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2025年4月4日 星期五
农业生物技术学报  2021, Vol. 29 Issue (9): 1698-1709    DOI: 10.3969/j.issn.1674-7968.2021.09.005
  研究论文与报告 本期目录 | 过刊浏览 | 高级检索 |
烟草NtMYB4a基因CRISPR/Cas9敲除载体构建与遗传转化
蒋悦1, 罗倩1, 杨琴2, 李红强1, 谢瑞莹1, 聂琼1,2,*
1 贵州大学 烟草学院/贵州省烟草品质研究重点实验室,贵阳 550025;
2 贵州大学 农学院,贵阳 550025
Construction and Genetic Transformation of Tobacco (Nicotiana tabacum) NtMYB4a Gene CRISPR/Cas9 Knockout Vector
JIANG Yue1, LUO Qian1, YANG Qin2, LI Hong-Qiang1, XIE Rui-Ying1, NIE Qiong1,2,*
1 College of Tobacco Science/Key Laboratory of Tobacco Quality in Guizhou Province, Guizhou University, Guiyang 550025, China;
2 College of Agriculture, Guizhou University, Guiyang 550025, China
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摘要 MYB转录因子在植物生长发育和抵御胁迫中起着重要的作用。为开展烟草(Nicotiana tabacum) NtMYB4a的功能研究和新种质的创制,本研究以NtMYB4a为靶基因,根据其CDS序列在第1和第3外显子上选定两个靶标Y1和B1,分别合成Oligo二聚体后和CRISPR载体连接,转化大肠杆菌(Escherichia coil)感受态DH5α后提取质粒并测序,将测序正确的质粒Y1-1和B1-1经LguⅠ酶切后用T4连接酶进行重组连接,成功构建了CRISPR/Cas9编辑系统的双靶标敲除载体,利用农杆菌(Agrobacterium tumefaciens)介导的叶盘法来转化烟草,经PCR和测序鉴定获得29株阳性植株,阳性率为24.37%,其中15株在靶点和非靶点处发生了不同程度的突变,共有4种突变类型,突变率为51.72%。初步观察发现NtMYB4a编辑T0代植株出现不同程度的矮化和花色变异,甚至死亡。qRT-PCR分析发现T0代植株花、茎、叶中NtMYB4a表达量不同程度下调。本研究为后续开展NtMYB4a的功能和以NtMYB4a为节点的分子调控机制研究提供了基础资料。
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蒋悦
罗倩
杨琴
李红强
谢瑞莹
聂琼
关键词 烟草NtMYB4a基因基因敲除载体构建遗传转化CRISPR/Cas9    
Abstract:MYB transcription factors play an important role in plant growth and development and resistance to stress. In order to carry out the functional study of the tobacco (Nicotiana tabacum) NtMYB4a gene and the creation of a new germplasm, this study took the tobacco NtMYB4a as the target gene, selected two targets Y1 and B1 on exons 1 and 3 based on its CDS sequence, synthesized oligo dimers separately and connected with CRISPR vector, transformed Escherichia coil competent DH5α, extracted the plasmid and sequenced, and the correct Y1-1 and B1-1 were digested with LguⅠ and then recombined with T4 ligase. The CRISPR/Cas9 editing system double target knockout vector was successfully constructed, and through Agrobacterium tumefaciens mediated leaf disc method to transform tobacco. After PCR and sequencing, 29 positive plants containing Cas9 and Hyg resistance genes and target genes were obtained, and the positive rate was 24.37%. The PCR products of 29 positive tobacco strains were sequenced. Among them, 15 strains had different degrees of mutations at the target and non-target sites. There were 4 mutation types, and the mutation rate was 51.72%. The results showed that plants in the edited NtMYB4a T0 generation appeared dwarfing and flower color variation, and some even died. qRT-PCR analysis showed that the expression of NtMYB4a gene was down-regulated in the flowers, stems and leaves of tobacco plants of the T0 generation to varying degrees. This study provides basic information for the further study on the function of NtMYB4a and the molecular regulation mechanism of NtMYB4a.
Key wordsTobacco    NtMYB4a gene    Gene knockout    Vector construction    Genetic transformation    CRISPR/Cas9
收稿日期: 2020-12-28     
ZTFLH:  S572  
基金资助:贵州省科技计划项目(黔科合基础[2019]1409); 贵州大学人才引进项目(2018) 38号
通讯作者: * nqiong10@163.com   
引用本文:   
蒋悦, 罗倩, 杨琴, 李红强, 谢瑞莹, 聂琼. 烟草NtMYB4a基因CRISPR/Cas9敲除载体构建与遗传转化[J]. 农业生物技术学报, 2021, 29(9): 1698-1709.
JIANG Yue, LUO Qian, YANG Qin, LI Hong-Qiang, XIE Rui-Ying, NIE Qiong. Construction and Genetic Transformation of Tobacco (Nicotiana tabacum) NtMYB4a Gene CRISPR/Cas9 Knockout Vector. 农业生物技术学报, 2021, 29(9): 1698-1709.
链接本文:  
http://journal05.magtech.org.cn/Jwk_ny/CN/10.3969/j.issn.1674-7968.2021.09.005     或     http://journal05.magtech.org.cn/Jwk_ny/CN/Y2021/V29/I9/1698
 
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