Efficient CELI endonuclease production in Nicotiana benthamiana through transient expression and applications in detections of mutation and gene editing events
文献类型：期刊论文
| 作者 | Yao, Xue-Feng; Wu, Shengyang; Guo, Lei5; Liu, Chun-Ming
|
| 刊名 | PLANT SCIENCE
 |
| 出版日期 | 2020 |
| 卷号 | 296 |
| 关键词 | Production of CELI endonuclease Optimization of CELI digestion Transient expression Detections of mismatch and gene editing |
| ISSN号 | 0168-9452 |
| DOI | 10.1016/j.plantsci.2020.110469 |
| 文献子类 | Article |
| 英文摘要 | Rapid and low-cost methods of detecting mutations and polymorphisms are crucial for genotyping applications including mutagenesis and gene editing. S1 family endonucleases such as T7E1, EndoV and CELI can potentially be used in enzymatic mismatch detection. Among them, CELI has been shown to be effective in detecting mutations in Targeting Induced Local Lesions IN Genomes (TILLING). However, current method of CELI purification from celery is laborious, and challenging for many non-biochemical laboratories, and the presence of post-translational modifications hinders efficient production of the enzyme in E. coli. Here, we report an efficient system for bulk production of enzymatically active CELI endonuclease through transient expression in a model plant Nicotiana benthamiana. We also optimized the reaction buffer, by additions of Mn2+ and DTT, with enhanced mismatch cleavage activity. Using the new CELI production and reaction system, we were able to routinely detect mismatches in 1/32 mixed mutant and wildtype DNA samples. We believe the newly established system has many applications in characterization of mutations occurred in natural variations, mutagenized populations and gene editing. |
| 学科主题 | Biochemistry & Molecular Biology ; Plant Sciences |
| 出版地 | CLARE |
| WOS关键词 | PLANTS ; CRISPR/CAS9 ; GENERATION ; PROTOCOL ; SYSTEM |
| WOS研究方向 | Biochemistry & Molecular Biology ; Plant Sciences |
| 语种 | 英语 |
| WOS记录号 | WOS:000541125700004 |
| 出版者 | ELSEVIER IRELAND LTD |
| 资助机构 | National Transgenic Research Projects of China [2019ZX08010003-002-009, 2016ZX08009-001-006-002, 2016ZX08009-003-005] |
| 源URL | [http://ir.ibcas.ac.cn/handle/2S10CLM1/21715]  |
| 专题 | 中科院植物分子生理学重点实验室 |
| 作者单位 | 1.Wu, Shengyang 2.Chinese Acad Agr Sci, Inst Crop Sci, Beijing 100081, Peoples R China 3.Univ Chinese Acad Sci, Beijing 100049, Peoples R China 4.Chinese Acad Sci, Inst Bot, Key Lab Plant Mol Physiol, Beijing 100093, Peoples R China 5.[Yao, Xue-Feng |
| 推荐引用方式 GB/T 7714 | Yao, Xue-Feng,Wu, Shengyang,Guo, Lei,et al. Efficient CELI endonuclease production in Nicotiana benthamiana through transient expression and applications in detections of mutation and gene editing events[J]. PLANT SCIENCE,2020,296. |
| APA | Yao, Xue-Feng,Wu, Shengyang,Guo, Lei,&Liu, Chun-Ming.(2020).Efficient CELI endonuclease production in Nicotiana benthamiana through transient expression and applications in detections of mutation and gene editing events.PLANT SCIENCE,296. |
| MLA | Yao, Xue-Feng,et al."Efficient CELI endonuclease production in Nicotiana benthamiana through transient expression and applications in detections of mutation and gene editing events".PLANT SCIENCE 296(2020). |
入库方式： OAI收割
来源：植物研究所
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