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园艺学报 ›› 2021, Vol. 48 ›› Issue (5): 873-882.doi: 10.16420/j.issn.0513-353x.2020-1829

• 研究论文 • 上一篇    下一篇

梨愈伤组织双靶点CRISPR/Cas9基因编辑系统的建立

杨锋, 杨钦淞, 高雨豪, 马云晶, 许英, 滕元文, 白松龄*()   

  1. 浙江大学农业与生物技术学院园艺系,农业部园艺植物生长发育与品质调控重点实验室,杭州 310058
  • 收稿日期:2020-10-10 修回日期:2021-01-26 出版日期:2021-05-25 发布日期:2021-06-07
  • 通讯作者: 白松龄 E-mail:songlingbai@zju.edu.cn
  • 基金资助:
    国家重点研发计划项目(2018YFD1000100);国家自然科学基金项目(31772272)

Establishment of Dual-cut CRISPR/Cas9 Gene Editing System in Pear Calli

YANG Feng, YANG Qinsong, GAO Yuhao, MA Yunjing, XU Ying, TENG Yuanwen, BAI Songling*()   

  1. Department of Horticulture,College of Agriculture and Biotechnology,Zhejiang University,State Agriculture Ministry Key Laboratory of Horticultural Plant Growth,Development & Quality Improvement,Hangzhou 310058,China
  • Received:2020-10-10 Revised:2021-01-26 Online:2021-05-25 Published:2021-06-07
  • Contact: BAI Songling E-mail:songlingbai@zju.edu.cn

摘要:

为了在梨愈伤组织中建立CRISPR/Cas9基因编辑体系,将proDAM3:GUS转入‘茄梨’愈伤组织,通过构建双靶点的CRISPR/Cas9基因编辑载体,利用农杆菌介导的遗传转化法侵染proDAM3:GUS转基因梨愈伤组织,通过测序及GUS染色的方法检验敲除效果,分析靶基因突变类型。结果表明,共有4个转基因株系在靶位点处发生了基因突变,编辑效率为66.7%。对所有基因编辑株系的有效克隆进行分析,结果显示sgRNA1在靶位点GUST1的突变频率达到71.4%,高于sgRNA2在靶位点GUST2突变频率的46.4%,说明sgRNA1可以更有效地与靶位点结合。测序结果表明,4个基因编辑株系中GUS基因均被敲除,基因突变的类型包含碱基缺失、插入及两个靶点之间大片段的缺失,不存在碱基替换。GUS染色结果显示,基因敲除后的GUS转基因愈伤完全呈白色,说明利用CRISPR/Cas9多靶点基因编辑系统可以在梨愈伤组织中实现高效的基因敲除。

关键词: 梨, 愈伤组织, 基因编辑, 多靶点, CRISPR/Cas9

Abstract:

This work aims to establish the CRISPR/Cas9 gene editing system using European pear calli,and provides a technical platform for functional genomics study and molecular breeding of pear. We edited theGUS gene in proDAM3:GUS transgenic pear calli using a dual-cut strategy. Based on the sequence characteristics of the GUS gene,two sgRNAs were designed to target the second exon of the GUS gene. The corresponding GUS-pYLCRISPR/Cas9 plasmids were constructed and transformed into the proDAM3:GUS transgenic calli by Agrobacterium-mediated transformation. The results from sequencing data revealed that four out of six independent transgenic lines carried mutations at the target sites,with mutation rates of 66.7%. A total of 56 successfully sequenced clones from the gene edited lines were generated,which indicated that sgRNA1 target had the higher mutation frequency(71.4%)than that of sgRNA2 target(46.4%). We further analyzed the mutation types of the four GUS editing lines,and found that #1,#2,#3 lines showed deletion and insertion of nucleotides,while #5 lines showed deletion of large fragments between two target sites,whereas no nucleotide substitution was observed in any edited line. To check the phenotype,GUS staining showed that the control calli were blue in color,while the four gene editing lines were white,indicating that the CRISPR/Cas9 system was a powerful and precise method to induce targeted mutagenesis in pear calli.

Key words: pear, calli, gene editing, multiple target sites, CRISPR/Cas9

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