增强VIGE诱导靶基因突变的拟南芥Cas9表达株系筛选

doi:10.3969/j.issn.1674-7968.2023.07.019

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摘要植物病毒介导的基因编辑(virus-mediated gene editing, VIGE)体系可以快速筛选出高效的单向导 RNA (single guide RNA, sgRNA)用于植物目标基因的靶向编辑,为定向创制遗传突变体材料提供了基因编辑工具。为了在拟南芥(Arabidopsis thaliana)中筛选出能高效敲除目标基因的 VIGE 受体材料,本研究以拟南芥 AtBRI1 (brassinosteroid insensitive 1)和 AtGL2 (GLABRA 2)基因为靶标,利用基于棉花叶皱缩病毒(Cotton leaf crumple virus, CLCrV)介导的 VIGE 系统,探究了以不同组织特异性 Cas9 超表达(Cas9-OE) 株系作为 VIGE 受体对这 2 个基因的编辑效率。qPCR 检测了 Cas9-OE 株系中 Cas9 基因的表达量,发现 Cas9 基因在不同组织特异性 Cas9-OE 株系中稳定遗传表达。利用农杆菌(Agrobacterium)介导的瞬时转化法将 CLCrV-AtU6-26::AtBRI1-sgRNA 和 CLCrV-AtU6-26::AtGL2-sgRNA 接种不同组织特异性 Cas9-OE 植株叶片。突变检测结果表明,3 种组织特异性 Cas9-OE 株系作为 VIGE 受体均可以实现 AtBRI1 和 AtGL2 基因的靶向编辑,在这 2 个基因靶序列区域出现了不同碱基插入、替换和缺失的突变类型,证明了这 3 种组织特异性 Cas9-OE 株系作为 VIGE 受体的有效性。进一步对每一株接种 CLCrV-AtU6-26::AtBRI1-sgRNA 和 CLCrV-AtU6-26::AtGL2-sgRNA 的单株进行突变检测,突变分析结果显示,ProCDC45::Cas9 和 ProYao:: Cas9 对 AtBRI1 和 AtGL2 基因的编辑效率相对较高,编辑效率为 50%~81.25%,表明 ProCDC45:: Cas9 和 ProYao::Cas9 转基因拟南芥可作为理想的 VIGE 受体用于拟南芥基因编辑的研究。本研究为进一步提升拟南芥中VIGE系统的基因编辑效率和高效创制拟南芥突变体材料提供了参考。

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关键词 ：病毒介导的基因编辑(VIGE), 拟南芥, 棉花叶皱缩病毒(CLCrV), 组织特异性, Cas9超表达株系

Abstract：Plant virus-mediated gene editing (VIGE) system can rapidly screen high-efficiency sgRNA (single guide RNA) for targeted editing of plant genes, providing a gene editing tool for the targeted creation of genetic mutant materials. In order to screen the VIGE receptor materials capable of efficiently knockout the target genes in Arabidopsis thaliana, this study using the AtBRI1 (brassinosteroid insensitive 1) and AtGL2 (GLABRA 2) as target genes, and explored the editing efficiency of different tissue-specific Cas9 overexpression (Cas9-OE) lines as VIGE receptors for these 2 genes based on the Cotton leaf crumple virus (CLCrV)-mediated VIGE system. The expression of Cas9 gene in different tissue-specific Cas9-OE lines was examined by qPCR, and it was found that Cas9 gene was stably genetically expressed in different tissue-specific Cas9-OE lines. The leaves of different tissue-specific Cas9-OE plants were inoculated with CLCrV- AtU6-26:: AtBRI1-sgRNA and CLCrV-AtU6-26:: AtGL2-sgRNA by Agrobacterium-mediated transient transformation. Mutation detection results showed that the 3 tissue-specific Cas9-OE lines as VIGE receptors could achieve targeted editing of AtBRI1 and AtGL2 genes, and different base insertions, substitutions and deletion mutation types appeared in the target sequence regions of these 2 genes, demonstrating the effectiveness of these 3 tissue-specific Cas9-OE lines as VIGE receptors. Further mutation detection was performed on each individual plant inoculated with CLCrV-AtU6-26:: AtBRI1-sgRNA and CLCrV-AtU6-26::AtGL2-sgRNA. The mutation analysis showed that ProCDC45:: Cas9 and ProYao:: Cas9 had relatively high editing efficiency for AtBRI1 and AtGL2 genes with editing efficiency of 50%~81.25%, which proved that ProCDC45:: Cas9 and ProYao:: Cas9 transgenic Arabidopsis can be used as ideal VIGE receptors for gene editing research in Arabidopsis. The study provides a basis for further improving the gene editing efficiency of the VIGE system in Arabidopsis and efficiently creating Arabidopsis mutant materials.

Key words： Virus-mediated gene editing (VIGE) Arabidopsis Cotton leaf crumple virus (CLCrV) Tissue- specificity Cas9 over-expression lines (Cas9-OE) Mutant

收稿日期: 2022-07-31

基金资助:新疆维吾尔自治区高校基本科研业务费科研项目(XJEDU2022J007)；新疆农业大学大学生创新项目(dxscx2023013)

通讯作者: **kyleijianfeng@163.com

作者简介: *同等贡献作者

引用本文:

彭静, 麦尔哈巴·海散, 陈林羊, 卡米燃·买吐送, 林嘉豪, 刘壮, 雷建峰. 增强VIGE诱导靶基因突变的拟南芥Cas9表达株系筛选[J]. 农业生物技术学报, 2023, 31(7): 1547-1554.
PENG Jing ,MAIERHABAHai-San ,CHEN Lin-Yang,KAMIRANMai-Tu-Song,LIN Jia-Hao,LIU Zhuang,LEI Jian-Feng . Screening of Arabidopsis thaliana Cas9-expressing Lines with Enhanced VIGE-induced Target Gene Mutations. 农业生物技术学报, 2023, 31(7): 1547-1554.

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http://journal05.magtech.org.cn/Jwk_ny/CN/10.3969/j.issn.1674-7968.2023.07.019 或 http://journal05.magtech.org.cn/Jwk_ny/CN/Y2023/V31/I7/1547

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