and there was no insertion of foreign vector in their genomes.Different types of targeted mutagenesis of PDS gene were detected in these two seedlings compared with the wild type.In conclusion,gene editing can be achieved in B.campestris by CRISPR/Cas9 through in planta tissue culture-independent transformation,and particularly non-transgenic editing mutants without exogenous insertion can be obtained directly.

关 键 词：菜薹 无筛选标记 真空渗透转化 不依赖组织培养 CRISPR/Cas9 

分 类 号：S634.5[农业科学—蔬菜学] Q943.2[农业科学—园艺学]